Intraperitoneal administration of nanoparticles containing tocopheryl succinate prevents peritoneal dissemination.

Intraperitoneal administration of anticancer nanoparticles is a rational strategy for preventing peritoneal dissemination of colon cancer due to the prolonged retention of nanoparticles in the abdominal cavity. However, instability of nanoparticles in body fluids causes inefficient retention, reducing its anticancer effects. We have previously developed anticancer nanoparticles containing tocopheryl succinate, which showed high in vivo stability and multifunctional anticancer effects. In the present study, we have demonstrated that peritoneal dissemination derived from colon cancer was prevented by intraperitoneal administration of tocopheryl succinate nanoparticles. The biodistribution of tocopheryl succinate nanoparticles was evaluated using inductively coupled plasma mass spectroscopy and imaging analysis in mice administered quantum dot encapsulated tocopheryl succinate nanoparticles. Intraperitoneal administration of tocopheryl succinate nanoparticles showed longer retention in the abdominal cavity than by its intravenous (i.v.) administration. Moreover, due to effective biodistribution, tumor growth was prevented by intraperitoneal administration of tocopheryl succinate nanoparticles. Furthermore, the anticancer effect was attributed to the inhibition of cancer cell proliferation and improvement of the intraperitoneal microenvironment, such as decrease in the levels of vascular endothelial growth factor A, interleukin 10, and M2-like phenotype of tumor-associated macrophages. Collectively, intraperitoneal administration of tocopheryl succinate nanoparticles is expected to have multifaceted antitumor effects against colon cancer with peritoneal dissemination.

Tocopheryl Phosphate Inhibits Rheumatoid Arthritis-Related Gene Expression In Vitro and Ameliorates Arthritic Symptoms in Mice.

Anti-rheumatoid arthritis (RA) effects of α-tocopherol (α-T) have been shown in human patients in a double-blind trial. However, the effects of α-T and its derivatives on fibroblast-like synoviocytes (FLS) during the pathogenesis of RA remain unclear. In the present study, we compared the expression levels of genes related to RA progression in FLS treated with α-T, succinic ester of α-T (TS), and phosphate ester of α-T (TP), as determined via RT-PCR. The mRNA levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-3, and MMP-13 were reduced by treatment with TP without cytotoxicity, while α-T and TS did not show such effects. Furthermore, intraperitoneal injection of TP ameliorated the edema of the foot and joint and improved the arthritis score in laminarin-induced RA model mice. Therefore, TP exerted anti-RA effects through by inhibiting RA-related gene expression.

α-Tocopheryl succinate stabilizes the structure of tumor vessels by inhibiting angiopoietin-2 expression.

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.

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.

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.

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.

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.

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.

Electric stimulus opens intercellular spaces in skin.

Iontophoresis is a technology for transdermal delivery of ionic small medicines by faint electricity. Since iontophoresis can noninvasively deliver charged molecules into the skin, this technology could be a useful administration method that may enhance patient comfort. Previously, we succeeded in the transdermal penetration of positively charged liposomes (diameters: 200-400 nm) encapsulating insulin by iontophoresis (Kajimoto, K., Yamamoto, M., Watanabe, M., Kigasawa, K., Kanamura, K., Harashima, H., and Kogure, K. (2011) Int. J. Pharm. 403, 57-65). However, the mechanism by which these liposomes penetrated the skin was difficult to define based on general knowledge of principles such as electro-repulsion and electro-osmosis. In the present study, we confirmed that rigid nanoparticles could penetrate into the epidermis by iontophoresis. We further found that levels of the gap junction protein connexin 43 protein significantly decreased after faint electric stimulus (ES) treatment, although occludin, CLD-4, and ZO-1 levels were unchanged. Moreover, connexin 43 phosphorylation and filamentous actin depolymerization in vivo and in vitro were observed when permeation of charged liposomes through intercellular spaces was induced by ES. Ca(2+) inflow into cells was promoted by ES with charged liposomes, while a protein kinase C inhibitor prevented ES-induced permeation of macromolecules. Consequently, we demonstrate that ES treatment with charged liposomes induced dissociation of intercellular junctions via cell signaling pathways. These findings suggest that ES could be used to regulate skin physiology.

