Enhancing pressure consistency and transferability of structure-based coarse-graining.

Coarse-graining, which models molecules with coarse-grained (CG) beads, allows molecular dynamics simulations to be applied to systems with large length and time scales while preserving the essential molecular structure. However, CG models generally have insufficient representability and transferability. A commonly used method to resolve this problem is multi-state iterative Boltzmann inversion (MS-IBI) with pressure correction, which matches both the structural properties and pressures at different thermodynamic states between CG and all-atom (AA) simulations. Nevertheless, this method is usually effective only in a narrow pressure range. In this paper, we propose a modified CG scheme to overcome this limitation. We find that the fundamental reason for this limitation is that CG beads at close distances are ellipsoids rather than isotropically compressed spheres, as described in conventional CG models. Hence, we propose a method to compensate for such differences by slightly modifying the radial distribution functions (RDFs) derived from AA simulations and using the modified RDFs as references for pressure-corrected MS-IBI. We also propose a method to determine the initial non-bonded potential using both the target RDF and pressure. Using n-dodecane as a case study, we demonstrate that the CG model developed using our scheme reproduces the RDFs and pressures over a wide range of pressure states, including three reference low-pressure states and two test high-pressure states. The proposed scheme allows for accurate CG simulations of systems in which pressure or density varies with time and/or position.

Effect of High Fructose-Induced Metabolic Syndrome on Tissue Vitamin E and Lipid Peroxide Levels in Rats.

In the present study, we examined the effect of high fructose-induced metabolic syndrome (MetS) on tissue vitamin E and lipid peroxide (LPO) levels in rats. Feeding of a diet containing 60% fructose (HFD) to Wistar rats for 2, 4, and 6 wk caused week-dependent increases in HOMA-IR score and serum insulin, triglyceride, total cholesterol, and free fatty acid concentrations. Each week HFD feeding increased serum vitamin E concentration. Six-week HFD feeding reduced vitamin E status (the serum ratio of vitamin E/triglyceride+total cholesterol). Four- and 6-wk HFD feeding increased serum LPO concentration. Two-week HFD feeding increased liver, heart, kidney, and skeletal muscle (SM) vitamin E contents and decreased white adipose tissue (WAT) vitamin E content. Four- and 6-wk HFD feeding further reduced WAT vitamin E content without affecting the increased kidney and SM vitamin E contents. Six-week HFD feeding reduced the increased liver and heart vitamin E contents below the level of non-HFD feeding. Four-week HFD feeding increased heart and WAT LPO contents. Six-week HFD feeding increased liver LPO content and further increased heart and WAT LPO contents. Kidney and SM LPO contents remained unchanged. These results indicate that HFD-rats with early MetS have increased liver, kidney, heart, and SM vitamin E contents and decreased WAT vitamin E content under unchanged tissue LPO content and vitamin E status, while HFD-fed rats with progressed MetS have both decreased liver, heart, and WAT vitamin E contents under increased tissue LPO content and disrupted vitamin E status.

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.

Anisotropic Shear Viscosity of Photoaligned Liquid Crystal Confined in Submicrometer-to-Nanometer-Scale Gap Widths Revealed with Simultaneously Measured Molecular Orientation.

