Intradermal Delivery of a Liposomal Formulation Encapsulating Amphiphilic Ascorbic Acid by Iontophoresis for Promotion of Collagen Synthesis

Journal of Drug Delivery Science and Technology


Development of functional chimeric nanoparticles by membrane fusion of small extracellular vesicles and drug-encapsulated liposomes

Since small extracellular vesicle (sEVs) are involved in cell-to-cell communication via transfer of certain bioactive molecules and have the capability to overcome biological barriers against drug transport, their use as a drug delivery system (DDS) has been demonstrated in treatment of a diverse range of diseases. However, some issues in drug encapsulation have been pointed out, including low encapsulation efficiency and poor reproducibility. It was previously reported that liposomes containing phosphatidylserine (PS) can fuse together in the presence of calcium ion, which allows for drug encapsulation into the resultant liposomes (i.e., calcium fusion method). On the other hand, PS is reportedly present in lipid membrane of sEVs as a distinct lipid composition. We therefore hypothesized that PS-mediated membrane fusion of sEVs with PS-liposomes encapsulating therapeutic agents via the calcium fusion method can be applied to convenient drug encapsulation into sEVs. Membrane fusion of PS-liposomes and sEVs derived from murine melanoma B16F1 cells (B16-sEVs) was firstly confirmed. The obtained nanoparticles, termed chimeric nanoparticles (CM-NP), showed comparable cellular uptake to B16-sEVs into B16F1 cells. Moreover, CM-NP encapsulating an anticancer drug doxorubicin (DOX) (CM-NP-DOX) could be prepared by membrane fusion of PS-liposomes encapsulating DOX (PS-Lipo-DOX) and B16-sEVs. CM-NP-DOX exhibited a superior anticancer effect on B16F1 cells in vitro compared with PS-Lipo-DOX. These findings suggest that the calcium fusion method could be applied for membrane fusion of sEVs and PS-liposomes, and that this approach would likely be useful for efficient drug encapsulation into sEVs, as well as increasing liposome functionality.


Development of a novel tocopheryl ester for suppression of lipid accumulation without cytotoxicity by optimization of dicarboxylic ester moiety


Iontophoresis-mediated direct delivery of nucleic acid therapeutics, without use of carriers, to internal organs via non-blood circulatory pathways

Nanoparticle drug carriers have been employed to achieve systemic delivery of nucleic acid therapeutics, including small interfering RNA (siRNA); however, non-specific distribution and immune-related events often cause undesired adverse effects. Thus, there is a need for a new technology capable of specifically delivering nucleic acid therapeutics to desired sites. We demonstrated the utility of iontophoresis (IP) using weak electric current (0.3-0.5 mA/cm) as a local drug delivery technology. Our previous studies revealed that IP allows for transdermal permeation of nucleic acid therapeutics via induction of intercellular junction cleavage initiated by Ca influx-mediated cellular signaling activation, and subsequent cytoplasmic delivery through a unique endocytosis process in both skin and other cells. Based on these findings, we hypothesized that IP may enable direct delivery of nucleic acid therapeutics to internal organs through non-blood circulatory pathways without the use of delivery carriers. Permeation of fluorescent-labeled nucleic acids administered via IP applied to the surface of the liver and pancreas was observed in both organs, but not with topical application. IP-mediated local delivery of siRNA into the liver and pancreas significantly suppressed target mRNA expression in each organ. Moreover, IP administration of therapeutic siRNA against the molecules responsible for liver steatosis and fibrosis significantly inhibited lipid accumulation and fibrotic hepatic damage in individual model mice. These findings suggest that IP may be a useful technology to directly deliver nucleic acid therapeutics to internal organs without use of drug delivery carriers via non-blood circulatory pathways.


