Kentaro Kogure, Akira Tokumura and Kenji Fukuzawa : ビタミンE研究の進歩 X, KYORITSU SHUPPAN.CO.,LTD, Tokyo, 2002.
12.
濱 進, Kentaro Kogure, 森田 元喜, Akira Tokumura and Kenji Fukuzawa : ビタミンE研究の進歩 X, KYORITSU SHUPPAN.CO.,LTD, Tokyo, 2002.
Academic Paper (Judged Full Paper):
1.
Susumu Hama, Yuriko Okamura, Kazuho Kamei, Saki Nagao, Mari Hayashi, Maeda Shizuka, Kenji Fukuzawa and Kentaro Kogure : α-Tocopheryl succinate stabilizes the structure of tumor vessels by inhibiting angiopoietin-2 expression, Biochemical and Biophysical Research Communications, 2020.
(Summary)
α-Tocopheryl succinate (TS) is a tocopherol derivative and has multifaceted anti-cancer effects; TS not only causes cancer cell-specific apoptosis but also inhibits tumor angiogenesis. Although TS has the potential to be used as a well-tolerated anti-angiogenic drug, it is still unclear which step of the angiogenic process is inhibited by TS. Here, we show that TS inhibits the expression of angiopoietin (Ang)-2, which induces destabilization of vascular structure in the initial steps of the angiogenic process. In mouse melanoma cells, TS treatment decreased mRNA and extracellular protein levels of Ang-2; however, the mRNA level of Ang-1, which stabilizes the vascular structure, remained unchanged. Furthermore, aorta ring and Matrigel plug angiogenesis assays indicated that the conditioned medium from TS-treated cells (CM-TS) inhibited neovascularization and blood leakage from the existing blood vessels, respectively. Following immunohistochemical staining of the vessels treated with CM-TS, imaging studies showed that the vascular endothelial cells were highly packed with pericytes. In conclusion, we found that TS inhibits Ang-2 expression and, consequently, stabilizes the vascular structure during the initial step of tumor angiogenesis.
MIka Adachi, Gou Horiuchi, Natsuki Ikematsu, Tamotsu Tanaka, Shigeki Sano, Kenji Fukuzawa and Akira Tokumura : Intragastrically administrered lysophospatidic acid protect against gastric ulcer in rats under water-immersion restraint stress, Digestive Diseases and Sciences, Vol.56, No.8, 2252-2261, 2011.
(Summary)
Lysophosphatidic acid exerts important physiological effects on many types of animal cells through its specific binding to several G protein-coupled receptors. In particular, its potent wound-healing effect has attracted much attention. To determine whether lysophosphatidic acids in a foodstuff and Chinese medicine are effective in protecting against gastric ulcer, we subjected rats to water-immersion restraint stress. Three direct administrations of a solution of lysophosphatidic acid with a C18 fatty acyl group to the rat stomach in a concentration range of 0.001-0.1 mM resulted in a significant reduction in the number of gastric ulcers induced during water-immersion restraint stress, and the potencies were as follows: linoleoyl species=α-linolenoyl species>oleoyl species. Intragastric administrations of a solution of highly purified lysophosphatidic acid from soybean lecithin significantly protected against the stress-induced gastric ulcers at lower concentrations than partially purified lysophosphatidic acid from soybean lecithin did. In addition, administration of a decocted solution of antyu-san, and lysophosphatidic acid-rich Chinese medicine, to the stomach was more effective in protecting against stress-induced ulcer than decoctations of antyu-san lacking the corydalis tuber component that is rich in lysophosphatidic acid. These results clearly show that lysophosphatidic acid is the effective component of soybean lecithin and antyu-san in protection against stress-induced gastric ulcer in the rat model, and suggest that daily intake of lysophosphatidic acid-rich foods or Chinese medicines may be beneficial for prevention of stress-induced gastric ulcer in human subjects.
Kaori Ueda, Masanori Yoshihara, Michiyasu Nakao, Tamotsu Tanaka, Shigeki Sano, Kenji Fukuzawa and Akira Tokumura : Evaluation of inhibitory actions of flavonols and related substances on lysophospholipase d activity of serum autotaxin by a convenient assay using a chromogenic substrate., Journal of Agricultural and Food Chemistry, Vol.58, No.10, 6053-6063, 2010.
(Summary)
Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be utilized to ameliorate the LPA-related pathological conditions. In this study, a new assay was devised to quantify p-nitrophenol from hydrolysis of chromogenic substrate by serum lysoPLD without tedious lipid extraction procedures. Flavonols, phenolic acids, free fatty acids, and N-acyltyrosines inhibited lysoPLD activity in a micromolar range. They were classified into competitive, noncompetitive, or mixed type inhibitors. The results show that the low hydrophobicity of an inhibitor is a critical factor in its preference for the binding to a noncatalytic binding site over a catalytic binding site. Considering its reported bioavailability and the low dependency of its inhibitory activity on serum dilution, flavonol is likely to be a more effective lysoPLD inhibitor in human blood circulation in vivo than the other inhibitors including LPA.
Akira Tokumura, Tetsuya Kume, Kenji Fukuzawa, Masahiro Tahara, Keiichi Tasaka, Junken Aoki, Hiroyuki Arai, Katsuhiko Yasuda and Hideyo Kanzaki : Peritoneal fluids from patients with certain gynecologic tumor contain elevated levels of bioactive lysophospholipase D activity, Life Sciences, Vol.80, No.18, 1641-1649, 2007.
(Summary)
Levels of lysophosphatidic acid (LPA), an important phospholipid mediator, in serum and ascitic fluid from ovarian cancer patients were shown to be higher than those from healthy women and from patients with other type of cancer, respectively. Although LPA in human serum seems mainly to be generated by lysophospholipase D (lysoPLD), the source and pathway for LPA in the ascitic fluid remain still obscure. In this study, we examined whether lysoPLD activity producing bioactive LPA in human peritoneal fluid was significantly elevated under pathological statuses. Lysophospholipase D activity in human peritoneal fluids was measured by quantifying choline released from exogenous lysophosphatidylcholine on their incubation at 37 degrees C. We also compared the activity of lysoPLD in sera from patients with different gynecologic diseases. We found relatively high lysoPLD activity in peritoneal fluids from patients with ovarian cancer, dermoid cyst or mucinous cystadenoma, whereas there were no significant differences in the serum lysoPLD activity among clinical groups and healthy subjects. The lysoPLD in the peritoneal fluid was found to have similar substrate specificity and metal ion requirement to those of serum lysoPLD, that has been identified as autotaxin, a tumor cell-motility stimulating protein. Our results suggest that increased lysoPLD activity in peritoneal fluid from patients with certain gynecologic tumors might be relevant to its potential of tumor progression.
Junichi Morishige, Kanako Touchika, Tamotsu Tanaka, Kiyoshi Satouchi, Kenji Fukuzawa and Akira Tokumura : Production of bioactive lysophosphatidic acid by lysophospholipase D in hen egg white, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1771, No.4, 491-499, 2007.
(Summary)
Lysophosphatidic acid (LPA), a lysophospholipid mediator, is produced extracellularly by lysophospholipase D (lysoPLD) secreted in several animal body fluids including blood plasma. Previously, we reported that hen egg white contains polyunsaturated fatty acid-rich LPA. In this study, we examined whether lysoPLD is involved in the production of LPA in hen egg white. LysoPLD activity was measured by determining LPA and choline by mass spectrometric and enzyme-linked fluorometric analyses, respectively. LysoPLD increased with increased dilution of egg white, indicating that one or more components of egg white strongly inhibit its lysoPLD activity. This dilution-dependent increase in the lysoPLD activity was masked by co-incubation of the egg white with lysozyme, a major protein in hen egg white. Furthermore, addition of Zn(2+), Mn(2+), Ni(2+), or Co(2+) to diluted egg white altered preference patterns of lysoPLD toward choline-containing substrates. In particular, the egg white lysoPLD activity was greatly increased when Co(2+) was added. The cation-requirement of lysoPLD activity in hen egg white resembled that of plasma autotaxin (ATX)/lysoPLD. Western blot analysis revealed that egg white contained a protein that was immunostained with anti-ATX antibody. These results suggested that LPA in hen egg white is produced from lysophospholipids, especially LPC, by the action of ATX/lysoPLD, possibly originating from hen oviduct fluid.
Toshihiko Tsutsumi, Junichi Morishige, Kenji Fukuzawa and Akira Tokumura : Movememt of monoglyceride derived from hydrolysis of fluorescence-labeled lyso platelet-activating factor by lysophospholipase C through plasma membranes of porcinr kidney epithelial cell line LLC-PK1, Prostaglandins & Other Lipid Mediators, Vol.83, No.1-2, 33-41, 2007.
(Summary)
To investigate the mechanisms of the release of lyso platelet-activating factor (PAF), an alkyl ether-linked lysophosphatidylcholine, from the kidney epithelial cell line LLC-PK1, the cell monolayer was incubated with a fluorescence-labeled lysoPAF analog, Bodipy-lysoPAF, on either the basolateral or apical side. The fluorescent lipids in the culture media mixed with or without bovine serum albumin at a final concentration of 2% were analyzed by thin layer chromatography. In both cases, two major bands, assignable to Bodipy-lysoPAF and Bodipy-monoglyceride (MG), were detected in the culture medium to which Bodipy-lysoPAF had been added, whereas the culture medium at the opposite side exhibited only the major band of Bodipy-MG. Our results suggest that lysoPAF was degraded by high ecto-lysophospholipase C activity. The possible physiological significance of this metabolic pathway is discussed.
Kenji Fukuzawa, Aya Fujisaki, Kaori Akai, Akira Tokumura, Junji Terao and Jansuz M. Gebicki : Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay, Analytical Biochemistry: Methods in the Biological Sciences, Vol.359, No.1, 18-25, 2006.
(Summary)
Formation of a colored complex between ferric iron and xylenol orange (XO) has been used for the determination of hydroperoxides (FOX method). Original or modified FOX methods were performed on aqueous or organic solutions consisting of a single phase. However, for lipid peroxides in heterogeneous samples, such as biological materials, much of the lipid is sequestered in a separate phase. Organic solvent extraction of these lipids is often incomplete and may result in additional peroxidation during the extraction procedure. In this study, we applied the FOX assay for measurement of the membrane phosphatidylcholine hydroperoxides (PC-OOH) in separated phases. The presence of membranous egg yolk phosphatidylcholine (EYPC) in 60% MeOH shifted the broad peak at 560 nm of Fe(3+)-XO complex to 610 nm with a sharp peak associated with the increased intensity of the absorbance. The shift of the peak is useful to measure the unknown amounts of Fe(3+) because the uncomplexed XO considerably contributed to the absorbance of the peak at 560 nm but did not affect the absorbance at 610 nm. EYPC was required to form the membranes to shift the peak because the shift occurred in 60% MeOH but did not by the treatment with detergents or in 90% MeOH in which EYPC did not form the membranes. The molar absorption coefficient (epsilon 610) was 32,700 M(-1) cm(-1), which was about twice the molar absorption co efficient (epsilon 560) reported. We applied this method to the assay of PC-OOH prepared from EYPC and obtained the molar absorption coefficients (epsilon 610), which were 79,100 and 115,700 M(-1) cm(-1) in the presence and absence of BHT, respectively. This finding allows the determination of PC-OOH concentration even in chemically complex systems.
Kentaro Kogure, Sachie Manabe, Ichiro Suzuki, Akira Tokumura and Kenji Fukuzawa : Cytotoxicity of α-tocopheryl succinate, malonate and oxalate in normal and cancer cells in vitro and their anti-cancer effects on mouse melanoma in vivo, Journal of Nutritional Science and Vitaminology, Vol.51, No.6, 392-397, 2005.
(Summary)
alpha-Tocopheryl succinate (TS), which is known to induce apoptosis selectively in cancer cells, has attracted attention as a chemotherapeutic agent. Recently, we found that alpha-tocopheryl malonate (TM) and alpha-tocopheryl oxalate (TO), among the alpha-tocopheryl esters tested, have high apoptogenic activity as well as TS. In this study, we investigated the characteristics of their cytotoxicity on normal cells and cancer cells in vitro, and their anti-cancer effects on mice inoculated with melanoma B16-F1 cells in vivo. The order of in vitro cytotoxicity was TO > or = TM > TS in all cell lines examined. Addition of exogenous superoxide dismutase (SOD) and the antioxidant N-acetyl cysteine (NAC) inhibited TS- and TM- but not TO-induced cell deaths. A selective cytotoxic effect on cancer cells was observed with TS but not with TM or TO. c-Jun N-terminal kinase (JNK) inhibitor II prevented cell death induced by TS but did not prevent cell deaths induced either by TM or TO. Intravenous administration of vesiculated TS and TM to mice inoculated with melanoma B16-F1 cells prevented tumor growth and enhanced the mean survival time, but TO administration killed the mice due to its acute high toxicity. From these results, we discussed the characteristics of their selective cytotoxicity toward tumor cells in vitro and anti-cancer effects in vivo.