Resistin regulates the expression of plasminogen activator inhibitor-1 in 3T3-L1 adipocytes.

Resistin and plasminogen activator inhibitor-1 (PAI-1) are adipokines, which are secreted from adipocytes. Increased plasma resistin and PAI-1 levels aggravate metabolic syndrome through exacerbation of insulin resistance and induction of chronic inflammation. However, the relationship between resistin and PAI-1 gene expression remains unclear. Previously, we found that resistin regulates lipid metabolism via carbohydrate responsive element-binding protein (ChREBP) during adipocyte maturation (Ikeda et al., 2013) [6]. In this study, to clarify the relationship between expression of resistin and PAI-1, PAI-1 expression in differentiated 3T3-L1 adipocytes was measured after transfection with anti-resistin siRNA. We found that PAI-1 gene expression and secreted PAI-1 protein were significantly decreased by resistin knockdown. Furthermore, phosphorylation of Akt, which can inhibit PAI-1 expression, was accelerated and the activity of protein phosphatase 2A (PP2A) was suppressed in resistin knockdown 3T3-L1 adipocytes. In addition, the expression of glucose transporter type 4, a ChREBP target gene, was reduced and was associated with inhibition of PP2A. The addition of culture medium collected from COS7 cells transfected with a resistin expression plasmid rescued the suppression of PAI-1 expression in resistin knockdown 3T3-L1 adipocytes. Our findings suggest that resistin regulates PAI-1 expression in 3T3-L1 adipocytes via Akt phosphorylation.

A novel effector secretion mechanism based on proton-motive force-dependent type III secretion apparatus rotation.

The type III secretion apparatus (T3SA) participates in the secretion of bacterial proteins called effectors, although the detailed mechanism of effector secretion remains unclear. T3SA and flagellum were shown to branch from a common ancestor and also show structural similarity. In addition, both T3SA-dependent effector secretion and flagellar rotation were reported to require proton-motive force (PMF) for activity. From these reports, we hypothesized that T3SA, like the flagellum, would rotate via PMF and that this rotation is responsible for effector secretion. To observe T3SA rotation, we constructed a novel observation system by modifying the tip of T3SA on bacterial cell membranes with an observation probe, which allowed documentation of T3SA rotation for the first time. T3SA rotation was stopped by the addition of a protonophore that decreases PMF. Moreover, increased viscosity of the observation medium inhibited both rotation of T3SA associated with beads and effector secretion. These results suggested that effector secretion would follow the PMF-dependent rotation of T3SA and could be inhibited by preventing T3SA rotation. Moreover, the motion-track analysis of bead rotation suggested that the T3SA needle might be flexible. Consequently, we propose a "rotational secretion model" as a novel effector secretion mechanism of T3SA.

Selenate induces epithelial-mesenchymal transition in a colorectal carcinoma cell line by AKT activation.

In addition to potent anticancer effects of selenite, a modest therapeutic effect of sodium selenate has been demonstrated in prostate cancer patients. Selenate acts by activating protein phosphatase 2A, which inhibits various signal transduction cascades, including the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The human colorectal carcinoma cell line DLD-1 harbors a constitutive active mutation in PIK3CA encoding the PI3K p110α catalytic subunit. Thus, we examined the anticancer effect of sodium selenate in DLD-1 cells. As expected, selenate significantly decreased cell viability and increased apoptosis at a 50% inhibitory concentration (IC50) of 0.88mM, whereas selenite was much more potent at an IC50 of 0.0061mM. Surprisingly, at lower concentrations (0.04-0.16mM), selenate induced changes in cell morphology and motility that are characteristic of the epithelial-mesenchymal transition (EMT). Moreover, selenate-induced EMT was associated with AKT activation, increased expression of the EMT-inducing transcription factor TWIST1 and the mesenchymal cell-specific intermediate filament vimentin, and decreased expression of the epithelial cell-specific adhesion molecule E-cadherin. The critical role of AKT activation in selenate-induced EMT was identified using the AKT inhibitor Akti-1/2, which suppressed EMT-associated cell motility and invasion. These results suggest that although sodium selenate is a potential anticancer drug, deleterious effects of EMT induction should be taken into careful consideration.