Effective Anticancer Therapy by Combination of Nanoparticles Encapsulating Chemotherapeutic Agents and Weak Electric Current

Delivery of medicines using nanoparticles via the enhanced permeability and retention (EPR) effect is a common strategy for anticancer chemotherapy. However, the extensive heterogeneity of tumors affects the applicability of the EPR effect, which needs to overcome for effective anticancer therapy. Previously, we succeeded in the noninvasive transdermal delivery of nanoparticles by weak electric current (WEC) and confirmed that WEC regulates the intercellular junctions in the skin by activating cell signaling pathways (J. Biol. Chem., 289, 2014, Hama et al.). In this study, we applied WEC to tumors and investigated the EPR effect with polyethylene glycol (PEG)-modified doxorubicin (DOX) encapsulated nanoparticles (DOX-NP) administered via intravenous injection into melanoma-bearing mice. The application of WEC resulted in a 2.3-fold higher intratumor accumulation of nanoparticles. WEC decreased the amount of connexin 43 in tumors while increasing its phosphorylation; therefore, the enhancing of intratumor delivery of DOX-NP is likely due to the opening of gap junctions. Furthermore, WEC combined with DOX-NP induced a significant suppression of tumor growth, which was stronger than with DOX-NP alone. In addition, WEC alone showed tumor growth inhibition, although it was not significant compared with non-treated group. These results are the first to demonstrate that effective anticancer therapy by combination of nanoparticles encapsulating chemotherapeutic agents and WEC.


Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities.

Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids.

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences


Low level electricity increases the secretion of extracellular vesicles from cultured cells

Exosomes, a type of extracellular vesicles, can be collected from the conditioned medium of cultured cells, and are expected to be used in disease therapy and drug delivery systems. However, since the yield of exosomes from conditioned medium is generally low, investigations to develop new methods to increase exosome secretion and to elucidate the secretion mechanism have been performed. Our previous studies demonstrated that activation of intracellular signaling including Rho GTPase and subsequent endocytosis of extraneous molecules in cells could be induced by low level electricity (0.3-0.5 mA/cm). Since exosomes are produced in the process of endocytosis and secreted by exocytosis via certain signaling pathways, we hypothesized that low level electric treatment (ET) would increase exosome secretion from cultured cells via intracellular signaling activation. In the present study, the influence of ET (0.34 mA/cm) on extracellular vesicle (EV) secretion from cultured cells was examined by using murine melanoma and murine fibroblast cells. The results showed that the number of EV particles collected by ultracentrifugation was remarkably increased by ET in both cell lines without cellular toxicity or changes in the particle distribution. Also, protein amounts of the collected EVs were significantly increased in both cells by ET without alteration of expression of representative exosome marker proteins. Moreover, in both cells, the ratio of particle numbers to protein amount was not significantly changed by ET. Rho GTPase inhibition significantly suppressed ET-mediated increase of EV secretion in murine melanoma, indicating that Rho GTPase activation could be involved in ET-mediated EV secretion in the cell. Additionally, there were almost no differences in uptake of each EV into each donor cell regardless of whether the cells had been exposed to ET for EV collection. Taken together, these results suggest that ET could increase EV secretion from both cancer and normal cells without apparent changes in EV quality.


Characteristics of unique endocytosis induced by weak current for cytoplasmic drug delivery


Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables

Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables.

Journal of Nutritional Science and Vitaminology


No enhancing effects of plasmid-specific histone acetyltransferase recruitment system on transgene expression in vivo

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [ 123, 277-280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.

Nucleosides, Nucleotides & Nucleic Acids


Low electric treatment activates Rho GTPase via heat shock protein 90 and protein kinase c for intracellular delivery of siRNA

Low electric treatment (LET) promotes intracellular delivery of naked siRNA by altering cellular physiology. However, which signaling molecules and cellular events contribute to LET-mediated siRNA uptake are unclear. Here, we used isobaric tags in relative and absolute quantification (iTRAQ) proteomic analysis to identify changes in the levels of phosphorylated proteins that occur during cellular uptake of siRNA promoted by LET. iTRAQ analysis revealed that heat shock protein 90 (Hsp90)α and myristoylated alanine-rich C-kinase substrate (Marcks) were highly phosphorylated following LET of NIH 3T3 cells, but not untreated cells. Furthermore, the levels of phosphorylated Hsp90α and protein kinase C (PKC)γ were increased by LET both with siRNA and liposomes having various physicochemical properties used as model macromolecules, suggesting that PKCγ activated partly by Ca influx as well as Hsp90 chaperone function were involved in LET-mediated cellular siRNA uptake. Furthermore, LET with siRNA induced activation of Rho GTPase via Hsp90 and PKC, which could contribute to cellular siRNA uptake accompanied by actin cytoskeleton remodeling. Collectively, our results suggested that LET-induced Rho GTPase activation via Hsp90 and PKC would participate in actin-dependent cellular uptake of siRNA.