In the context of the use of liquid crystals (LCs) as lubricants and lubricant additives, this study investigates the anisotropic shear viscosity of LCs confined in nanometer-sized gap widths subject to both shearing and photoalignment. The photoalignment is achieved using anisotropically dimerized polyvinyl cinnamate (PVCi) films coated on substrates. We simultaneously measure the viscosity and order parameter of a liquid crystal (4-cyano-4'-pentylbiphenyl) confined and sheared in the gap range of 500 nm down to a few nm. We achieve this simultaneous measurement using an original method that combines a highly sensitive viscosity measurement and a sensitive birefringence measurement. When the LC is sheared in the same direction as the photoalignment (parallel shearing), the order parameter, which is around 0.3 in the bulk state, increases up to around 0.4 at a gap width of less than 50 nm and the viscosity is smaller than half the bulk viscosity. We consider that this increase in the order parameter is due to the highly ordered photoaligned LC layer near the PVCi film, and the viscosity decrease is due to shear thinning of this layer enhanced by both confinement and molecular ordering. In addition, we observe a gradual decrease in viscosity starting at a gap of less than around 300 nm in the parallel shearing. Based on the apparent slip model, we show that the LC layer near the PVCi film can also cause this gradual viscosity decrease. In contrast, when the LC is sheared in the direction perpendicular to the photoalignment direction (perpendicular shearing), the viscosity increases as the gap decreases. We speculate that this is due to the rotational motion of the LC molecules caused by the competing effect of shear alignment and photoalignment. We believe our findings can significantly contribute to a better understanding of the confined LCs utilized for lubrication.

Effect of Dietary Vitamin E Supplementation on Liver Oxidative Damage in Rats with Water-Immersion Restraint Stress.

We examined how dietary supplementation of vitamin E protects against liver oxidative damage in rats with water-immersion restraint stress (WIRS). Before WIRS exposure, rats received a normal diet (ND) or vitamin E-supplemented diet (VESD) (500 IU α-tocopherol/kg diet) at a mean dose of 15 g/animal/d for 4 wk. The two diet groups had serum transaminases and lactate dehydrogenase activities and adrenocorticotropic hormone, corticosterone, and glucose levels to a similar extent. VESD-fed rats had higher liver α-tocopherol concentrations and lower liver ascorbic acid, total coenzyme Q9 (CoQ9), reduced CoQ9, reduced CoQ10, and lipid peroxide (LPO) concentrations than ND-fed rats. When the two diet groups were exposed to 6 h of WIRS, the serum liver cell damage index enzyme activities increased more greatly in ND-fed rats than in VESD-fed rats but the serum stress marker levels increased to a similar extent. The WIRS exposure caused no change in liver LPO concentration with the further increase in liver α-tocopherol concentration in VESD-fed rats but increased liver LPO concentration without changing liver α-tocopherol concentration in ND-fed rats. Upon the WIRS exposure, liver reduced glutathione concentration decreased with the further decrease in liver ascorbic acid concentration in VESD-fed rats and those concentrations decreased in ND-fed rats. The WIRS exposure recovered the decreased liver total CoQ9 and reduced CoQ9 concentrations in VESD-fed rats but decreased liver total CoQ9, reduced CoQ9, and reduced CoQ10 concentrations in ND-fed rats. These results indicate that dietary vitamin E supplementation protects against liver oxidative damage without affecting the stress response in rats with WIRS.

Boundary condition at a two-phase interface in the lattice Boltzmann method for the convection-diffusion equation.

A boundary scheme in the lattice Boltzmann method (LBM) for the convection-diffusion equation, which correctly realizes the internal boundary condition at the interface between two phases with different transport properties, is presented. The difficulty in satisfying the continuity of flux at the interface in a transient analysis, which is inherent in the conventional LBM, is overcome by modifying the collision operator and the streaming process of the LBM. An asymptotic analysis of the scheme is carried out in order to clarify the role played by the adjustable parameters involved in the scheme. As a result, the internal boundary condition is shown to be satisfied with second-order accuracy with respect to the lattice interval, if we assign appropriate values to the adjustable parameters. In addition, two specific problems are numerically analyzed, and comparison with the analytical solutions of the problems numerically validates the proposed scheme.