Nanoparticles consisting of tocopheryl succinate are a novel drug-delivery system with multifaceted antitumor activity.

Tumor heterogeneity hampers the clinical efficacy of cancer chemotherapy. Therefore, it is necessary to develop a multifaceted, rational treatment strategy with the potential to modulate overall tumor heterogeneity. Since combination therapy using several drugs has been shown to have enhanced therapeutic effects compared with monotherapy, combining agents with different antitumor effects would be a multifaceted form of therapy to overcome tumor heterogeneity. Therefore, the development of effective drug-delivery system (DDS) carriers for combination therapy is required. The ideal DDS carrier for combination therapy should itself have antitumor activity in addition to the ability to deliver drugs to tumors. α-Tocopheryl succinate (TS), a succinic acid ester of α-tocopherol, has attracted attention as a unique antitumor agent, and TS itself can form nanoparticles. In this review, we introduce nanoparticles consisting of TS as a novel DDS carrier with multifaceted antitumor effects for combination therapy.

Retinoids ameliorate insulin resistance in a leptin-dependent manner in mice.

UNLABELLED: Transgenic mice expressing dominant-negative retinoic acid receptor (RAR) α specifically in the liver exhibit steatohepatitis, which leads to the development of liver tumors. Although the cause of steatohepatitis in these mice is unknown, diminished hepatic expression of insulin-like growth factor-1 suggests that insulin resistance may be involved. In the present study, we examined the effects of retinoids on insulin resistance in mice to gain further insight into the mechanisms responsible for this condition. Dietary administration of all-trans-retinoic acid (ATRA) significantly improved insulin sensitivity in C57BL/6J mice, which served as a model for high-fat, high-fructose diet-induced nonalcoholic fatty liver disease (NAFLD). The same effect was observed in genetically insulin-resistant KK-A(y) mice, occurring in concert with activation of leptin-signaling pathway proteins, including signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2. However, such an effect was not observed in leptin-deficient ob/ob mice. ATRA treatment significantly up-regulated leptin receptor (LEPR) expression in the livers of NAFLD mice. In agreement with these observations, in vitro experiments showed that in the presence of leptin, ATRA directly induced LEPR gene expression through RARα, resulting in enhancement of STAT3 and insulin-induced insulin receptor substrate 1 phosphorylation. A selective RARα/ß agonist, Am80, also enhanced hepatic LEPR expression and STAT3 phosphorylation and ameliorated insulin resistance in KK-A(y) mice. CONCLUSION: We discovered an unrecognized mechanism of retinoid action for the activation of hepatic leptin signaling, which resulted in enhanced insulin sensitivity in two mouse models of insulin resistance. Our data suggest that retinoids might have potential for treating NAFLD associated with insulin resistance.

Development of a novel nanoparticle by dual modification with the pluripotential cell-penetrating peptide PepFect6 for cellular uptake, endosomal escape, and decondensation of an siRNA core complex.

Development of novel devices for effective nucleotide release from nanoparticles is required to improve the functionality of nonviral delivery systems, because decondensation of nucleotide/polycation complexes is considered as a key step for cytoplasmic delivery of nucleotides. Previously, PepFect6 (PF6) comprised chloroquine analog moieties and a stearylated cell-penetrating peptide to facilitate endosomal escape and cellular uptake, respectively, was developed as a device for efficient siRNA delivery. As PF6 contains bulky chloroquine analog moieties, the polyplexes are expected to be loose structure, which facilitates decondensation. In the present study, siRNA was electrostatically condensed by PF6, and the PF6/siRNA complexes were coated with lipid membranes. The surface of the nanoparticles encapsulating the PF6/siRNA core (PF6-NP) was modified with PF6 for endosomal escape (PF6/PF6-NP). The RNAi effect of PF6/PF6-NP was compared with those of stearylated cell-penetrating peptide octaarginine (R8)-modified PF6-NP, R8-modified nanoparticles encapsulating the R8/siRNA core (R8-NP) and PF6-modified R8-NP. Nanoparticles encapsulating the PF6 polyplex, especially PF/PF-NP, showed a significant knockdown effect on luciferase activity of B16-F1 cells stably expressing luciferase. siRNA was widely distributed within the cytoplasm after transfection of the nanoparticles encapsulating the PF6 polyplex, while siRNA encapsulated in the R8-presenting nanoparticles was localized within the nuclei. Thus, the siRNA distribution was dependent on the manner of peptide-modification. In conclusion, we have successfully developed PF6/PF6-NP exhibiting a potent RNAi effect resulting from high cellular uptake, efficient endosomal escape and decondensation of the polyplexes based on the multifunctional cell penetrating peptide PF6. PF6 is therefore a useful pluripotential device for siRNA delivery.

Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

Peroxisome proliferator-activated receptor γ (PPARγ) plays indispensable roles in adipogenesis, which is frequently impaired under pathological conditions such as non-alcoholic steatohepatitis (NASH). Thus, a potent PPARγ antagonist, T0070907 is known as a useful tool for understanding such pathological conditions, while T007097 was also suggested to have PPARγ-independent actions. In the present study, we found that T0070907 inhibited adipogenesis concomitantly with the induction of rapid apoptosis of immature adipocytes within 2 h, whereas another PPARγ antagonist, SR-202 did not show such cytotoxicity. However, T0070907 did not affect the viabilities of pre-adipocytes, mature adipocytes, and NIH-3T3 fibroblasts. The cytotoxic effect of T0070907 was not inhibited by GW1929, a PPARγ agonist, but was inhibited by α-tocopherol, which was previously shown to provide clinical benefit to NASH patients. Interestingly, treatment with high amounts of α-tocopherol alone slightly increased the cellular lipid content in mature adipocytes, but did not affect PPARγ-dependent luciferase reporter expression in COS-7 cells. Moreover, other lipophilic antioxidants, such as tocotrienols, tert-butylhydroquinone, and butylated hydroxyanisole, also inhibited T0070907-induced apoptosis like α-tocopherol. Consequently, it is suggested that T0070907 efficiently inhibits adipogenesis, not only via PPARγ-dependent manner, but also through the induction of apoptosis specifically against immature adipocytes via oxidative stress in a PPARγ-independent manner.

Presence of glyceraldehyde-derived advanced glycation end-products in the liver of insulin-resistant mice.

Insulin resistance is a fundamental feature of metabolic disorders such as metabolic syndrome. The formation of advanced glycation end-products (AGEs) is increased in patients with hyperglycemia, which results in the loss of protein function. Therefore, considerable attention has been paid to the pathological significance of AGEs in diseases associated with insulin resistance. We previously demonstrated that all-trans-retinoic acid (ATRA) ameliorated insulin resistance in mice that were fed a high-fat, high-fructose (HFHFr) diet. However, it is unclear whether the HFHFr diet increases the production of AGEs in the liver, and whether ATRA affects this production. In the present study, we investigated the production of glyceraldehyde-derived AGEs (Glycer-AGEs) in the liver of HFHFr diet-induced insulin-resistant mice using an antibody against Glycer-AGEs. We noted a remarkable formation of Glycer-AGEs with estimated molecular weights of approximately 265, 282, and 312 kDa in the liver of the insulin-resistant mice; however, the production of Glycer-AGEs was limited in the control. In accordance with previous observations, these Glycer-AGEs in mice disappeared after treatment with ATRA. These results suggest that hepatic Glycer-AGEs can be useful markers for the diagnosis and therapeutic evaluation of insulin resistance and may play a pathological role in the development of insulin resistance.

Scavenging of hydroxyl radicals in aqueous solution by astaxanthin encapsulated in liposomes.