Efficacy of high-affinity liposomal astaxanthin on up-regulation of age-related markers induced by oxidative stress in human corneal epithelial cells

Decreases in tear volume, unstable tear films and excessive tear evaporation are known to cause desiccation and hyperosmolar stress. These, in turn, induce oxidative stress that is thought to cause dry eye, which is also considered to be age-related disease. We hypothesized that oxidative stress induces up-regulation of age-related markers, and that the antioxidant astaxanthin prepared as a liposomal formulation may be a candidate for the treatment of dry eye. Herein, we examined age-related markers in an dry eye model, and evaluated the efficacy of high-affinity liposomes containing astaxanthin. The dry eye model showed desiccation time-dependent increases in reactive oxygen species. We confirmed the up-regulation of p53, p21 and p16 as a function of desiccation time. Pretreatment with both neutral and slightly-positively-charged astaxanthin liposomal formulations showed significant suppression of up-regulation of all markers, with the positively-charged liposomes exhibiting the greatest efficacy. Furthermore, positively-charged liposomes labeled with fluorescent dyes demonstrated much higher affinity to normal human corneal epithelial cells (HCECs) than neutral liposomes. Taken together, we confirmed the up-regulation of age-related markers, especially p16, in an dry eye model, and demonstrated the potential of high-affinity liposomal astaxanthin for the treatment of dry eye.

Journal of Clinical Biochemistry and Nutrition


Carotenoid Stereochemistry Affects Antioxidative Activity of Liposomes Co-encapsulating Astaxanthin and Tocotrienol

We previously found that antioxidative activity of liposomes co-encapsulating astaxanthin (Asx) and tocotrienols (T3s) was higher than the calculated additive activity, which results from intermolecular interactions between both antioxidants (J. Clin. Biochem. Nutr., 59, 2016, Kamezaki et al.). Herein, we conducted experiments to optimize Asx/α-T3 ratio for high antioxidative activity, and tried to elucidate details of intermolecular interaction of Asx with α-T3. Higher activity than calculated additive value was clearly observed at an Asx/α-T3 ratio of 2 : 1, despite two α-T3 would potentially interact with two terminal rings of one Asx. The synthetic Asx used in this study was a mixture of three stereoisomers, 3R,3'R-form (Asx-R), 3S,3'S-form (Asx-S) and 3R,3'S-meso form (Asx-meso). The calculated binding energy of the Asx-S/α-T3 complex was higher than those of Asx-R/α-T3 and Asx-meso/α-T3, suggesting that Asx-S and α-T3 is the most preferable combination for the intermolecular interaction. The optimal Asx-S/α-T3 ratio for antioxidation was shown to be 1 : 2. These results suggest that the Asx stereochemistry affects the intermolecular interaction of Asx/α-T3. Moreover, the absorption spectrum changes of Asx-S upon co-encapsulation with α-T3 in liposomes indicate that the electronic state of Asx-S is affected by intermolecular interactions with α-T3. Further, intermolecular interactions with α-T3 affected the electronic charges on the C9, C10 and C15 atoms in the polyene moiety of Asx-S. In conclusion, the intermolecular interaction of Asx/T3 depends on the Asx stereochemistry, and caused a change in the electronic state of the Asx polyene moiety by the presence of double bond in the T3 triene moiety.