A kinetic study of the radical-scavenging action of tocotrienols in the membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the second-order rate constants (ks) for the reaction of tocotrienol homologues (α-, ß-, γ-, and δ-Toc-3Hs) with the aroxyl radical (ArO•) used as a model for lipid peroxyl radicals (LOO•) in the membranes of egg yolk phosphatidylcholine (EYPC) vesicles by stopped-flow spectrophotometry, and compared them to those of tocopherol homologues (α-, ß-, γ-, and δ-TocHs). The relative rate constants of Toc-3H homologues to α-Toc-3H in membranes (α/ß/γ/δ=100/83.7/63.2/20.2) were not much different to those of TocH homologues to α-TocH (α/ß/γ/δ=100/88.4/83.8/17.3). Each ks value of Toc-3H homologues in membranes was 60-80% of that of the corresponding TocH homologues except for the almost identical ks values of δ-homologues, but there was no difference in EtOH solution between each ks value of the corresponding homologues of Toc-3H and TocH. These results indicate that the difference of the alkyl-side chain structure of vitamin E causes a change in the mobility of vitamin E molecules and/or the location of their antioxidant OH-groups in membranes, resulting in lowered radical-trapping rates of Toc-3Hs. By use of the ratio of the kinh value of α-TocH with LOO• (3.20×10(6) M(-1)s(-1)) to the ks value of α-TocH with ArO• (8.05×10(4) M(-1)s(-1)) in chlorobenzene (that is, 39.8), the kinh value for the reaction of α-TocH with LOO• in membrane was estimated to be 1.03×10(5) M(-1)s(-1).

Vitamin D3 derivatives increase soluble CD14 release through ERK1/2 activation and decrease IL-8 production in intestinal epithelial cells.

Dysfunction of the innate immune system has been reported to cause intestinal inflammation. Vitamin D3 is known to be an important immune system regulator and exerts anti-inflammatory effects. We investigated in vitro effects of vitamin D3 and its derivatives on the innate immune system in HT-29 cells, a line of human colon adenocarcinoma cells. Among the innate immune-related receptors such as Toll-like receptor (TLR) 1, 2, 4, 6, and CD14 examined by flow cytometry, only CD14 was up-regulated by vitamin D3 derivatives. Release of soluble form CD14 (sCD14) was also increased by vitamin D3 derivatives. The 1α,25-dihydroxy-22-oxavitamin D3 (Oxa-D3) induced-sCD14 release was inhibited by U0126 (a specific inhibitor of extracellular signal-regulated kinase; ERK1/2) but not by SB203580 (a specific inhibitor of p38 MAPK), and ERK1/2 phosphorylation was accelerated by Oxa-D3. These results indicate that Oxa-D3 facilitates the release of sCD14 through ERK1/2 activation. IL-8 production stimulated with LPS was diminished by vitamin D3 derivatives. Recombinant sCD14 also lowered the LPS-stimulated IL-8 production, suggesting neutralization of LPS by sCD14. The anti-inflammatory effect of vitamin D3 derivatives was thus associated with diminution of IL-8 production due to increased release of sCD14.

Structure-based coarse-graining for inhomogeneous liquid polymer systems.

The iterative Boltzmann inversion (IBI) method is used to derive interaction potentials for coarse-grained (CG) systems by matching structural properties of a reference atomistic system. However, because it depends on such thermodynamic conditions as density and pressure of the reference system, the derived CG nonbonded potential is probably not applicable to inhomogeneous systems containing different density regimes. In this paper, we propose a structure-based coarse-graining scheme to devise CG nonbonded potentials that are applicable to different density bulk systems and inhomogeneous systems with interfaces. Similar to the IBI, the radial distribution function (RDF) of a reference atomistic bulk system is used for iteratively refining the CG nonbonded potential. In contrast to the IBI, however, our scheme employs an appropriately estimated initial guess and a small amount of refinement to suppress transfer of the many-body interaction effects included in the reference RDF into the CG nonbonded potential. To demonstrate the application of our approach to inhomogeneous systems, we perform coarse-graining for a liquid perfluoropolyether (PFPE) film coated on a carbon surface. The constructed CG PFPE model favorably reproduces structural and density distribution functions, not only for bulk systems, but also at the liquid-vacuum and liquid-solid interfaces, demonstrating that our CG scheme offers an easy and practical way to accurately determine nonbonded potentials for inhomogeneous systems.

Vitamin E depletion enhances liver oxidative damage in rats with water-immersion restraint stress.