Astaxanthin (Asx) is known to be a potent quencher of singlet oxygen and an efficient scavenger of superoxide anion. Therefore, Asx would be expected to be a useful antioxidant for the prevention of oxidative stress, a causative factor in severe diseases such as ischemic reperfusion injury. However, it is still unclear whether Asx has scavenging capability against the most potent reactive oxygen species (ROS), hydroxyl radical, because the hydrophobicity of Asx prevents analysis of hydroxyl radical scavenging ability in aqueous solution. In this study, to solve this problem, liposomes containing Asx (Asx-lipo), which could be dispersed in water, were prepared, and the scavenging ability of Asx-lipo for the hydroxyl radical was examined. The liposomal formulation enabled encapsulation of a high concentration of Asx. Asx-lipo gave a dose-dependent reduction of chemiluminescence intensity induced by hydroxyl radical in aqueous solution. Hydroxyl radical scavenging of Asx was more potent than α-tocopherol. The absorbance of Asx in the liposome decreased after reduction of hydroxyl radicals, indicating the direct hydroxyl radical scavenging by Asx. Moreover, Asx-lipo prevented hydroxyl radical-induced cytotoxicity in cultured NIH-3T3 cells. In conclusion, Asx has potent scavenging capability against hydroxyl radicals in aqueous solution, and this paper is the first report regarding hydroxyl radial scavenging by Asx.

Quantitative comparison of adipocytokine gene expression during adipocyte maturation in non-obese and obese rats.

Adipocytokines secreted from adipocytes have been extensively analyzed due to their role as key factors in various complications of obesity, including arterial sclerosis, liver steatosis, insulin resistance, and diabetes. Several in vivo and in vitro studies have suggested that adipocyte maturation is related to fluctuations in adipocytokine secretion. However, the relationship between adipocyte maturation and adipocytokine levels has not been fully elucidated. Therefore, we sought to clarify the link between adipocytokine gene expression and adipocyte maturation through systematic analysis. We quantified mRNA for six adipocytokine genes: adiponectin, resistin, leptin, plasminogen activator inhibitor 1 (PAI-1), heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and visfatin, in adipose tissue, in primary cultured adipocytes obtained from an obese Zucker rat, and in the preadipocyte cell line 3T3-L1. Moreover, to elucidate the role of adipocytokines in adipocyte maturation, adipocytokine expression levels were analyzed during maturation. Although fluctuations in adipocytokine gene expression were heterogeneous, gene expression was highly similar during maturation of primary cultured adipocytes from obese and non-obese rats, suggesting that the maturation process is independent from processes that lead to obesity. Moreover, the expression patterns of adiponectin, resistin and leptin mRNA in 3T3-L1 cells were highly similar to those in primary cultured adipocytes, indicating that these adipocytokines could be common maturation markers for primary cultured adipocytes obtained from obese and non-obese rats, and for preadipocyte cell lines.

A new method to evaluate the unfolding activity of chaperone unit ClpA based on Fe-S cluster disruption.

ATP-dependent proteases unfold their substrates and then refold (via chaperone activity) or degrade (via protease activity) them. The proteases choose between these two activities by selecting their substrates; however, little is known about their substrate selection mechanism. The present study attempts to clarify this mechanism by investigating the role of the Escherichia coli (E. coli) ATP-dependent protease ClpAP. To address this, a reaction system that can measure both chaperone and protease activities simultaneously must be constructed. However, the chaperone activities cannot be evaluated in the presence of protease units. Green fluorescent protein (GFP) is usually used as a model substrate of ClpAP; the fluorescence decrease reflects the degradation of substrates. However, it is difficult to evaluate the chaperone activity of ClpAP using this system, because it cannot distinguish between intact and refolded substrates. Therefore, it is necessary to evaluate the exact unfolding activity while avoiding restoration of substrate spectroscopic characteristics due to chaperone activity. In this study, E. coli Ferredoxin (Fd) was used as a new model substrate for ClpAP to evaluate its unfolding activity. Intact and refolded substrates may be distinguished by the existence of an Fd Fe-S cluster. To verify this hypothesis, the absorption spectrum of Fd complexed with ClpA, the chaperone unit of ClpAP, was measured. A decrease in two peaks derived from the Fe-S cluster was observed, indicating that the Fe-S cluster of Fd was disrupted by the ClpA chaperone. This reaction system should prove useful to evaluate the exact unfolding activity of ATP-dependent proteases.