Lysophosphatidic acid in medicinal herbs enhances prostaglandin E2 and protects against indomethacin-induced gastric cell damage in vivo and in vitro

Lysophosphatidic acid (LPA) is a bioactive phospholipid that induces diverse biological responses. Recently, we found that LPA ameliorates NSAIDs-induced gastric ulcer in mice. Here, we quantified LPA in 21 medicinal herbs used for treatment of gastrointestinal (GI) disorders. We found that half of them contained LPA at relatively high levels (40-240 μg/g) compared to soybean seed powder (4.6 μg/g), which we previously identified as an LPA-rich food. The LPA in peony (Paeonia lactiflora) root powder is highly concentrated in the lipid fraction that ameliorates indomethacin-induced gastric ulcer in mice. Synthetic 18:1 LPA, peony root LPA and peony root lipid enhanced prostaglandin E production in a gastric cancer cell line, MKN74 cells that express LPA abundantly. These materials also prevented indomethacin-induced cell death and stimulated the proliferation of MKN74 cells. We found that LPA was present in stomach fluids at 2.4 μM, which is an effective LPA concentration for inducing a cellular response in vitro. These results indicated that LPA is one of the active components of medicinal herbs for the treatment of GI disorder and that orally administered LPA-rich herbs may augment the protective actions of endogenous LPA on gastric mucosa.


Prevention of UV-induced Melanin Production by Accumulation of Redox Nanoparticles in the Epidermal Layer via Iontophoresis

UV rays induce melanin production in the skin, which, from a cosmetic point of view, is problematic. Reactive oxygen species (ROS) generated in the skin upon UV irradiation are thought to be responsible for melanin production. Thus, effective antioxidants are recognized as useful tools for prevention of UV-induced melanin production. Redox nanoparticles (RNPs) containing nitroxide radicals as free radical scavengers were previously developed, and shown to be effective ROS scavengers in the body. RNPs are therefore expected to be useful for effective protection against UV-induced melanin production. However, as the sizes of RNPs are typically larger than the intercellular spaces of the skin, transdermal penetration is difficult. We recently demonstrated effective transdermal delivery and accumulation of nanoparticles in the epidermal layer via faint electric treatment, i.e., iontophoresis, suggesting that iontophoresis of RNPs may be a useful strategy for prevention of UV-induced melanin production in the skin. Herein, we performed iontophoresis of RNPs on the dorsal skin of hairless mice that produce melanin in response to light exposure. RNPs accumulated in the epidermal layer upon application of iontophoresis. Further, the combination of RNPs with iontophoresis decreased UV-induced melanin spots and melanin content in the skin. Taken together, we successfully demonstrated that iontophoresis-mediated accumulation of RNPs in the epidermis prevented melanin production.


Tumor microenvironment-sensitive liposomes penetrate tumor tissue via attenuated interaction of the extracellular matrix and tumor cells, and accompanying actin depolymerization

Delivery of anticancer drugs into tumor cores comprised of malignant cancer cells can result in potent therapeutic effects. However, conventional nanoparticle-based drug delivery systems used for cancer therapy often exhibit inefficient tumor-penetrating properties, thus, suggesting the need to improve the functional design of such systems. Herein, we focus on the interactions between cancer cells and the extracellular matrix (ECM) and demonstrate that liposomes modified with slightly acidic pH-sensitive peptide (SAPSp-lipo) can penetrate in vivo tumor tissue and in vitro spheroids comprised of cancer cells and ECM. We previously reported SAPSp-lipo, tumor microenvironment-sensitive liposomes, are effectively delivered to tumor tissue (Hama et al. J Control Release 2015, 206, 67-74). Compared with conventional liposomes, SAPSp-lipo could be delivered to deeper regions within both spheroids and tumor tissues. Furthermore, tumor penetration was found to be promoted at regions where actin depolymerization was induced by SAPSp-lipo and inhibited by the polymerization of actin. In addition, SAPSp-lipo attenuated the interaction between cancer cells and ECM, contributing to the penetration of SAPSp-lipo. These results suggest that SAPSp-lipo penetrates tumors via the interspace route and is accompanied by actin depolymerization. Taken together, SAPSp-lipo demonstrates potential as a novel tumor-penetrable drug carrier for induction of therapeutic effects against malignant cells that comprise tumor cores.