We examined the effect of vitamin E depletion on liver oxidative damage in rats with water-immersion restraint stress (WIRS). Male Wistar rats were fed a normal diet (N) or vitamin E-depleted diet (VE-D) for 4 wk. N- and VE-D-fed rats were exposed to WIRS for 6 h. The activities of serum transaminases and lactate dehydrogenase and serum ascorbic acid concentration were similar in both diet groups. WIRS exposure increased these serum enzyme activities and the serum ascorbic acid concentration in both diet groups but the ratios of these increases were higher in VE-D-fed rats than in N-fed rats. Serum and liver α-tocopherol concentrations in VE-D-rats were approximately 50% and 30% of those in N-fed rats, respectively. WIRS exposure reduced liver α-tocopherol concentration in VE-D-fed rats, but not in N-fed rats. Liver ascorbic acid and reduced glutathione concentrations were higher in the VE-D-fed group than in the N-fed group. WIRS exposure reduced liver ascorbic acid and reduced glutathione concentrations in both diet groups. There were no differences in liver concentrations of coenzyme Q9 or coenzyme Q10 in the reduced form between the N- and VE-D-fed groups. WIRS exposure reduced liver concentrations of coenzyme Q9 and coenzyme Q10 in the reduced form in both diet groups. Liver lipid peroxide concentration was higher in the VE-D-fed group than in the N-fed group. WIRS exposure raised liver lipid peroxide concentration more in the VE-D-fed group than in the N-fed group. These results indicate that vitamin E depletion enhances liver oxidative damage in rats with WIRS.

Involvement of intestinal intraepithelial lymphocytes in turnover of intestinal epithelial cells: morphological and functional alterations due to daily administration of FK506.

To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14days were investigated. IELs, particularly TCR-γδ⁺ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover.

A single exposure of rats to water-immersion restraint stress induces oxidative stress more severely in the thymus than in the spleen.

OBJECTIVES: We examined whether a single exposure of rats to water-immersion restraint stress (WIRS) induces oxidative stress in the thymus and spleen. METHODS: Vitamin E, ascorbic acid, reduced glutathione (GSH), and lipid peroxide (LPO) were assayed in the thymus and spleen of rats with and without 6 hours of WIRS. RESULTS: In unstressed rats, vitamin E, ascorbic acid, GSH, and LPO levels were higher in the thymus than in the spleen. Thymic ascorbic acid level was lower in stressed rats than in unstressed rats. Splenic ascorbic acid level was similar in both groups. Thymic and splenic GSH levels were lower in stressed rats than in unstressed rats but the reduced amount of GSH was lower in the spleen than in the thymus. Thymic vitamin E level was lower in stressed than in unstressed rats. Splenic vitamin E level was higher in stressed rats than in unstressed rats. Thymic and splenic LPO levels were higher in stressed rats than in unstressed rats but the increased amount of LPO was higher in the thymus than in the spleen. CONCLUSION: It is indicated that a single expose of rats to WIRS induces oxidative stress more severely in the thymus than in the spleen.

Development of a novel drug delivery system consisting of an antitumor agent tocopheryl succinate.

We have developed a novel drug delivery system (DDS) using an antitumor agent, α-tocopheryl succinate (TS). TS has attracted attention as a unique anti-cancer drug for its ability to induce apoptosis in various cancer cells. Furthermore, TS itself readily forms nanovesicles (TS-NVs) and is a prospective tool for use as an antitumor DDS. However, TS-NVs are unstable for encapsulating drugs and passive targeting delivery to tumor tissue via enhanced permeation and retention effect. Therefore, to improve the stability of vesicles, we developed a novel nanovesicle consisting of TS and egg phosphatidylcholine (TS-EPC-NVs). The stability of vesicles of TS-EPC-NVs was significantly higher than that of TS-NVs. As a result, the in vivo antitumor activity of TS-EPC-NVs was more potent than that of TS-NVs. The enhanced antitumor activity of TS-EPC-NVs was found to be due to its effective intratumoral distribution. Moreover, the in vitro anticancer efficiency of TS-EPC-NVs increased seven-fold. We suggest that the improvement is due to homogenous cellular uptake and enhanced cytosolic delivery of the nanovesicles via alteration of intracellular trafficking. Furthermore, TS-EPC-NVs encapsulating siRNA showed significant knockdown efficiency. In summary, TS-EPC-NVs represent a novel and attractive drug delivery system. The system shows antitumor activity of the encapsulated drug and the carrier itself.