Koichiro Tsuchiya, Kaori Akai, Akira Tokumura, Shinji Abe, Toshiaki Tamaki, Yoshiharu Takiguchi and Kenji Fukuzawa : Oxygen radical photo-induced by ferric nitrilotriacetate complex, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1725, No.1, 111-119, 2005.
(Summary)
This study examined the photo-induced generation of reactive oxygen species (ROS) by the carcinogenic iron(III)-NTA complex. Iron(III)-NTA complex (1:1) has three conformations (type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10) with two pK(a) values (pK(a1) approximately 4, pK(a2) approximately 8). The iron(III)-NTA complex was reduced to iron(II) under cool-white fluorescent light without the presence of any reducing agent, and the reduction rates of the three conformations of iron(III)-NTA were in the order type (a)>type (n)>type (b) as reported previously (Akai K. et al., Free Radic. Res. 38, 951-962, 2004). ROS generation was investigated by electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. Apparent EPR signals attributed to PBN/*(13)CH(3) and PBN/*OCH(3) spin adducts were observed after incubation of the iron(III)-NTA complex was mixed with alpha-phenyl-tert-butylnitrone (PBN) and (13)C-DMSO in an aerobic condition. The addition of catalase effectively attenuated the PBN adducts, but superoxide dismutase enhanced them. Taken together, these results indicate that the iron(III)-NTA complex is spontaneously reduced to the iron(II)-NTA complex by light under acidic to neutral pH, and in turn transfers an electron to molecular oxygen to form ROS.
Kenji Fukuzawa, Yasuaki Saitoh, Kaori Akai, Kentaro Kogure, Satoru Ueno, Akira Tokumura, Masaki Otagiri and Akira Shibata : Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide, Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol.1668, No.1, 145-155, 2005.
(Summary)
Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.
Kentaro Kogure, Aiko Yamauchi, Akira Tokumura, Kyoko Kondou, Naonobu Tanaka, Yoshihisa Takaishi and Kenji Fukuzawa : Novel antioxidants isolated from plants of the genera Ferula, Inula, Prangos and Rheum collected in Uzbekistan, Phytomedicine, Vol.11, No.7-8, 645-651, 2004.
(Summary)
We examined the effects of 48 compounds isolated from Ferula pallida, F. penninervis, Inula macrophylla, Prangos pabularia, P. tschimganica and Rheum maximowiczii collected in Uzbekistan on ADP/Fe2+-induced lipid peroxidation of egg yolk phosphatidylcholine liposomes. Of those compounds, 23 inhibited ADP/Fe2+-induced lipid peroxidation and nine showed especially strong inhibition of lipid peroxidation. Most compounds that inhibited peroxidation scavenged the 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radical, indicating that the inhibition was due to radical scavenging. However, some compounds did not scavenge DPPH but inhibited lipid peroxidation significantly, suggesting that their inhibitory effect was not due to radical scavenging but to some other mechanism, such as prevention of Fe2+ function. Thus, we found various new antioxidants, some of which had a unique mechanism of action, in Ferula, Inula, Prangos and Rheum plants collected in Uzbekistan as seeds used in medicine.
Mamoru Okasaka, Yoshihisa Takaishi, Kentaro Kogure, Kenji Fukuzawa, Hirofumi Shibata, Tomihiko Higuti, Gisho Honda, Michiho Ito, Olimjon K. Kodzhimatov and Ozodbek Ashurmetov : New Stilbene Derivatives from Calligonum leucocladum, Journal of Natural Products, Vol.67, No.6, 1044-1046, 2004.
(Summary)
Two new stilbene derivatives, (E)-resveratrol 3-(6' '-galloyl)-O-beta-D-glucopyranoside (1) and (E)-resveratrol 3-(4' '-acetyl)-O-beta-D-xylopyranoside (2), and five known stilbene derivatives (3-7) were isolated from the dried aerial parts of Calligonum leucocladum. Their structures were established on the basis of spectroscopic evidence. Compound 1 showed antioxidant activity and a restorative effect of the inhibition of oxacillin to oxacillin/methicillin-resistant Staphylococcus aureus.
Kentaro Kogure, Susumu Hama, Mayumi Kisaki, Hideaki Takemasa, Akira Tokumura, Ichiro Suzuki and Kenji Fukuzawa : Structural characteristic of terminal dicarboxyric moiety required for apoptogenic activity of α-Tocopheryl esters., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1672, No.2, 93-99, 2004.
(Summary)
alpha-Tocopheryl succinate (TS) is known to induce apoptosis in various cells and has attracted attention as a chemotherapeutic agent. Recently, we reported the structural significance of the terminal dicarboxylic moiety for the action of TS [J. Nutr. Sci. Vitaminol. 49 (2003) 310-314]. In this study, to determine details of the relationship between the structure and the function of the terminal ester moiety of alpha-tocopherol (alpha-T), we synthesized four novel esters, alpha-tocopheryl oxalate (TO), alpha-tocopheryl malonate (TM), alpha-tocopheryl pimelate (TP) and alpha-tocopheryl succinate ethyl ester (TSE), and compared their apoptogenic activities with those of TS, alpha-T, gamma-tocopherol (gamma-T) and two commercially available alpha-T derivatives, alpha-tocopheryl nicotinate (TN) and alpha-tocopheryl acetate (TA), in vascular smooth muscle cells and a mouse breast cancer cell line C127I. TO and TM in addition to TS, but not the others, induced apoptosis in both cells. Particularly, TO was the most potent of all alpha-T derivatives used. The addition of exogenous superoxide dismutase (SOD) significantly prevented the apoptosis induced by TM as well as that by TS as reported previously, but did not affect TO-induced apoptosis. These results suggest that O(2)(-) generated exogenously participates in TM-induced apoptosis but not in TO-induced apoptosis. The difference in their apoptotic effects is attributed to structural properties of the terminal dicarboxylic moiety, which has an inflexible plane conformation in TO, while it is highly flexible in TM and TS.
Kentaro Kogure, Sawa Nakashima, Akiko Tsuchie, Akira Tokumura and Kenji Fukuzawa : Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and thair degradation product lysophosphatidylcholine., Chemistry and Physics of Lipids, Vol.126, No.1, 29-38, 2003.
(Summary)
To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.
Kentaro Kogure, Susumu Hama, Satoru GOTO, Tatsuo Munakata, Akira Tokumura and Kenji Fukuzawa : alpha-Tocopheryl succinate activates protein kinase C in cellular and cell-free systems, Journal of Nutritional Science and Vitaminology, Vol.49, No.5, 310-314, 2003.
(Summary)
The effect of alpha-tocopheryl succinate (TS) on protein kinase C (PKC) activity was examined. TS increased the auto-phosphorylation of PKC in vascular smooth muscle cells. Furthermore TS activated isolated PKC-like phorbol 12-myristate 13-acetate (PMA), although it was required at a significantly higher concentration than PMA for PKC activation. Molecular superimposition of the TS on PMA by computation suggested that TS took an active binding conformation to the PKC-like PMA, but that the conformational population was about 1/1.000. Consequently, we conclude that TS interacts directly with PKC, and activates it by taking an active conformation like PMA.
(Keyword)
Animals / Cell-Free System / Enzyme Activation / Models, Molecular / Molecular Conformation / Muscle, Smooth, Vascular / phosphorylation / Protein Kinase C / Rats / Tetradecanoylphorbol Acetate / Tocopherols / vitamin E
Kentaro Kogure, Koichiro Tsuchiya, Kazutoyo Abe, Michinori Akasu, Toshiaki Tamaki, Kenji Fukuzawa and Hiroshi Terada : Direct radical scavenging by the bisbenzylisoquinoline alkaloid cepharanthine., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1622, No.1, 1-5, 2003.
(Summary)
Cepharanthine (Ceph) is known as a potent antiperoxidative agent. Recently, we characterized the antiperoxidative effects of Ceph [Biochim. Biophys. Acta 1426 (1999) 133]. However, it was not clear whether the antiperoxidative effect is really due to its direct radical scavenging activity. Therefore, we studied the interaction of Ceph with the hydroxyl radical (*OH) by the electron paramagnetic resonance (EPR) technique. Results showed that Ceph actually scavenged *OH derived by the Fenton reaction. We also found that Ceph radicals were generated on interaction of Ceph with *OH in neutral aqueous solution, but not in acidic solution, consistent with the pH-dependent anti-lipid peroxidation activity of Ceph. Hence, we concluded that anti-lipid peroxidation by Ceph is due to its direct radical scavenging activity.
Kentaro Kogure, Sachie Manabe, Susumu Hama, Akira Tokumura and Kenji Fukuzawa : Potentiation of anti-cancer effect by intravenous administration of vesiculated α-tocopheryl hemisuccinate on mouse melanoma in vivo., Cancer Letters, Vol.192, No.1, 19-24, 2003.
(Summary)
We examined the effect of alpha-tocopheryl hemisuccinate (TS) on the growth of mouse melanoma cells B16-F1 inoculated on the back of hairless mice by two administration procedures of TS, i.p. administration of TS dissolved with dimethyl sulfoxide (TS i.p.) and i.v. administration of TS vesicles (TS-vesicle i.v.). TS i.p. significantly prevented the tumor growth of only half the mice in the group. However, TS-vesicle i.v. almost completely inhibited the tumor growth of all mice. Furthermore, the mean survival of the TS-vesicle i.v. group was 1.4-fold those of the control and TS i.p. groups.
Kentaro Kogure, Susumu Hama, Sachie Manabe, Akira Tokumura and Kenji Fukuzawa : High cytotoxicity of α-tocopheryl hemisuccinate to cancer cells is due to failure of their antioxidative defense systems., Cancer Letters, Vol.186, No.2, 151-156, 2002.
(Summary)
Alpha-tocopheryl hemisuccinate (TS) has been reported to induce apoptosis in various cells, and to show higher toxicity to cancer cells than to normal cells. In this study, although TS induced apoptosis in both a mouse breast normal cell line NMuMG and a mouse breast cancer cell line C127I, the latter were more susceptible to TS. TS-induced apoptosis in C127I was inhibited by superoxide dismutase, alpha-tocopherol and butylated hydroxyanisol. From these results, superoxide (O(2)(-)) itself and reactive oxygen species derived from O(2)(-) and/or free radicals are assumed to be associated with TS toxicity, and the high toxicity of TS to cancer cells is suggested to be due to failure of their antioxidative defense systems.
Akira Tokumura, Yumi Kanaya, Maki Miyake, Shuji Yamano, Minoru Irahara and Kenji Fukuzawa : Increased Production of Bioactive Lysophosphatidic Acid by Serum Lysophospholipase D in Human Pregnancy, Biology of Reproduction, Vol.67, No.5, 1386-1392, 2002.
(Summary)
Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of lysophospholipase D in blood might participate in maintenance of pregnancy.
Akira Tokumura, Eiji Majima, Yuko Kariya, Kyoko Tominaga, Kentarou Kogure, KAtuhiko Yasuda and Kenji Fukuzawa : Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase, The Journal of Biological Chemistry, Vol.277, No.42, 39436-39442, 2002.
(Summary)
We purified human plasma lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K(m) value for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.
Akira Tokumura, Junya Shinomiya, Seishi Kishimoto, Tamotsu Tanaka, Kentaro Kogure, Takayuki Sugiura, Kiyoshi Satouchi, Keizo Waku and Kenji Fukuzawa : Human platelets respond differentially to lysophosphatidic acids having a highly unsaturated fatty acyl group and alkyl ether-linked lysophosphatidic acids, The Biochemical Journal, Vol.365, No.Pt 3, 617-628, 2002.