Distribution of glycosylinositol phosphoceramide-specific phospholipase D activity in plants

Previously, we detected an unknown sphingophospholipid in cabbage leaves and identified it as phytoceramide-1-phosphate (PC1P). We also found an enzyme activity that produces PC1P by glycosylinositol phosphoceramide (GIPC)-specific hydrolysis in cabbage leaves. To characterize the GIPC-specific phospholipase D (GIPC-PLD) activity, we investigated distributions of GIPC-PLD activity in 25 tissues of 10 plants. In most plants, the GIPC-PLD activity was the highest in roots. Young leaves of cabbage and Welsh onion had higher activities than corresponding aged outer leaves. The GIPC-PLD activities in leaves, stems and roots of mung bean were higher in the sprouting stage than in more mature stages. We also examined the distribution of substrate GIPC and product PC1P and found that GIPC was ubiquitously distributed at 50-280 nmol/g (wet wt) in tissues of plants, whereas PC1P was detectable (3-60 nmol/g wet wt.) only in tissues showing considerable GIPC-PLD activity. These results suggest a possibility that GIPC-PLD activity is involved in plant growth.


Development of flexible nanocarriers for siRNA delivery into tumor tissue

Various non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.


Concentrated phosphatidic acid in cereal brans as potential protective agents against indomethacin-induced stomach ulcer.

One of complications associated with long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) is peptic ulcer. Recently, we found that orally administered phosphatidic acid (PA) ameliorated aspirin-induced stomach lesions in mice. In this study, we identified PA-rich food sources and examined the effects of the food materials on indomethacin-induced stomach ulcer. Among examined, buckwheat (Fagopyrum esculentum) bran contained the highest level of PA (188 mg/100 g). PA was the richest phospholipid (25%) in the lipid fraction of the buckwheat bran. Administration of the lipid extracts of buckwheat bran significantly ameliorated indomethacin-induced stomach lesions in mice. In contrast, wheat (Triticum durum) bran lipids (PA, 4%) and soybean (Glycine max) lipids (PA, 3%) were not associated with ameliorative effects. These results indicated that PA-rich lipids can be used as an effective supplement for prevention of NSAID-induced stomach ulcer.

Journal of Agricultural and Food Chemistry


The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.

Science and Technology of Advanced Materials


Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH

Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects.


Synergistic antioxidative effect of astaxanthin and tocotrienol by co-encapsulated in liposomes

Astaxanthin and vitamin E are both effective antioxidants that are frequently used in cosmetics, as food additives, and in to prevent oxidative damage. A combination of astaxanthin and vitamin E would be expected to show an additive anntioxidative effect. In this study, liposomes co-encapsulating astaxanthin and the vitamin E derivatives α-tocopherol (α-T) or tocotrienols (T3) were prepared, and the antioxidative activity of these liposomes toward singlet oxygen and hydroxyl radical was evaluated in vitro. Liposomes co-encapsulating astaxanthin and α-T showed no additive anntioxidative effect, while the actual scavenging activity of liposomes co-encapsulating astaxanthin and T3 was higher than the calculated additive activity. To clarify why this synergistic effect occurs, the most stable structure of astaxanthin in the presence of α-T or α-T3 was calculated. Only α-T3 was predicted to form hydrogen bonding with astaxanthin, and the astaxanthin polyene chain would partially interact with the α-T3 triene chain, which could explain why there was a synergistic effect between astaxanthin and T3 but not α-T. In conclusion, co-encapsulation of astaxanthin and T3 induces synergistic scavenging activity by intermolecular interactions between the two antioxidants.

Journal of Clinical Biochemistry and Nutrition


Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules.


Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery

Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.


Transmission of external environmental pH information to the inside of liposomes via pore-forming proteins embedded within the liposomal membrane

Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems. We hypothesize that embedding pore-like "access points" into the liposomal membrane could allow for the transmission of information between the internal and external liposomal environments and thus overcome this inherent limitation of conventional liposomes. To investigate this, we evaluated whether a change in the pH of an external solution could be transmitted to the inside of liposomes through the pore-forming protein, yeast voltage-dependent anion channel (VDAC). Transmission of a pH change via VDAC was evaluated using a polyglutamic acid/doxorubicin complex (PGA/Dox) as an internal pH sensor. Upon encapsulation into conventional liposomes, PGA/Dox exhibits no pH sensitivity due to isolation from the external environment. On the other hand, PGA/Dox was found to retain its pH sensitivity upon encapsulation into VDAC-reconstituted liposomes, suggesting that VDAC facilitated the transmission of information on the pH of the external environment to the inside of the liposomes. In conclusion, we successfully demonstrated the transmission of information between the external and internal liposomal environments by a stable pore-like structure embedded into the liposomal membranes, which serve as access points.


食品に含まれるグリコシルイノシトールホスホセラミドおよびフィトセラミド-1-リン酸


Analysis of molecular species profiles of ceramide-1-phosphate and sphingomyelin using MALDI-TOF mass spectrometry

Ceramide-1-phosphate (C1P) is a potential signaling molecule that modulates various cellular functions in animals. It has been known that C1P with different N-acyl lengths induce biological responses differently. However, molecular species profiles of the C1P in animal tissues have not been extensively examined yet. Here, we developed a method for determination of the molecular species of a C1P using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with Phos-tag, a phosphate capture molecule. The amounts of total C1P in skin, brain, liver, kidney and small intestine of mice were determined to be 344, 151, 198, 96 and 90 pmol/g wet weight, respectively. We found a C1P species having an α-hydroxypalmitoyl residue (h-C1P, 44 pmol/g wet weight) in mouse skin. The h-C1P was detected only in the skin, and not other tissues of mice. The same analysis was applied to sphingomyelin after conversion of sphingomyelin to C1P by Streptomyces chromofuscus phospholipase D. We found that molecular species profiles of sphingomyelin in skin, kidney and small intestine of mice were similar to those of C1P in corresponding tissues. In contrast, molecular species profiles of sphingomyelin in liver and brain were quite different from those of C1P in these tissues, indicating selective synthesis or degradation of C1P in these tissues. The method described here will be useful for detection of changes in molecular species profiles of C1P and sphingomyelin.


MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion.

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Biochimica et Biophysica Acta (BBA) - Biomembranes


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

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.

Journal of Nutritional Science and Vitaminology


Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

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.


Novel antioxidants isolated from plants of the genera Ferula, Inula, Prangos and Rheum collected in Uzbekistan

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.


Ability of ferric nitrilotriacetate complex with three pH-dependent conformations to induce lipid peroxidation.


New Stilbene Derivatives from Calligonum leucocladum

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.


Structural characteristic of terminal dicarboxyric moiety required for apoptogenic activity of α-Tocopheryl esters.

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.

Biochimica et Biophysica Acta (BBA) - General Subjects


Two lignan dimers from bamboo stems (Phyllostachys edulis)

Phyllostadimers A and B, two bis-lignans in which the two lignan units are directly connected by a C-C bond, were isolated from stems of bamboo, Phyllostachys edulis. Their structures were determined on the basis of spectral evidence. In addition, 14 known compounds were also obtained throughout the investigation. Phyllostadimer A significantly inhibited liposomal lipid peroxidation.


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.

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.


alpha-Tocopheryl succinate activates protein kinase C in cellular and cell-free systems

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.

Journal of Nutritional Science and Vitaminology


Direct radical scavenging by the bisbenzylisoquinoline alkaloid cepharanthine.

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.


Potentiation of anti-cancer effect by intravenous administration of vesiculated α-tocopheryl hemisuccinate on mouse melanoma in vivo.

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.


High cytotoxicity of α-tocopheryl hemisuccinate to cancer cells is due to failure of their antioxidative defense systems.

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.