(Summary)
Lysophosphatidic acid (LPA) is a physiological agonist that is produced by lysophospholipase D, phospholipase A(1) and phospholipase A(2) in the blood of animals. It exerts diverse biological actions on a broad range of animal cells. Specific receptors for this important agonist have been characterized. In this investigation, for the first time we prepared LPAs having a highly unsaturated fatty acyl group, such as the eicosapentaenoyl or docosahexaenoyl residue, and their acetylated derivatives. Human platelets aggregated more potently in response to the highly unsaturated acyl-LPAs than to LPAs with a C(18) fatty acyl group, such as an oleoyl group, while alkyl ether-linked LPAs (alkyl-LPA) had much stronger aggregating activity. Two positional isomers of LPAs with an arachidonoyl, eicosapentaenoyl or docosahexaenoyl group had equipotent aggregatory activity as well as the positional isomers of their acetylated analogues, indicating that putative LPA receptors could not distinguish the difference between the positional isomers. We found that platelet preparations from two individuals showed no aggregatory response to alkyl-LPAs, although they contained mRNAs for known LPA receptors in the following order of expression level: endothelial differentiation gene (Edg)-4>Edg-7>Edg-2. We also obtained evidence that 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082), a phospholipase A(2) inhibitor, potentiated alkyl-LPA-induced platelet aggregation, but inhibited highly unsaturated acyl-LPA-induced platelet aggregation. These results indicated that human platelets express acyl-LPA-selective and alkyl-LPA-selective receptors on their plasma membrane.
Kentaro Kogure, Motoki Morita, Susumu Hama, Sawa Nakashima, Akira Tokumura and Kenji Fukuzawa : Enhancement by α-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon-γ through up-regulation of protein kinase C in rat vascular smooth muscle cells., European Journal of Biochemistry, Vol.269, No.9, 2367-2372, 2002.
(Summary)
The effect of alpha-tocopheryl hemisuccinate (TS) on lipopolysaccharide (LPS)/interferon-gamma (IFN)-induced nitric oxide production in rat vascular smooth muscle cells (VSMC) was examined. The LPS/IFN-induced NO production was enhanced by TS but not by the other alpha-tocopherol (alpha-T) derivatives alpha-tocopheryl acetate (TA) and alpha-tocopheryl nicotinate (TN), or alpha-T itself. alpha-T, TA and TN inhibited the enhancement by TS of LPS/IFN-induced NO production. The enhancing effect of TS was observed in the presence of LPS, but not IFN, suggesting that TS participates in the LPS-stimulated signal pathway leading to NO production. Protein kinase C (PKC) inhibitors, but not protein kinase A inhibitors, inhibited the enhancing effect of TS on LPS/IFN-induced NO production. Furthermore, TS enhanced the amount of PKCalpha in VSMC. From these results, we concluded that the enhancing effect of LPS/IFN-induced NO production was caused by upregulation of PKC in VSMC.
(Keyword)
Animals / Cyclic AMP-Dependent Protein Kinase Type II / Cyclic AMP-Dependent Protein Kinases / Interferon-gamma / Lipopolysaccharides / Muscle, Smooth, Vascular / nitric oxide / Protein Kinase C / Rats / Tocopherols / Up-Regulation / vitamin E
Kyoko Nishi, Masaaki Uno, Kenji Fukuzawa, Hidehisa Horiguchi, Kiyohito Shinno and Shinji Nagahiro : Clinicopathological significance of lipid peroxidation in carotid plaques., Atherosclerosis, Vol.160, No.2, 289-296, 2002.
(Summary)
Several reports have suggested an association between lipid peroxidation and human carotid atherosclerosis, but few reports have demonstrated a link between lipid peroxidation and carotid plaques in humans. In this study, we investigated the relationship between clinical features, histopathological characteristics and lipid peroxidation in patients undergoing carotid endarterectomy (CEA). Forty-one carotid plaques were obtained. A portion of the most severe lesions was subjected to histopathologic examination, and the remainder of the plaques examined for lipid peroxidation. Thiobarbituric acid-reactive substances (TBARS) values were determined as a marker for lipid peroxidation. The lipid-rich core (LC) and macrophage infiltration (Mphi) component as a percentage of total plaque area were measured morphometrically. Based on the results, all plaques were classified into four groups. Group I (GI): LC <10%; Group IIa (GIIa): LC 10-30%, Mphi <5%; Group IIb (GIIb): LC 10-30%, Mphi < or = 5%, and Group III (GIII): LC < or =30%. The plaque TBARS values of GIII were significantly higher than those of GI, GIIa, and GIIb. The TBARS values of GIIb were one-and-a-half times higher than those of GIIa. Our results show that lipid peroxidation in carotid plaques is significantly associated with carotid atherosclerosis, especially plaque instability. These findings provide direct evidence of an association between lipid peroxidation and human atherosclerosis.
Yumi Kanaya, Akira Tokumura, Masaki Kitahara, Maki Miyake, Yasuko Yoshioka and Kenji Fukuzawa : Increased formation of lysophosphatidic acids by lysophospholipase D in serum of hypercholesterolemic rabbits., Journal of Lipid Research, Vol.43, No.2, 307-315, 2002.
(Summary)
Lysophosphatidic acid (LPA) is a biologically active phospholipid that has been identified as a vasoactive principle in incubated plasma and serum of mammals. Previously, we found that mammalian plasma and serum contain a lysophospholipase D, which hydrolyzes lysophosphatidylcholines (LPCs) with different fatty acyl groups to the corresponding LPAs during its incubation at 37 degree C. In this study, we examined whether lysophospholipase D activity and levels of LPCs in rabbit serum were modulated by feeding rabbits a high cholesterol diet. Results showed that the serum levels of LPCs increased gradually in animals fed a high cholesterol diet for 12 weeks. We found that the levels of individual LPAs formed on incubation of serum for 24 h increased with an increase in the period of feeding of rabbits a high cholesterol diet. LPA with a linoleate residue was the most abundant LPA, followed in order by 16:0-, 18:1- and 18:0-LPAs. LPA was found to increase attachment of the monocytic cell line THP-1 to vascular endothelial cells pre-stimulated with tumor necrosis factor-alpha. These results indicated that increases in the levels of LPAs generated by lysophospholipase D in the blood of hypercholesterolemic rabbits may be relevant to attachment of monocytes to vascular walls, a key phenomenon observed at an early stage of atherosclerosis.
Akira Tokumura, Kyoko Tominaga, Yasuda Katsuhiko, Hideharu Kanzaki, Kentaro Kogure and Kenji Fukuzawa : Lack of significant differences in the corrected activity of lysophospholipase D, producer of phospholipid mediator lysophosphatidic acid, in incubated serum from women with and without ovarian tumors, Cancer, Vol.94, No.1, 141-151, 2002.
(Summary)
Several studies have shown that lysophosphatidic acid (LPA), a phospholipidic chemical mediator, is relevant to the pathogenesis of ovarian carcinoma. Higher plasma levels of LPA have been reported in patients with ovarian carcinoma than in healthy patients, and LPA is known to activate ovarian carcinoma cells. To determine the reason for the increased plasma LPA levels in ovarian carcinoma patients, we compared the activities of serum lysophospholipase D, a novel LPA-producing metallo-enzyme, in healthy volunteers, patients with benign ovarian tumor, and patients with ovarian carcinoma. Lysophospholipase D activity was assessed by measuring the percentage conversion of [14C]palmitoyl-lysophosphatidylcholine (LPC) added to human serum. The apparent enzyme activities were corrected based on the serum levels of palmitoyl-LPC determined by gas-liquid chromatography after its purification and conversion to fatty acid methyl esters. The apparent activity of lysophospholipase D in serum preparations from four patients with ovarian carcinoma at Stage IV was significantly higher than those from five healthy subjects, five patients with benign ovarian tumors, and fourteen patients with ovarian carcinoma at Stages I (n = 5), II (n = 4), and III (n = 5). The serum levels of LPC, an endogenous substrate of lysophospholipase D, in ovarian carcinoma patients were less than those in patients with benign ovarian tumors. There were no significant differences in the corrected lysophospholipase D activity for the LPC levels in healthy women, patients with benign ovarian tumors, and patients with ovarian carcinoma at various stages. The current results suggest that lysophospholipase D is not associated with the elevated plasma levels of LPA in ovarian carcinoma patients previously reported, although only a limited number of patients were analyzed.
Akira Shibata, Yuriko Kiba, Nobuyasu Akati, Kenji Fukuzawa and Hiroshi Terada : Molecular characteristics of astaxanthin and β-carotene in the phospholipid monolayer and their distributions in the phospholipid bilayer, Chemistry and Physics of Lipids, Vol.113, No.1-2, 11-22, 2001.
(Summary)
The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.
Kentaro Kogure, Motoki Morita, Sawa Nakashima, Susumu Hama, Akira Tokumura and Kenji Fukuzawa : Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1528, No.1, 25-30, 2001.
(Summary)
We investigated the mechanism of cell toxicity of alpha-tocopheryl hemisuccinate (TS). TS concentration- and time-dependently induced the lactate dehydrogenase release and DNA fragmentation of rat vascular smooth muscle cells (VSMC). Exogenous addition of superoxide dismutase, but not catalase, significantly inhibited the cell toxicity of TS. The NADPH-dependent oxidase activity of VSMC was stimulated by TS treatment. The cell toxicity of TS was inhibited by NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride. Consequently, TS-induced apoptosis of VSMC was suggested to be caused by exogenous O(2)(-) generated via the oxidase system activated with TS.
(Keyword)
Animals / Apoptosis / Ascorbic Acid / Catalase / Cells, Cultured / Cytochrome c Group / DNA Fragmentation / Dose-Response Relationship, Drug / Enzyme Activation / L-Lactate Dehydrogenase / Muscle, Smooth, Vascular / NADPH Oxidase / Oxygen / Rats / Superoxide Dismutase / Superoxides / Tocopherols / Vitamin E
Kenji Fukuzawa, Akira Tokumura, Kentaro Kogure, Masahito Iemura, Naoto Gondoh, Masanobu Fujii, Satoru Ueno and Akira Shibata : A comparative study of the ability of ferric nitrilotriacetate and other iron chelators to assist membrane lipid peroxidation by superoxide radicals, Chemistry and Physics of Lipids, Vol.110, No.1, 69-84, 2001.
(Summary)
This study examined some of the variables determining the efficiency of lipid peroxidation in egg yolk phosphatidylcholine liposomes and in microsomes exposed to enzymatically-generated superoxide radicals. The initiation of peroxidation required the presence of preformed lipid peroxides and a chelated metal catalyst. Comparison of the relative effectiveness of four iron chelating agents showed that the chelate must bind to the membrane by coulombic attraction between the charged membrane and a chelate carrying an opposite net charge. Of the chelates tested, only the carcinogenic ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) was an effective catalyst of oxidation of all membranes, whether carrying a net charge, or not. We postulate that the unique catalytic capacity of the ferric nitrilotriacetate [corrected] (Fe(3+)-NTA) can be explained by its existence in two forms at neutral pH, each binding to oppositely charged membranes and initiating their peroxidation. This gives the complex the unique ability to bind to any membrane, which may be a factor in its carcinogenicity.
Takashi Chikawa, Takaaki Ikata, Shinsuke Katoh, Yoshitaka Hamada, Kentaro Kogure and Kenji Fukuzawa : Preventive effects of lecithinized superoxide dismutase and methylprednisolone on spinal cord injury in rats, --- Transcriptional regulation of inflammatory and neurotrophic genes ---, Journal of Neurotrauma, Vol.18, No.1, 93-103, 2001.
(Summary)
The effects of lecithinized superoxide dismutase (PC-SOD) and/or methylpredisolone (MP) in preventing secondary pathological changes after spinal cord injury (SCI) were investigated in rats with reference to recovery of hindlimb motor function and expression of mRNA of pro-inflammatory and neurotrophic genes. Hindlimb motor function was assessed as the BBB open field locomotor scores. The BBB scores of three groups treated with either PC-SOD (40,000 units/kg), MP (30 mg/kg), or a combination of PC-SOD and MP (PC-SOD+MP) increased with time until 3 days after SCI, and were significantly higher than that of the control group (p < 0.05). Thereafter, the score of the PC-SOD group increased, whereas that of the MP group showed a temporary decrease from day 3 to 5 and then it gradually recovered. The scores in all groups reached a plateau about 18 days after SCI. The PC-SOD+MP group did not show a synergism but a tendency similar to that of the MP group. PC-SOD and MP had down-regulatory effects on mRNA expression of pro-inflammatory substances such as interleukin-1beta (IL-1beta), intercellular adhesion molecule-1 (ICAM-1), and inducible-nitric oxide synthetase (i-NOS) after spinal cord compression at 3, 6, and 24 h, respectively, as judged by a semiquantitative reverse transcription-polymerase chain reaction and on the lipid peroxide (LPO) level 1 h after injury as determined by thiobarbituric acid-reactive substances. The suppression of pro-inflammatory genes expression, especially IL-1beta were greater in the MP group than in the PC-SOD group, while suppression of LPO level was similar in these two groups. PC-SOD+MP treatment augmented the suppression of all three pro-inflammatory genes expression and the decrease of the LPO level. The level of neurotrophin-3 (NT-3) mRNA increased from 6 h after SCI and reached a maximum after 48 h. NT-3 mRNA level was enhanced by PC-SOD treatment, but not by MP treatment. Thus, the effect of MP in suppressing these pro-inflammatory genes expression was more than that of PC-SOD. The difference in motor function in the early and later stage may be partially due to differences in expression of IL-1beta and NT-3 after either treatment, through an IL-1beta-dependent or NT-3-mediated repair response.
Akira Tokumura, T. Sumida, M. Toujima, Kentaro Kogure, Kenji Fukuzawa, Y. Takahashi and S. Yamamoto : Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase., Journal of Lipid Research, Vol.41, No.6, 953-962, 2000.
(Summary)
Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.
Akira Tokumura, Maki Miyake, Yuko Nishioka, Shuji Yamano, Toshihiro Aono and Kenji Fukuzawa : Production of Lysophosphatidic Acids by Lysophospholipase D in Human Follicular Fluids of In Vitro Fertilization Patients, Biology of Reproduction, Vol.61, No.1, 195-199, 1999.
(Summary)
Lysophosphatidic acids (LPAs) are known to be normal constituents of mammalian serum, and they mimic some biological effects of the serum. We previously reported that lysophospholipase D (LPLD) was involved in the accumulation of LPAs in incubated rat plasma and serum. In this study we detected, by gas-liquid chromatography, various molecular species of LPA in follicular fluids collected from women programmed for in vitro fertilization. When the follicular fluid was incubated at 37 degrees C for 48 h, persistent increases in the amounts of LPAs were observed concomitant with decreases in the amounts of the corresponding lysophosphatidylcholines (LPCs), although the concentrations of saturated LPCs increased in the first 6 h of incubation. These results suggest that human follicular fluid has LPLD activity, and this was confirmed by experiments with follicular fluids mixed with an exogenous radioactive LPC. The LPLD showed preference for unsaturated over saturated LPCs, similar to plasma LPLD, indicating that it originated from the circulation.
Akira Tokumura, Hiroaki Fujimoto, Osamu Yoshimoto, Yuko Nishioka, Maki Miyake and Kenji Fukuzawa : Production of lysophosphatidic acid by lysophospholipase D in incubated plasma of spontaneously hypertensive rats and Wistar Kyoto rats, Life Sciences, Vol.65, No.3, 245-253, 1999.
(Summary)
Lysophosphatidic acid has been identified as a vasopressor principle in incubated mammalian plasma and sera, and shown to be generated extracellulary by lysophospholipase D-like activity. In this study, we monitored the time course of changes in the major phospholipid fractions during incubation of plasma, and found that polyunsaturated lysophosphatidic acids accumulate more rapidly than saturated lysophosphatidic acids at expense of the corresponding lysophosphatidylcholines. We compared the phospholipase activities for producing bioactive LPA in age-matched spontaneously hypertensive rats and Wistar Kyoto rats. The lysophospholipase D activity in rat plasma was found to be independent of strain and age. We suggest that lysophospholipase D functions in rat for persistent production of bioactive LPA in the circulation throughout life. However, our finding that production of LPA in spontaneously hypertensive rats was not greater than that in Wistar Kyoto rats does not seem to support the idea that increased production of LPA is involved in the pathogenesis of hypertension.
(Keyword)
aging / Animals / Chromatography, Gas / Chromatography, Thin Layer / Fatty Acids / In Vitro Techniques / Lysophospholipids / Male / Phosphatidylcholines / Phosphoric Diester Hydrolases / Rats / Rats, Inbred SHR / Rats, Inbred WKY / Species Specificity / Time Factors
Akira Tokumura, Yuko Nishioka, Osamu Yoshimoto, Junya Shinomiya and Kenji Fukuzawa : Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1437, No.2, 235-245, 1999.
(Summary)
Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor.
Koichiro Tsuchiya, Jiang Jin-Jie, Masanori Yoshizumi, Toshiaki Tamaki, Hitoshi Houchi, Kazuo Minakuchi, Kenji Fukuzawa and P.Mason Ronald : Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD., Free Rad. Biol. Med., Vol.27, 347-355, 1999.
39.
Akira Tokumura, Maki Miyake, Osamu Yoshimoto, Mayumi Shimizu and Kenji Fukuzawa : Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma, Lipids, Vol.33, No.10, 1009-1015, 1998.
(Summary)
We found that lysophospholipase D (LPLD) in rat plasma prefers unsaturated to saturated lysophosphatidylcholines as substrates, generating a biologically active lipid, lysophosphatidic acid, but it does not hydrolyze diacyl-phospholipids. In this study, this LPLD required a metal ion for activity, Co2+ being the most effective, followed in order by Zn2+, Mn2+, and Ni2+. This metal-ion-stimulated LPLD with unique substrate specificity, which has not been described previously, was susceptible to thiol-blocking reagents and serine esterase inhibitors, but not to a histidine-modifying reagent. Consistent with results using thiol-modifying agents, short-chain fatty aldehydes, secondary products of lipid peroxidation, were found to inhibit LPLD. Addition of dibutylhydroxytoluene or butylhydroxyanisole to the plasma increased the activity of this enzyme, probably in a manner independent of its antioxidant activity, since another antioxidant, propyl gallate, was rather inhibitory. These results suggest that rat plasma contains an active LPLD that differs in some properties from other members of the known phospholipase D family detected in animal tissues and body fluids.
Kenji Fukuzawa, Yasuki Inokami, Akira Tokumura, Junji Terao and Asahi Suzuki : Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and alpha-tocopherol in liposomes, Lipids, Vol.33, No.8, 751-756, 1998.
(Summary)
The (1)O2 quenching rate constants (kQ) of alpha-tocopherol (alpha-Toc) and carotenoids such as beta-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of alpha-Toc and carotenoids in inhibiting (1)O2-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC50](-1)) were also measured in liposomes and ethanol solution and compared with their kQ values. The kQ and [IC50](-1) values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate (1)O2 at different sites in membranes. The kQ values of alpha-Toc were 2.9 x 10(8) M(-1) s(-1) in ethanol solution and 1.4 x 10(7) M(-1) s(-1) (RB system) or 2.5 x 10(6) M(-1) s(-1) (PDA system) in liposomes. The relative [IC50](-1) value of alpha-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of alpha-Toc in membranes, the kQ value of alpha-Toc in liposomes was recalculated as 3.3 x 10(6) M(-1) s(-1) in both the RB and PDA systems. The kQ values of all the carotenoids tested in two photosensitizing systems were almost the same. The kQ value of alpha-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600-1200 times less than those in ethanol solution. The [IC50](-1) value of alpha-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60-170 times less those in ethanol solution. There were no great differences (less than twice) in the kQ and [IC50](-1) values of any carotenoids. The kQ values of all carotenoids were 40-80 times higher than that of alpha-Toc in ethanol solution but only six times higher that of alpha-Toc in liposomes. The [IC50](-1) values of carotenoid were also higher than that of alpha-Toc in ethanol solution than in liposomes, and these correlated well with the kQ values.
Akira Tokumura, Masaaki Okuno, Kenji Fukuzawa, Hitoshi Houchi, Koichiro Tsuchiya and Motoo Oka : Positive and negative controls by protein kinases of sodium-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells stimulated by lysophosphatidic acid, Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol.1389, No.1, 67-75, 1998.
(Summary)
We previously found that lysophosphatidic acid (LPA), a bioactive phospholipid, induced Na+-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells, possibly by activating a Na+/Ca2+ exchanger. The present study on the structure-activity relationship of its action revealed that 1-acyl type LPAs were stronger stimulants than the corresponding 1-O-alkyl type LPAs having a long alkyl moiety with the same chain length. Lysophosphatidylglycerol, suramin and N-palmitoyl-tyrosine phosphoric acid have all been reported to inhibit the action of LPA in some animal cells and platelets, but only lysophosphatidylglycerol was found to inhibit selectively LPA-induced Ca2+ efflux from chromaffin cells. LPA-induced Ca2+ extrusion was suggested to be involved in both acceleration of return of intracellular Ca2+ in Fura 2-loaded bovine chromaffin cells after addition of carbachol, and inhibition of carbachol-induced catecholamine release when the cells were co-incubated with LPA. The Ca2+ efflux from chromaffin cells stimulated by LPA was augmented by their pretreatment with staurosporine or calphostin C, inhibitors of protein kinase C, but reduced by their preincubation with phorbol 12-myristate 13-acetate. Furthermore, the response to LPA was potentiated by sodium vanadate, a protein tyrosine phosphatase inhibitor, but inhibited by genistein, an inhibitor of protein tyrosine kinase. These results suggest that protein kinase C and protein tyrosine kinase are involved negatively and positively, respectively, in the signal transduction triggered by LPA, leading to activation of the Na+/Ca2+ exchanger.
Kenji Fukuzawa, Yasuki Inokami, Akira Tokumura, Junji Terao and Asahi Suzuki : Singlet oxygen scavenging by lpha-tocopherol and eta-carotene: Kinetic studies in phospholipid membranes and ethanol solution, BioFactors, Vol.7, No.1-2, 31-40, 1998.
(Summary)
The rate constants (ks) of 1O2 scavenging for alpha-tocopherol (alpha-Toc) and beta-carotene (beta-Car) were measured in liposome membranes, and compared with those in EtOH solution. 1O2 was site-specifically generated by photoirradiation using two photosensitizers, water-soluble Rose bengal (RB) and lipid-soluble 12-(1-pyrene)-dodecanoic acid (PDA). The ks value for beta-Car in EtOH solution was 1.3 x 10(10) M-1 s-1, which was 36 times that for alpha-Toc (3.6 x 10(8) M-1 s-1), but there was no difference between their ks values in liposomes (1.8 x 10(7) M-1 s-1 for beta-Car and 1.2 x 10(7) M-1 s-1 for alpha-Toc). In the liposomes, the ks value for alpha-Toc was affected by the membrane site where 1O2 was generated, which depended on the localization of the photosensitizer, being high at the membrane surface in the RB-system and low in the inner region of the membrane in the PDA-system. In contrast, the ks value for beta-Car was not affected by the 1O2-generating site. These differences were supposed to be caused by differences in the relative concentrations of 1O2 and active sites of alpha-Toc and beta-Car in the membranes. alpha-Toc and beta-Car inhibited 1O2-dependent peroxidation of egg yolk phosphatidylcholine (egg PC). The concentrations of alpha-Toc required for 50% inhibition of lipid peroxidation (IC50) were higher than those of beta-Car, being more than 6 times higher in EtOH solution and less than 2 times higher in liposomes. The ratio of the antioxidant activity of beta-Car to that of alpha-Toc was more in EtOH solution than in liposomes, and was well correlated with the ratio of their 1O2 scavenging rate constants.
Kenji Fukuzawa, Kayo Matsuura, Akira Tokumura, Asahi Suzuki and Junji Terao : Kinetics and Dynamics of Singlet Oxygen Scavenging by -Tocopherol in Phospholipid Model Membranes, Free Radical Biology and Medicine, Vol.22, No.5, 923-930, 1997.
(Summary)
Scavenging of singlet oxygen (1O2) by alpha-tocopherol (alpha-Toc) was investigated in liposomes. 1O2 was generated by photoirradiation in the presence of two photosensitizers, water-soluble methylene blue (MB) and lipid-soluble 12-(1-pyrene)dodecanoic acid (PDA). The rates of oxidation of alpha-Toc differed depending on the photosensitizing dye and the membrane charge: in the MB-system, alpha-Toc was oxidized fast in negatively charged dimyristoylphosphatidylcholine (DMPC) liposomes containing dicetylphosphate (DCP) and slowly in neutrally charged DMPC liposomes and positively charged DMPC liposomes containing stearylamine (SA), but in the PDA-system, the oxidation rate was independent of the membrane charge. The charge-dependent difference in the MB-system would be due to the site of 1O2 generation depending on the charge-dependent distribution of MB, because positively charged MB increased the zeta-potential of DCP-DMPC liposomes by its interaction with DCP at the membrane surface, but changed the zeta-potentials of DMPC and SA-DMPC liposomes less because of its location in the bulk water phase. The oxidation rate of alpha-Toc in liposomes was different from that in EtOH solution: in the MB system, the oxidation rate was faster in EtOH solution than in DMPC or SA-DMPC liposomes but the same as that in DCP-DMPC liposomes. However, in the PDA system, the oxidation rate was slower in EtOH solution than in DMPC liposomes with or without a charge. Membrane fluidity changed the rate of alpha-Toc oxidation in liposomes, the rate being higher in the liquid crystalline phase than the gel phase, as judged by the higher rate in DMPC liposomes than in dipalmitoylphosphatidylcholine (DPPC) liposomes at 30 degrees C. The rate constants of alpha-Toc for scavenging, the chemical reaction and physical quenching of 1O2 were determined in membranes using DCP-DMPC liposomes labeled with 1,3-diphenyl-isobenzofuran (DPBF), which traps 1O2. These constants differed in the two photosensitizing systems, being higher in the MB-system than in the PDA-system, and were lower than those in EtOH solution.