Mechanism of potent antiperoxidative effect of capsaicin

The effect of a pungent ingredient of red pepper, capsaicin, on lipid peroxidation of rat liver mitochondria (RLM) induced by ADP/Fe(2+) was studied. Capsaicin inhibited the lipid peroxidation significantly, being more effective than the well-known antioxidant alpha-tocopherol. Capsaicin was also found to scavenge 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radicals both in solution and in membranes, especially the latter. Capsaicin was found to scavenge radicals both at/near the membrane surface and in the interior of the membrane. The phenolic OH-group of capsaicin remained intact after reaction with DPPH radicals, indicating that the hydroxyl group is not associated with the radical scavenging reaction. From the results of quantum chemical calculations of various radical intermediates derived from the model compound N-vanillylacetamide, and the findings that vanillin and 8-methyl-6-noneamide were major reaction products of capsaicin with DPPH radicals, it was concluded that the radical scavenging site of capsaicin is the C7-benzyl carbon.


Human platelets respond differentially to lysophosphatidic acids having a highly unsaturated fatty acyl group and alkyl ether-linked lysophosphatidic acids

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.


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.

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.


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

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.


Superoxide is responsible for apoptosis in rat vascular smooth muscle cells induced by α-tocopheryl hemisuccinate.

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.

Biochimica et Biophysica Acta (BBA) - General Subjects


Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin

The effects of the carotenoids beta-carotene and astaxanthin on the peroxidation of liposomes induced by ADP and Fe(2+) were examined. Both compounds inhibited production of lipid peroxides, astaxanthin being about 2-fold more effective than beta-carotene. The difference in the modes of destruction of the conjugated polyene chain between beta-carotene and astaxanthin suggested that the conjugated polyene moiety and terminal ring moieties of the more potent astaxanthin trapped radicals in the membrane and both at the membrane surface and in the membrane, respectively, whereas only the conjugated polyene chain of beta-carotene was responsible for radical trapping near the membrane surface and in the interior of the membrane. The efficient antioxidant activity of astaxanthin is suggested to be due to the unique structure of the terminal ring moiety.

Biochimica et Biophysica Acta (BBA) - Biomembranes


A comparative study of the ability of ferric nitrilotriacetate and other iron chelators to assist membrane lipid peroxidation by superoxide radicals

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.


Preventive effects of lecithinized superoxide dismutase and methylprednisolone on spinal cord injury in rats --- Transcriptional regulation of inflammatory and neurotrophic genes

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.


Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase.

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.


Potent anti-peroxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine: The amine moiety is responsible for its pH-dependent radical scavenge activity

The bisbenzylisoquinoline alkaloid cepharanthine, which has been considered to exhibit antiperoxidation activity due to its membrane stabilizing effect, was found to scavenge radicals such as .OH and DPPH (1,1-diphenyl-2-picrylhydrazyl) in solution, and to inhibit lipid peroxidation in mitochondria and liposomes by Fe2+/ADP. The antiperoxidation activity of cepharanthine in rat liver mitochondria initiated by Fe2+/ADP at pH 7.4 was much greater than that of alpha-tocopherol, its half-inhibitory concentration being about 23 microM. However, cepharanthine was effective only at neutral pH values such as pH 7.4, not in a moderately acidic pH region below pH 6.5. Accordingly, the neutral form of the deprotonated amine moiety in the tetrahydroisoquinoline ring is concluded to be responsible for the radical scavenging activity of cepharanthine. There are two amine moieties in the cepharanthine molecule, but we specified the effective amine moiety from the antiperoxidation activities of the imine analogs of cepharanthine.


Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene

In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression


Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell line

The steady state transcript levels of two hexokinase isozymes and type 1 glucose transporter in human tumor cell lines were analyzed. In HepG2 cells, both type II hexokinase and type 1 glucose transporter were highly expressed. However, in cell lines A431 and HeLa, in which the expression level of type 1 glucose transporter was lower than that in HepG2 cells, the amount of type II hexokinase transcript was almost negligible.


リン脂質酸化二次作物による血管平滑筋細胞のアポトーシス誘導とビタミンEによる抑制作用


抗ガン作用の増強を志向したビタミンEコハク酸ベシクル

抗ガン作用の増強を志向したビタミンEコハク酸ベシクル


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

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


モルモット腹腔洗浄液のリゾホスホリパーゼD活性

モルモット腹腔洗浄液のリゾホスホリパーゼD活性


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


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

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


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

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