(Keyword)
electrochemistry / Ethanol / Free Radical Scavengers / Free Radicals / In Vitro Techniques / Kinetics / Lauric Acids / liposomes / Membrane Fluidity / Methylene Blue / oxidation and reduction / Oxygen / Phosphatidylcholines / Photosensitizing Agents / Singlet Oxygen / Solutions / vitamin E
Akira Tokumura, M Toujima, Yasuko Yoshioka and Kenji Fukuzawa : Lipid peroxidation in low density lipoproteins from human plasma and egg yolk promotes accumulation of 1-acyl analogues of platelet-activating factor-like lipids, Lipids, Vol.31, No.12, 1251-1258, 1996.
(Summary)
Oxidative modification of low density lipoprotein (LDL) is known to be a key event for induction of atherosclerosis. However, there has been little progress in structural elucidation of oxidized lipids, especially oxidatively fragmented phospholipids retaining a glycerol backbone. In this study, we found that LDL derived from egg yolk has no platelet-activating factor (PAF) acetylhydrolase activity, and that prolonged incubation of egg yolk LDL with Cu2+ resulted in the formation of various PAF-like lipids: 1-acyl type phosphatidylcholines with an sn-2-short-chain dicarboxylate or monocarboxylate group. Only a very small amount of the PAF-like lipid having an sn-2-short-chain monocarboxylate group was detected by gas chromatography-mass spectrometry in Cu(2+)-oxidized LDL from human plasma with high PAF-acetylhydrolase activity, which has been reported to hydrolyze PAF-like lipids to lysophosphatidyl-cholines. Preincubation of plasma LDL with diisopropyl fluorophosphate dose-dependently inhibited PAF-acetylhydrolase activity, resulting in accumulation of the PAF-like lipids when the LDL was oxidized with Cu2+. As well as PAF and lysophosphatidylcholines, several PAF-like lipids were found to inhibit [3H]thymidine incorporation into cultured vascular smooth muscle cells derived from rat aorta. The possible formation of PAF-like lipids by lipid peroxidation in LDL is discussed as well as its possible significance for induction of atherosclerosis.
Akira Tokumura, Masaaki Okuno, Kenji Fukuzawa, Hitoshi Houchi and Motoo Oka : Two effects of lysophosphatidic acid on Ca(2+)-movement in cultured bovine adrenal chromaffin cells, Journal of Lipid Mediators and Cell Signalling, Vol.14, No.1-3, 127-135, 1996.
(Summary)
We found that lysophosphatidic acid (LPA) exerted two effects on Ca(2+)-movement in cultured bovine adrenal chromaffin cells. At concentrations of above 10(-5) M, it induced slight, but significant 45Ca2+ influx, resulting in release of a small portion of stored catecholamine. At high concentrations it also significantly increased the intracellular Ca2+ concentration in Fura-2-loaded cells. At concentrations as low as 10(-7) M, it stimulated extracellular Na(+)-dependent 45Ca2+ efflux, possibly by increasing Na+/Ca2+ exchange. The maximal efflux of Ca2+ attained with 10(-5) M LPA was inhibited by tyrosine kinase inhibitors, but augmented by a protein kinase C inhibitor. These results suggest that LPA-induced Ca2+ efflux is controlled positively and negatively by mechanisms involving tyrosine kinase and protein kinase C, respectively.
Kenji Fukuzawa, Masahito Iemura and Akira Tokumura : Lipid peroxidation in egg phosphatidylcholine liposomes: comparative studies on the induction systems Fe2+/ascorbate and Fe3+-chelates/xanthine-xanthine oxidase., Biological & Pharmaceutical Bulletin, Vol.19, No.5, 665-671, 1996.
(Summary)
Two typical systems of lipid peroxidation in egg yolk phosphatidylcholine (egg PC) liposomes were compared: an enzymic system involving superoxide (O2) generated by xanthine (X), xanthine oxidase (XO) and Fe(3+)-chelates (Fe(3+)-ADP and Fe(3+)-EDTA), and a non-enzymic system involving ascorbic acid (ASA) and Fe2+. These two systems exhibited a different pH-dependence: the rate in the enzymic system was maximal at pH 8-8.5, whereas that in the non-enzymic system was high below pH 7.4 and low above pH 7.6. The rates of lipid peroxidation differed with the membrane charge, and this charge-dependent phenomenon differed in the two peroxidation systems: in the Fe(3+)-chelates/X-XO-system, the rate was slow in neutrally charged egg PC liposomes and rapid in egg PC liposomes containing negatively charged dicetylphosphate (DCP) or positively charged stearylamine (SA), whereas in the Fe2+/AsA-system, the rate was rapid in neutral egg PC liposomes but no lipid peroxidation occurred in egg PC liposomes charged with DCP or SA. The decomposition rate of the hydroperoxide of PC (PC-OOH) incorporated into dimyristoyl-phosphatidylcholine (DMPC) liposomes differed depending on the membrane charge in the two systems and this charge-dependence of the rates correlated well with that of the initiation rate of lipid peroxidation dependent on membrane charge. In the Fe2+/AsA-system, lipid peroxidation depended on the endogenous presence of PC-OOH, and the amounts of PC-OOH required for initiation of the reaction differed depending on the membrane charge. However, in the Fe(3+)-chelates/X-XO-system, lipid peroxidation occurred very slowly in the absence of PC-OOH, but rapidly in its presence.
Kazushi Minami, Yasushi Hirata, Akira Tokumura, Yutaka Nakaya and Kenji Fukuzawa : Protein kinase c-independent inhibition of the Ca2+-activated K+ channel by angiotensin II and endothelin-1, Biochemical Pharmacology, Vol.49, No.8, 1051-1056, 1995.
(Summary)
We previously reported that the Ca(2+)-activated K+ channel (KCa-channel) in cultured smooth muscle cells from porcine coronary artery was inhibited by protein kinase C (C-kinase). In this study, inhibition of the KCa-channel by receptor-mediated vascular contractile agonists, such as angiotensin II (ANG II) and endothelin-1 (ET-1), was investigated by the patch-clamp technique. In cell-attached patches, addition of ANG II (500 nM) or ET-1 (50 nM) to the bath inhibited the KCa-channel activated by the calcium ionophore A23187 (10-20 microM). Phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, also decreased the open probability of the KCa-channel. The PMA-induced decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor, but the ANG II- and ET-1-induced decreases were not reversed by subsequent application of staurosporine (> 30 nM). Pretreatment of smooth muscle cells with 30 nM staurosporine, a protein kinase inhibitor, or 1 mM neomycin, an inhibitor of phospholipase C, also did not abolish the inhibition of the KCa-channel by ANG II. Furthermore, ANG II inhibited the KCa-channel in cells in which C-kinase was down-regulated. These results indicate that, in porcine coronary artery smooth muscle cells, ANG II and ET-1 inhibit the KCa-channel by a C-kinase-independent mechanism.
Kenji Fukuzawa, Keiji Soumi, Masahito Iemura, Satoru GOTO and Akira Tokumura : Dynamics of xanthine oxidase- and Fe^{3+}-ADP-dependent lipid peroxidation in negatively charged phospholipid vesicles, Archives of Biochemistry and Biophysics, Vol.316, No.1, 83-91, 1995.
(Summary)
Superoxide (O2-)-dependent lipid peroxidation on addition of xanthine oxidase (XO) and Fe(3+)-ADP was induced in egg phosphatidylcholine (PC) liposomes containing dicetylphosphate (DCP), which are negatively charged like biological membranes, but not in uncharged egg PC liposomes. Positively charged Fe(3+)-ADP interacted more with negatively charged egg PC-DCP liposomes than with uncharged egg PC liposomes. The activities of Fe(3+)-chelates for initiating O(2-)-dependent lipid peroxidation were in the order Fe(3+)-ADP > Fe(3+)-citrate > Fe(3+)-oxalate = Fe(3+)-malonate > Fe(3+)-EDTA = 0. This order was the same as that for the reduction rates of these Fe(3+)-chelates to Fe(2+)-chelates by O(2-)-generated by XO. Lineweaver-Burk plots showed that the chelators inhibited XO by different mechanisms: uncompetitively by ADP and adenosine and non-competitively by organic acid chelators (citrate and oxalate) and EDTA. These results suggest that ADP interacts with XO in a manner different from the other chelators. Lipid peroxidation by XO-xanthine and Fe(3+)-ADP was induced in egg PC liposomes containing a trace (0.31-0.35 mol%) of peroxidized egg PC (PC-OOH), but not in PC-OOH-free liposomes of egg PC obtained by their pretreatment with triphenylphosphine. PC-OOH incorporated into dimyristoyl phosphatidylcholine (DMPC) liposomes was degraded on addition of both XO-xanthine and Fe(3+)-chelate, but not of either one alone. alpha-Tocopherol in DMPC liposomes was oxidized on addition of XO-xanthine and Fe(3+)-chelates in the presence, but not in the absence of PC-OOH. Furthermore, PC-OOH was required for decrease of the ESR spectrum of the spin probe 12-(N-oxyl-4,4'-dimethyloxazolidin-2-yl)stearic acid, which labels the hydrophobic region of egg PC liposome membranes, on addition of XO-xanthine and Fe(3+)-chelates. These results indicate that the "induction message of lipid peroxidation," which is associated with reduction of Fe(3+)-ADP by O2- and concurrent degradation of PC-OOH, must be transferred from the membrane surface to the inner hydrophobic region of the membranes.
Hitoshi Houchi, Masaaki Okuno, Akira Tokumura, M Yoshizumi, Kenji Fukuzawa and Motoo Oka : Stimulatory effect of lysophosphatidic acid on calcium efflux from cultured bovine adrenal chromaffin cells, Life Sciences, Vol.57, 205-210, 1995.
50.
Akira Tokumura, Kazuya Morisawa, Akihiko Hiroshima, Toshihiko Tsutsumi, Kenji Fukuzawa and Hiroaki Tsukatani : Calcium movements in rabbit platelets induced by platelet-activating factor and its regulation by an antagonist of platelet-activating factor,, platelet-activating factor and its regulation by an antagonist of platelet-activating factor,, Vol.9, 63-71, 1990.
51.
Kenji Fukuzawa, Y Kurotori, Akira Tokumura and Hiroaki Tsukatani : Vitamin E. deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes, Lipids, Vol.24, No.3, 236-239, 1989.
(Summary)
Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6 min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129-240, 131-227 and 248-354 pmol/10(6) cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [3H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [3H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28 +/- 0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06 +/- 0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26 +/- 0.71 and 4.26 +/- 0.06 nmol/min/mg protein, respectively), measured as degradation of [3H]PAF to [3H]lysoPAF. In vitro addition of alpha-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, indicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by alpha-tocopherol. The acetyl-transferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 microM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 microM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.
(Keyword)
Acetyl Coenzyme A / Acetyltransferases / Animals / Enzyme Activation / Male / Neutrophils / Platelet Activating Factor / Rats / Rats, Inbred Strains / Time Factors / Vitamin E Deficiency
Kenji Fukuzawa, Katsuya Kishikawa, Toyohiro Tadokoro, Akira Tokumura, Hiroaki Tsukatani and Janusz M Gebicki : The effect of a-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles, Archives of Biochemistry and Biophysics, Vol.260, No.1, 153-160, 1988.
(Summary)
alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.
Akira Tokumura, Junichi Yoshida, Takayuki Maruyama, Kenji Fukuzawa and Hiroaki Tsukatani : Platelet aggregation induced by ether-linked phospholipids. 1. Inhibitory actions of bovine serum albumin and structural analogues of platelet activating factor, Thrombosis Research, Vol.46, No.1, 51-63, 1987.
(Summary)
Ether-linked lysophosphatidic acid was able to induce aggregation of platelets from various animals. Feline platelets were the most sensitive followed in decreasing order by human, bovine and rabbit platelets. ONO-6240 and CV-3988, which are antagonists of platelet activating factor, did not inhibit aggregation of feline platelets induced by ether-linked lysophosphatidic acid, indicating that the aggregation induced by the lysophosphatidic acid did not involve receptors for platelet activating factor. An addition of bovine serum albumin caused dose-dependent inhibitions on the aggregation of platelets by both ether-linked lysophosphatidic acid and platelet activating factor. Moreover, 0.5% bovine serum albumin reversed platelet aggregation by the active phospholipid before this aggregation reached a maximum. These results suggest that the lysophosphatidic acid exerts its stimulatory action extracellularly, probably by interacting with specific sites on the surface of platelet.
Akira Tokumura, Junichi Yoshida, Nobuko Okasaka, Kenji Fukuzawa and Hiroaki Tsukatani : Platelet aggregation induced by ether-linked phospholipids. 2. Mechanism of desensitization of rabbit platelets by platelet activating factor and reversibility of inhibitory actions of its antagonists, Thrombosis Research, Vol.46, No.1, 153-161, 1987.
(Summary)
Rabbit platelets pretreated with CV-3988 had reduced responsiveness to platelet activating factor(PAF) even after the platelets had been washed with a buffer. This was due to its tight binding to receptor sites for PAF, but not conformational changes of the receptor, because the platelets that were pretreated with CV-3988 and then washed with buffer containing 0.5% bovine serum albumin showed full responsiveness to PAF. In contrast, the reduced responsiveness of PAF-pretreated rabbit platelets was not reversed by washing with buffer containing 0.5% BSA. The PAF-pretreated platelets showed significantly lower responsiveness to 1-O-hexadecyl-2-O-monomethylaminocarbonyl-sn-glycero-3-phosphochol ine (MC-PAF) than to PAF, although MC-PAF had the same potency as PAF on inducing aggregation of normal rabbit platelets by interacting with receptor sites for PAF. These results suggest that the desensitization is accompanied by a decreased affinity of receptor sites for PAF or reduced efficiency in coupling of activated receptor to regulatory protein in plasma membranes rather than loss of receptor sites.
Junichi Yoshida, Akira Tokumura, Kenji Fukuzawa, Motomori Terao, Kenkichi Takauchi and Hiroaki Tsukatani : A platelet-aggregating and hypotensive phospholipid isolated from bovine brain, The Journal of Pharmacy and Pharmacology, Vol.38, No.12, 878-882, 1986.
(Summary)
A phospholipid that differs from known active lipids and causes potent platelet aggregation and weak hypotension has been isolated from bovine brain. Its platelet aggregating effect on heparinized platelet-rich plasma from rabbits, was at a threshold concentration of about 0.2 nmol ml-1 as phosphorus. The effect was inhibited by CV-3988. The phospholipid was converted by diazomethane treatment to another active lipid that caused short-term hypotension, but not platelet aggregation, rather it inhibited the aggregation of rabbit heparinized platelets induced by platelet-activating factor.
Akira Tokumura, Kengo Harada, Kenji Fukuzawa and Hiroaki Tsukatani : Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma., Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, Vol.875, No.1, 31-38, 1986.
Kenji Fukuzawa, K Kishikawa, Akira Tokumura, Hiroaki Tsukatani and M Shibuya : Fluorescent pigments by covalent binding of lipid peroxidation by-products to protein and amino acids, Lipids, Vol.20, No.12, 854-861, 1985.
(Summary)
The fluorescent products formed on reaction of 12-oxo-cis-9-octadecenoic acid (12-keto-oleic acid) with about 20 different amino acids, polylysine and bovine serum albumin (BSA) were studied. Besides glycine, only the basic amino acids histidine, lysine and arginine gave products with strong fluorescence. N-Acetylation of amino acids greatly reduced the fluorescence of their reaction products. The formation of fluorescent products was inhibited strongly by SH-amino acids such as N-acetyl-cysteine and glutathione. Polyacrylamide gel electrophoresis showed that BSA treated with 12-keto-oleic acid was more acidic than untreated or ricinoleic acid-treated BSA, indicating that basic amino acid residues in BSA were modified by reaction with the keto fatty acid. None of the structural analogs of 12-keto-oleic acid tested--12-oxo-trans-10-octadecenoic acid, 12-oxo-octadecanoic acid, 12-hydroxy-cis-9-octadecenoic acid (ricinoleic acid), cis-9-octadecenoic acid (oleic acid) and linoleic acid--reacted with glycine to give a fluorescent product. The fluorescent products formed on reaction of 12-keto-oleic acid methyl ester with benzyl amine and glycine methyl ester were shown to be 8-(N-substituted-4,5-dihydro-4-oxo-5-hexyl-5-hydroxy-2-pyrrolyl) octanoic acid methyl esters. The fluorescence properties of these compounds were attributed to the chromophobic system NC = CC = O which contains 6 pi electrons. This investigation contributes to insight of the mechanism of formation of fluorescent pigments, probably by a similar reaction of other compounds of the beta, gamma-unsaturated carbonyl type.
Akira Tokumura, T Maruyama, Kenji Fukuzawa and Hiroaki Tsukatani : Effects of lysophosphatidic acids and their structural analogs on arterial blood pressure of cats, Arzneimittel-Forschung, Vol.35, No.3, 287-292, 1985.
(Summary)
On intravenous injection into cats, lysophosphatidic acid elicited a biphasic change in arterial blood pressure: sharp hypotension followed immediately by hypertension. Respiration was greatly disturbed immediately after injection of lysophosphatidic acid, and remained stimulated for a long period, and the heart rate increased during the period of hypertension. Unsaturated lysophosphatidic acids were more potent in evoking hypertension than saturated ones. The hypotensive activity of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphate was about half that of 1-palmitoyl-2-lyso-sn-glycero-3-phosphate (lysophosphatidic acid). Elongation of the acyl chain at the sn-2-position of the glycerol moiety resulted in progressive reduction in the hypotensive activity. Chemically modified analogs with a head group, such as phosphoryl-methanol, ethanol, propanol and choline, had little or no activity. However, sn-2-acetyl-analogs of lyso-phosphatidyl cholines had hypotensive activity. 1-0-Hexadecyl-2-lyso-sn-glycero-3-phosphate, an alkyl-lyso-phosphatidic acid, had much stronger hypotensive activity than the corresponding acyl-lysophosphatidic acid, like its sn-2-0-acetyl analog. These structure-activity relationships of lysophosphatidic acids indicate the existence of their specific binding sites on cardiovascular cells.
Akira Tokumura, Kenji Fukuzawa and Hiroaki Tsukatani : Contractile action of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on strips of isolated rat intestine, The Journal of Pharmacy and Pharmacology, Vol.36, No.3, 210-212, 1984.
(Summary)
The contractile effects of AGEPC were examined on various regions of rat isolated intestine. The duodenum, jejunum and ileum showed only the tonic component of contraction to AGEPC at the low dose (less than 10(-9) M) but at the high dose (10(-7) M) biphasic contractions were induced, consisting of a phasic followed by a tonic component. In the colon, however, the AGEPC-induced maximum contraction was comparable in magnitude to that produced by acetylcholine; also the contraction profile was different from that elicited from the other regions of the intestine. Low doses of AGEPC caused a slow, sustained contraction and at high doses phasic and tonic components were not dissociated.
Akira Tokumura, K Harada, Kenji Fukuzawa and Hiroaki Tsukatani : Biphasic action of platelet-activating factor on isolated guinea-pig ileum, Lipids, Vol.18, No.11, 848-850, 1983.
(Summary)
1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10(-10)-10(-9) M induced slow contraction of isolated guinea-pig ileal muscles and the contraction persisted for a long time. At a higher concentration of 10(-7) M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10(-6)-10(-5) M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetyl-choline-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.
Akira Tokumura, Kenji Fukuzawa and Hiroaki Tsukatani : Contractile actions of lysophosphatidic acids with a chemically-defined fatty acyl group on longitudinal muscle from guinea-pig ileum., The Journal of Pharmacy and Pharmacology, Vol.34, No.8, 514-516, 1982.
(Summary)
Various lysophosphatidic acids caused phasic contractions of longitudinal muscle from guinea-pig ileum, followed by rhythmical contractions of lower magnitude. Polyunsaturated lysophosphatidic acids were more potent than saturated lysophosphatidic acids. Among the lysophosphatidic acids tested, linolenoyl-lysophosphatidic acid was the most active, but its activity was about 3-5 times less than that of prostaglandin F2 alpha. Contractile responses to various lysophosphatidic acids, were partially suppressed by tetrodotoxin, atropine and chlorpheniramine. In addition, lysophosphatidic acid-induced contractions were considerably reduced by pretreatment of the muscle with indomethacin.
(Keyword)
Animals / Chemical Phenomena / chemistry / Drug Interactions / Fatty Acids / Guinea Pigs / Ileum / In Vitro Techniques / Indomethacin / Lysophospholipids / Male / Muscle Contraction / Muscle, Smooth / Phosphatidic Acids / Prostaglandins
(Link to Search Site for Scientific Articles)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 6126563
Kenji Fukuzawa, Akira Tokumura, S Ouchi and Hiroaki Tsukatani : Antioxidant activities of tocopherols on Fe2+-ascorbate-induced lipid peroxidation in lecithin liposomes, Lipids, Vol.17, No.7, 511-513, 1982.
(Summary)
The antioxidant activities of 4 tocopherols, tocol, and a water-soluble model analog of alpha-tocopherol were compared. Egg lecithin liposomes were used and oxidation was catalyzed by Fe2+-ascorbate. The activities decreased in the order alpha greater than beta greater than gamma greater than delta-tocopherol greater than tocol, in agreement with their potencies in vivo. The water-soluble analog was the least effective. Activity depended on the molar ratio of antioxidant to unsaturated lipid, with one molecule each of the alpha-, beta-, gamma-, delta-tocopherol and tocol capable of protecting, respectively, 220, 120, 100, 30 and 20 molecules of polyunsaturated fatty acid. The mechanism of possible antioxidant effect of the compounds used is discussed.
(Keyword)
Ascorbic Acid / Dose-Response Relationship, Drug / Fatty Acids, Unsaturated / Lipid Peroxides / liposomes / Phosphatidylcholines / Time Factors / vitamin E
Akira Tokumura, Tetsuya Kume, Kenji Fukuzawa and Hiroaki Tsukatani : Cardiovascular effects of lysophosphatidic acid and its structural analogs in rats, The Journal of Pharmacology and Experimental Therapeutics, Vol.219, No.1, 219-224, 1981.
(Summary)
Injection of lysophosphatidic acid into anesthetized rats induced immediate hypertension, the effect depending on the structure of the sn-1-acyl moiety of the molecule. A hydroxyl group at the sn-2-position was not necessary, but a wedgeshaped structure was suitable for hypertensive activity. Most lysophospholipids with a chemical group attached to the phosphate portion had only weak hypotensive effects, but the sn-2-acetylated analogs of these depressor lysophospholipids elicited a hypotension at much lower doses. The durations of the hypotensions evoked by the sn-2-acetylated choline phospholipids were longer than those produced by 1-palmitoyl-2-acetoyl-sn-glycerol-3-phosphorylmethanol, ethanol and propanol.
Akira Tokumura, Kenji Fukuzawa, Junichi Isobe and Hiroaki Tsukatani : Lysophosphatidic acid-induced aggregation of human and feline platelets: Structure-activity relationship, Biochemical and Biophysical Research Communications, Vol.99, No.2, 391-398, 1981.
Akira Tokumura, Kenji Fukuzawa, S Yamada and Hiroaki Tsukatani : Stimulatory effect of lysophosphatidic acids on uterine smooth muscles of non-pregant rats, Archives internationales de Pharmacodymie et de Therapie, Vol.245, No.1, 74-83, 1980.
(Summary)
Lysophosphatidic acids stimulated isolated uterine smooth muscle dose-dependently. The contractions were not reduced by pretreatment with atropine or an anti-5-hydroxytryptaminic agent. The potency depended on the nature of the acyl chain in the molecule. Of the compounds with a saturated fatty acyl group tested, the most effective were myristoyl- and lauroyl-lysophosphatidic acid. In a series of unsaturated lysophosphatidic acids, the potency increased with the number of cis double bonds in the acyl chain, and linolenoyl-lysophosphatidic acid was the most active. When injected intravenously, these compounds induced an immediate rise in blood pressure and intrauterine pressure, like prostaglandin F2 alpha: The order of potency of their effects on the intact uterus was consistent with that of their effects on isolated uterine smooth muscle, but not with that of their hypertensive effects in rats.
Kenji Fukuzawa, H Ikeno, Akira Tokumura and Hiroaki Tsukatani : Effect of α-tocopherol incorporation on glucose permeability and phase transition of lecithin liposomes, Chemistry and Physics of Lipids, Vol.23, No.1, 13-22, 1979.
(Summary)
Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.
Akira Tokumura, Kenji Fukuzawa and Hiroaki Tsukatani : Effects of synthetic and natural lysophosphatidic acids on the arterial blood pressure of different animal species, Lipids, Vol.13, No.8, 572-574, 1978.
(Summary)
Intravenous injection of lysophosphatidic acid was found to cause hypertension in rats and guinea pigs, but hypotension in cats and rabbits. The potencies of the pressor and depressor effects of synthetic lysophosphatidic acids in rats and cats depended on their chain length and the degree of unsaturation of their fatty acyl moieties.
Akira Tokumura, Kenji Fukuzawa, Yasue Akamatsu, Sadaji Yamada, Tetsuya Suzuki and Hiroaki Tsukatani : Identification of vasopressor phospholipid in crude soybean lecithin, Lipids, Vol.13, No.7, 468-472, 1978.
(Summary)
The vasopressor phospholipid in crude soybean lecithin was isolated by column chromatography on Sephadex LH-20. It represented 0.1% of crude soybean lecithin. The isolated phospholipid was identified to be lysophosphatidic acid by gas chromatography-mass spectrometry analysis of TMS-deacylated product and acetolysis product. Nuclear magnetic resonance analysis favored the 1-monoacyl isomer over the 2-isomer. By enzymic determination with L-3-glycerophosphate dehydrogenase, the isolated phospholipid was identified as 1-monoacyl-L-3-glycerophosphate. Gas chromatographic examination revealed that it was composed of a large percentage of unsaturated fatty acids, especially linoleic acid. The activity of isolated lysophosphatidic acid was slightly less than that of synthetic 1-linoleoyl-L-3-glycerophosphate.
Kenji Fukuzawa, Akira Tokumura, Sadaji Yamada and Hiroaki Tsukatani : Oral Toxicity of Sodium Dodecyl Sulfate, Japanese Journal of Toxicology and Environmental Health, Vol.24, No.2, 107-110, 1978.
(Summary)
Weanling male rats were bred with a drinking water containing sodium dodecyl sulfate (SDS) at concentrations of 0.25,0.05,or 0% for 5 months. Increase of organ weights was observed in the lung and kidney, though body weight was not affected at a higher concentration (0.25%). Histopathology of major organs indicated the characteristic symptom to be bronchopneumonia by the continued ingestion of a small amount of SDS. Activities of serum enzymes, such as GOT, GPT, alkaline phosphatase, and cholinesterase, were not affected at the dosages used. In rats taking 0.25% of SDS, triglyceride level increased in the liver but decreased in serum, while hepatic and serum levels of cholesterol, phospholipid, and free fatty acid were unchanged.
Kenji Fukuzawa and Akira Tokumura : Glutathione peroxidase activity in tissues of vitamin E-deficient mice, Journal of Nutritional Science and Vitaminology, Vol.22, No.5, 405-407, 1976.
Junichi Morishige, Hideko Nagasawa, Hitoshi Hori, Kenji Fukuzawa and Akira Tokumura : ニワトリ卵白のリゾホスホリパーゼDによるリゾホスファチジン酸の産生, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.48, No.0, 91-94, 2006.
Akira Tokumura, 土江 明子, 平良 智 and Kenji Fukuzawa : ウサギ房水のリゾホスホリパーゼD活性によるリゾホスファチジルコリンの分解, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.46, 69-72, 2004.
Akira Tokumura, 平尾 恵理, 丸山 祐史, Kentaro Kogure, Kenji Fukuzawa, 安田 勝彦 and 神崎 秀暢 : 正常および異常妊娠時のヒト血清リゾホスホリパーゼD活性, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.45, No.0, 95-97, 2003.
Akira Tokumura, 富永 恭子, Kentaro Kogure, Kenji Fukuzawa, 安田 勝彦, 刈谷 優子 and 真島 英司 : 生理活性リン脂質LPAを産生するヒト血漿リゾホスホリパーゼDは多機能性ヌクレオチドホスジエステラーゼautotaxinである, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.44, 78-881, 2002.
10.
Akira Tokumura, 富永 恭子, 金谷 由美, Kentaro Kogure and Kenji Fukuzawa : モルモット腹腔洗浄液のリゾホスホリパーゼD活性, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.43, 82-85, 2001.
Kenji Fukuzawa, Kentaro Kogure, M Morita, S Hama, S Nakashima and Akira Tokumura : Enhancement by α-tocopherol hemisuccinate of nitric oxide production induced by lipopolysaccharide and interferon-γ through up-regulation of protein kinase C in rat vascular smooth muscle cells, Proc.Of the second China-Japan international Conference on Vitamins, 262-267, 2001.
12.
Akira Tokumura, Shuji Yamano, Toshihiro Aono and Kenji Fukuzawa : Lysophosphatidic Acids Produced by Lysophospholipase D in Mammalian Serum and Body Fluid, Annals of the New York Academy of Sciences, Vol.905, 347-350, 2000.
Akira Tokumura, 金谷 由美, 富永 恭子, Kentaro Kogure, Kenji Fukuzawa, 北原 真樹 and 安田 勝彦 : 血清リゾホスホリパーゼDによるリゾホスファチジン酸産生の病態下での変動, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.42, 41-44, 2000.
14.
Akira Tokumura, 四宮 淳也, Kentaro Kogure, Kenji Fukuzawa, 田中 保 and 里内 清 : 高度不飽和脂肪酸を有するリゾホスファチジン酸:血漿中での生成と血小板凝集活性, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.41, 193-195, 1999.
15.
Akira Tokumura, 住田 常喜, 東嶋 祐興, Kenji Fukuzawa, Shuji Yamano and Toshihiro Aono : リポキシゲナーゼによるホスファチジルコリンの酸化分解, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.40, 235-238, 1998.
Akira Tokumura, 三宅 真樹, Kenji Fukuzawa, Shuji Yamano and Toshihiro Aono : 妊婦血清リボホスホリパーゼD活性, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.39, 43-45, 1997.
Akira Tokumura, 吉本 修, 三宅 真樹 and Kenji Fukuzawa : ラット血漿のリゾホスファチジン酸生成康酵素活性, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.38, 251-254, 1996.
22.
Akira Tokumura, 東嶋 祐興 and Kenji Fukuzawa : LDLの過酸化により生成するPAF様脂質の分解, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.37, 203-206, 1995.
23.
Kenji Fukuzawa, T Hiramoto, T Fujii, K Kishikawa and Akira Tokumura : Lipid peroxidation induced by hydroxyl-and lipid free-radicals and iron-oxygen complexes,and its inhibition by a-tocopherol, Medical,Biochemical and Chemical Aspects of Free Radicals, 275-278, 1989.
24.
Kenji Fukuzawa, Yasuhiro Kurotori, Akira Tokumura and Hiroaki Tsukatani : Increased platelet-activating factor (PAF) synthesis in polymorphonuclear leukocytes of vitamin E-deficient rats, Annals of the New York Academy of Sciences, Vol.570, 449-454, 1989.
25.
Akira Tokumura, 高内 健吉, 吉田 淳一, 浅井 剛, Kenji Fukuzawa, 塚谷 博昭 and 小川 正 : 脳組織中のPAF及び関連リン脂質類, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.30, 384-386, 1988.
26.
Akira Tokumura, 堤 敏彦, 吉田 淳一, Kenji Fukuzawa and 塚谷 博昭 : PAFとそのリゾ体の血小板および好中球での膜内動態, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.30, 295-298, 1988.
27.
Kenji Fukuzawa, 藤井 智仁, 田所 豊博, Akira Tokumura and 塚谷 博昭 : [Fe2+錯体ー酸素]複合体によるリノール酸の過酸化誘導とα-トコフェロール(α-Toc)の抗酸化作用, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.29, 413-416, 1987.
28.
Akira Tokumura, 上安 功明, 浅井 剛, Kenji Fukuzawa and 塚谷 博昭 : ウシ脳組織中のPAF分子種の同定並びに定量, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.29, 333-336, 1987.
29.
Akira Tokumura, 吉田 淳一, 丸山 隆幸, Kenji Fukuzawa and 塚谷 博昭 : 血小板表面におけるエーテル結合型リン脂質の作用様式, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.28, 57-60, 1986.
30.
Kenji Fukuzawa, 大内 節博, Akira Tokumura and 塚谷 博昭 : リポソーム膜におけるビタミンE同族体の脂質抗酸化作用, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.23, 61-65, 1981.
31.
Kenji Fukuzawa, 千田 尚子, Akira Tokumura and 塚谷 博昭 : リポソーム膜における脂質過酸化反応とビタミンE, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.22, 229-232, 1980.
32.
Akira Tokumura, 久米 哲也, Kenji Fukuzawa and 塚谷 博昭 : リゾホスファチジン酸及びその構造類似体の心血管作用, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.22, 190-194, 1980.
33.
Kenji Fukuzawa, Akira Tokumura, 池野 宏彦, 塚谷 博昭 and 鈴木 旭 : 膜流動性および相転移に及ぼすトコフェロールの効果, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.21, 94-97, 1979.
34.
Akira Tokumura, Kenji Fukuzawa, 赤松 康江, 山田 貞二, 塚谷 博昭 and 鈴木 鉄也 : 市販大豆レシチン中の血圧活性脂質の単離と同定, Proceedings of the Japanese Conference on the Biochemistry of Lipids, Vol.20, 157-160, 1978.
真鍋 幸恵 and Kenji Fukuzawa : ビタミンE誘導体のアポトーシス誘導作用 : 構造-活性相関, Vitamins, Vol.78, No.3, 152-155, Mar. 2004.
6.
Kenji Fukuzawa, Kentaro Kogure, Motoki Morita, Susumu Hama and Akira Tokumura : Enhancement of Nitric Oxide and Superoxide Generations by α-Tocopheryl Succinate and Its Apoptotic and Anticancer Effects, Biochemistry (Moscow), Vol.69, No.1, 50-57, Jan. 2004.
(Summary)
Tocopheryl succinate (TS), a succinyl ester of alpha-tocopherol (alpha-T), has been reported to have various biological activities. In this communication, we review the current findings about TS including our recent studies of its effects on nitric oxide (NO) and superoxide (O2-) generations implicated in cancer and atherosclerosis. First, we investigated the effect of TS on NO production in vascular smooth muscle cells (VSMC) under atherosclerosis-like conditions using lipopolysaccharide (LPS) and interferon-gamma (IFN). TS enhanced LPS/IFN-dependent NO production, but alpha-T itself did not. The enhancement by TS of NO production was inhibited by alpha-T but not by antioxidants such as ascorbic acid and 2[3]-t-butyl-4-hydroxyanisole (BHA). TS enhanced the amount of protein kinase Calpha (PKCalpha) in VSMC, and PKC inhibitors inhibited TS-enhanced NO production, suggesting that the enhancing effect of TS on NO production is caused by up-regulation of PKC. Second, we found that TS induced apoptosis in VSMC associated with increase in O2- generation via NADPH-dependent oxidase. We further observed that a mouse breast cancer cell line C127I was more susceptible for TS-induced apoptosis than a mouse breast normal cell line NmuMG, and that superoxide dismutase, alpha-T, and BHA inhibited TS-caused morphological cell damage in C127I. From these results, O2- itself and/or other reactive oxygen species are assumed to associate with TS-induced cell toxicity, and antioxidative defense systems are supposed to be lowered in cancer cells. Finally, we found that intravenous injection of TS vesicles completely inhibited the growth of melanoma cells B16-F1 inoculated on the back of hairless mice and enhanced their survival time.
Kentaro Kogure and Kenji Fukuzawa : Tocopheryl succinate-versatile function due to its unique physicochemical properties, Journal of Clinical Biochemistry and Nutrition, Vol.35, No.1, 29-34, 2004.
Akira Tokumura, A. Tuchie, M. Imasaki and Kenji Fukuzawa : Generation of platelet-activating factor (PAF)-like phospholipids in blood circulation under elevated oxidative stress:its pathological significance on atherosclerosis, Journal of Clinical Biochemistry and Nutrition, Vol.34, No.1, 35-42, 2003.
森田 元喜 and Kenji Fukuzawa : 血管障害の抑制などγ-トコフェロールに新たに見いだされた作用, Vitamins, Vol.75, No.3, 132-134, Mar. 2001.
14.
Kenji Fukuzawa and Akira Tokumura : Kinetics and Dynamics of α-Tocopherol and Carotenoids for Deactivating Singlet Oxygen in Phospholipid Membranes, Vitamins, Vol.75, No.1, 1-13, Jan. 2001.
(Summary)
The singlet oxygen (^1O_2) deactivating rate constants (k_s) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin and canthaxanthin and their inhibiting activities for ^1O_2-dependentlipid peroxidation (IC_50' that is, the concentration required for 50% inhibition of lipid peroxidation) were investigated in liposomes and compared to those in EtOH solution. Liposomes were prepared from dimyristoylphosphatidylcholine to measure the k_s and egg yolk phosphatidylcholine to measure the IC_50 values. ^1O_2 was generated by photoirradiation using three photosensitizers, Rose bengal and methylene blue, which generate ^1O_2 at the membrane surface or in bulk water, and 2-(1-pyrene) dodecanoic acid, which generates ^1O_2 in the membrane hydrophobic inner region. The k_s and IC_50 values were determined in membranes considering the following two points: (1) antioxidants exist in membranes and their active moieties are distributed in different regions of membranes at different concentrations; (2) dynamics of ^1O_2 in membranes (generation site of ^1O_2 depending on the localization of photosensitizer in membranes, and solubility of ^1O_2 which differs in the site of membranes). In conclusion, the following three factors are experimentally confirmed to be important for the consideration of ^1O_2 deactivating activities of α-Toc and carotenoids in membranes:(1) the concentration of antioxidants (EtOH solution < membrane), especially the local concentration of their active moieties in membranes (in the case of OH-group of α-Toc, 0%, 50-60%, and 40-50% at polar zone, hydrogen belt, and hydrophobic core, respectively, of the membranes); (2) the mobility of antioxidants (EtOH solution > membrane), the mobility of membrane phospholipid (liquid crystalline state > gel state), the mobility of antioxidants in membranes (antioxidants located at one half of the bilayer membrane such as α-tocopherol > antioxidants located across the bilayer such as β-carotene; antioxidants interactive with polar head group of lipid in membrane such as astaxanthin < antioxidants nointeractive with polar head group of lipid in membrane such as β-carotene), and the local mobility of their active moieties (in case of OH-group of α-Toc, membrane surface < membrane inner region); (3) the dielectric constant (micropolarity) at the domains in membranes, that is higher in membrane surface than in membrane inner region, where the active moieties of antioxidants deactivate ^1O_2 (in case of α-Toc, reactivity is high in the solution with high dielectric constant).
Akira Tokumura, Tuneki Sumida, Masaoki Toujima, Kentaro Kogure and Kenji Fukuzawa : Platelet-activating factor (PAF)- like oxidized phospholipids: relevance to atherosclerosis, BioFactors, Vol.13, No.1-4, 29-33, 2000.
(Summary)
Lipid peroxidation is involved in the pathogenesis of chronic diseases including atherosclerosis. Oxidized lipoprotein has diverse biological activities and is believed to initiate atheroma formation and maturate fatty plaque. The active components of oxidized lipoproteins still remain to be clarified, but a likely candidate is the phosphatidylcholine (PC) having an sn-2-short-chain acyl group with a methyl, hydroxyl, aldehydic or carboxylic terminal. These unique PCs, formed by oxidative fragmentation of the polyunsaturated acyl group of the parent PC in liposomes, low density lipoproteins and blood plasma, induce platelet aggregation through the activation of the receptor for platelet-activating factor (PAF), due to their resemblance in structure with PAF. We have found that PAF-like lipids regulate DNA synthesis and production of nitric oxide independently of the activation of the PAF receptor in vascular smooth muscle cells. Regulation of vascular cell function through two distinct signaling pathways mediated by PAF-like lipids provides new insight into the mechanism of induction of atherosclerosis.
Akira Tokumura, Emi Kuwahara, Junichi Morishige, Kenji Fukuzawa and Shuji Yamano : Physiological and pathophysiological roles of lysophosphatidic acid production by by lysophospholipase D in the reproductive system, 3rd International Conference on phospholipase A2 and lipid mediators, Sorrento, May 2007.
2.
Junichi Morishige, Kenji Fukuzawa, Hideko Nagasawa, Hitoshi Hori and Akira Tokumura : Lysophosphatidic acid produced by lysophospholipase D in hen egg white induces blood vessel formation on hen chorioallantoic membrane, 3rd International Conference on phospholipase A2 and lipid mediators, Sorrento, May 2007.
3.
Akira Tokumura, Satoshi Taira, Kenji Fukuzawa, Toshihiko Tsutsumi, Tomoya Kitayama, K Morita and Toshihiro Dohi : LC-MS analysis on productions of bioactive lysophosphatidic acids from lysophosphatidylcholines in human mixed saliva, 47th International Conference on theBioscience of Lipids, Pecos, Sep. 2006.
4.
Kenji Fukuzawa, A. Fujiwara, Akira Tokumura, Junji Terao and Jansuz M. Gebicki : Measurement of phosphatidylcholine hydroperoxides in membranes by the ferric-xylenol orange (FOX) assay, XIII the Biennial Meeting of the Society for Free Radical Research International, Davos, Aug. 2006.
5.
Kenji Fukuzawa, Akira Shibata, Koichiro Tsuchiya, Akira Tokumura, H. Ichikawa and Jansuz M. Gebicki : Colored complex of ferric-xylenol orange/phosphatidylcholine vesicle in the Fox assay, XIII the Biennial Meeting of the Society for Free Radical Research International, Davos, Aug. 2006.
6.
Kenji Fukuzawa, Kentaro Kogure, Sachie Manabe and Akira Tokumura : In vitro cytotoxicity of alpha-tocopherol succinate, malonate and oxalate on cancer cells and their in vivo anti-cancer effects on mouse melanoma, XIII the Biennial Meeting of the Society for Free Radical Research International, Davos, Aug. 2006.
7.
Akira Tokumura, Emi Kuwahara, Junichi Morishige, Kenji Fukuzawa and Shuji Yamano : Lysophosphatidic acid stimulates hyaluronic acid production in mouse oocyte-cumulus cells complex in vitro, 20thIUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Jun. 2006.
8.
Kenji Fukuzawa, Kentaro Kogure, S. Manabe, Ichiro Suzuki and Akira Tokumura : Selective cytotoxicity to cancer cells of α-tocopheryl succinate analogues in vitro and their anti-cancer effects on mouse melanoma in vivo, International Interdisciplinary Conference on Vitamins, Coenzymes, and Biofactors 2005, Awaji, Hyogo, Nov. 2005.
K. Tsuchiya, K. Akai, Akira Tokumura, S. Abe, T. Tamaki, Y. Takiguchi and Kenji Fukuzawa : Oxygen radicals photo-induced by ferric nitrilotriacetate(Fe-NTA) complex, EPR 2005. The international Conference/Workshop on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems, Ohio,U.S.A., Sep. 2005.
11.
Akira Tokumura, S. Taira, J. Morishige, T. Tsutsumi and Kenji Fukuzawa : Lysophospholipase C and D produce lysolipids that may intercellularly mediate signals from mammalian cell lines, 46th International Conference on the Bioscience of Lipids, Ajaccio-Corsica, Sep. 2005.
12.
M. Watsky, W. Hatsuyama, T. Tsutsumi, Kenji Fukuzawa and Akira Tokumura : Lysophospholipase D and elevates lysophosphatidylcholine as sources of elevated aqeous humor LPA following corneal injury, ARVO 2005 Annual Meeting, Fort Lauderdale, Florida,U.S.A., May 2005.
13.
Akira Tokumura, C. Kageyama, W. Hatuyama, S. Taira, K. Touchika and Kenji Fukuzawa : Lysophosphatidic acid production by secretory lysophosholipase D:Physiological implications, 2nd International Conference on Phospholipase A2 and 8th International congress on Platelet-Activating Factor and Related Lipid Mediators, Berlin, Oct. 2004.
14.
Kenji Fukuzawa, Kentaro Kogure, M. Morita, S. Hama and Akira Tokumura : Enhancement by alpha-tocopheryl succinate of superoxide and nitric oxide generations in cultured cells, International Symposium: Reactive Oxygen and Nitrogen Species: Diagnostic,Preventive and Therapeutic Values, St. Petersburg-Kizhi-St.Petersburg Russia, Jul. 2002.
15.
Akira Tokumura, A. Tsuchie, Kentaro Kogure and Kenji Fukuzawa : Cigarette smoking increases plasma level of phosphatidylcholine having an oxidatively shortened fatty acyl group with a carboxyl terminal, 11th Biennial Scientific Meeting of the International Society for Free Radical Research, Paris, Jul. 2002.
16.
Kentaro Kogure, S. Hama, S. Manabe, M. Kisaki, Akira Tokumura and Kenji Fukuzawa : High toxicity of α-tocopheryl succinate to cancer cells is due to their failed antioxidative deffence system, 11th Biennial Scientific Meeting of the International Society for Free Radical Research, Paris, Jul. 2002.
17.
Kenji Fukuzawa, Y. Saitoh, K. Arai, Kentaro Kogure, Akira Tokumura and A. Shibata : Antioxidant effect of bovine serum albumin on iron-chelate superoxide-dependent membrane lipid peroxidation, 11th Biennial Scientific Meeting of the International Society for Free Radical Research, Paris, Jul. 2002.
18.
Kentaro Kogure, S. Hama, S. Manabe, Ichiro Suzuki, M. Shibuya, Akira Tokumura and Kenji Fukuzawa : Role of terminal structure of a-tocopheryl esters in nitric oxide production and apoptosis, The 9th Korea-Japan joint symposium of drug design and development, Seoul, May 2002.
19.
S. Hama, Kentaro Kogure, S. Manabe, Akira Tokumura and Kenji Fukuzawa : Anti-cancer effect of a-tocopheryl succinate mediated by apoptosis, The 9th Korea-Japan joint symposium of drug design and development, Seoul, May 2002.
20.
K. Tominaga, Kentaro Kogure, Kenji Fukuzawa and Akira Tokumura : Property of secretory phospholipase D which generates bioactive lysophosphatidic acid in guinea-pig peritoneal washing, The 1st Takeda Science Foundation Symposium on Pharma Sciences:Lipids in Signaling and Related Diseases, Tokyo, Oct. 2001.
21.
Kentaro Kogure, M. Morita, S. Nakashima, S. Hama, Akira Tokumura and Kenji Fukuzawa : Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate, 2nd China-Japan International Conference on Vitamins, Shanghai, Oct. 2001.
22.
Kenji Fukuzawa, Kentaro Kogure, M. Morita, S. Hama, S. Nakashima and Akira Tokumura : Enhancement by α-tocopheryl hemisuccinate of nitric oxide production indeced by lipopolysaccharide and interferon-γthrough up-regulation of protein kinase C in rat vascular smooth muscle cells, 2nd China-Japan International Conference on Vitamins, Shanghai, Oct. 2001.
23.
Yuji Mishima, Junko Motonaka, Ken-ichi Maruyama and Kenji Fukuzawa : Electiochemical evaluation of the hexacyanoferrate(III) modified titanium electrode for determination of hydrogen peroxide, Advanced Materials Development & Performance, Vol.2, 710-713, Tokushima, Nov. 1999.
盛重 純一, Hideko Nagasawa, Hitoshi Hori, Kenji Fukuzawa and Akira Tokumura : ニワトリ卵白のリゾホスホリパーゼDによるリゾホスファチジン酸の産生, 第48回日本脂質生化学会, Jun. 2006.
14.
Akira Tokumura, 平良 智, Kenji Fukuzawa, 堤 敏彦, 北山 友也 and 森田 克也 : ヒト口腔粘膜細胞のエクト型リゾホスホリパーゼDによるリン脂質性増殖因子(LPA)の産生, 日本薬学会第126年会, Mar. 2006.
15.
Akira Tokumura, Toshihiko Tsutsumi, Satoshi Taira, Kenji Fukuzawa and Mitchel Watsky : Lysophospholipase D and elevates lysophosphatidylcholine as sources of elevated aqueous humor LPA following rabbit corneal injury, 第78回日本生化学会, Oct. 2005.
16.
J. Morishige, T. Tanaka, K. Satouchi, Akira Tokumura and Kenji Fukuzawa : Production of lysophosphatidic acid by lysophospholipase D in hen egg white, 第78回日本生化学会, Oct. 2005.
17.
Akira Tokumura, Toshihiko Tsutsumi, Satoshi Taira, Kenji Fukuzawa and Michel Watsky : Lysophospholipase D and elevates lysophosphatidylcholine as sources of elevated aqueous humor LPA following rabbit corneal injury, 第78回日本生化学会, Oct. 2005.