Down-modulation of toll-like receptor 2 expression on granulocytes and suppression of interleukin-8 production due to in vitro treatment with cellulose acetate beads.

The Adacolumn, which is filled with cellulose acetate beads (CA beads), has been used as a medical device for inflammatory diseases. The CA beads selectively adsorb granulocytes and monocytes and remove them from the peripheral blood. The anti-inflammatory effects of the Adacolumn are possibly caused by removal of these cells but also due to the functional changes in the processed cells. In this study, we investigated the effects of CA beads treatment on modulation of the expression of innate immunity receptors such as the Toll-like receptor (TLR) family and production of an inflammatory cytokine, interleukin-8 (IL-8). Changes in the expressions of TLR1, 2, 4 and 6 in peripheral leukocytes exposed to CA beads were examined by flow cytometry. TLR2 expression on the surface of granulocytes exposed to CA beads was decreased, but the amount of intracellular TLR2 was increased, possibly by internalization. These changes were not observed in monocytes or lymphocytes. Peptidoglycan (PGN) treatment produced similar changes in TLR2 on granulocytes. We also measured the amounts of IL-8 in cultured blood treated with lipopolysaccharide (LPS) and PGN, which are known TLR agonists. PGN-induced IL-8 production was lower in CA beads-treated leukocytes than that in non-treated leukocytes, but LPS did not induce these changes. Based on these findings, we conclude that the down-modulation of TLR2 and suppression of IL-8 production on granulocytes by CA beads, may play an important role in the anti-inflammatory effects of the Adacolumn.

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, ß-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO·) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H(2)O=7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k(s)) for α-TocH in MeOH/H(2)O solution with EYPC vesicles were apparently 3.45×10(5)M(-1)s(-1), which was about 8 times higher than that (4.50×10(4)M(-1)s(-1)) in MeOH/H(2)O solution without EYPC vesicles. The corrected k(s) of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10mM EYPC vesicles than in the bulk MeOH/H(2)O solution, was 2.60×10(3)M(-1)s(-1), which was one-seventeenth that in MeOH/H(2)O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k(s) of vitamin E in membranes increased in the order of tocol<δ-TocH<γ-TocH∼ß-TocH<α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H(2)O solution among vitamin E analogues [k(s)(vesicle)/k(s) (MeOH/H(2)O)=7.7, 10.0, 9.5, 7.4, and 5.1 for α-, ß-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k(s)(micelle)/k(s)(EtOH)=100, 47, 41, 15, and 6.3 for α-, ß-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol<δ-TocH<γ-TocH∼ß-TocH<α-TocH.

Evaluation of inhibitory actions of flavonols and related substances on lysophospholipase d activity of serum autotaxin by a convenient assay using a chromogenic substrate.

Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be utilized to ameliorate the LPA-related pathological conditions. In this study, a new assay was devised to quantify p-nitrophenol from hydrolysis of chromogenic substrate by serum lysoPLD without tedious lipid extraction procedures. Flavonols, phenolic acids, free fatty acids, and N-acyltyrosines inhibited lysoPLD activity in a micromolar range. They were classified into competitive, noncompetitive, or mixed type inhibitors. The results show that the low hydrophobicity of an inhibitor is a critical factor in its preference for the binding to a noncatalytic binding site over a catalytic binding site. Considering its reported bioavailability and the low dependency of its inhibitory activity on serum dilution, flavonol is likely to be a more effective lysoPLD inhibitor in human blood circulation in vivo than the other inhibitors including LPA.

Measurement of lipid hydroperoxides by the ferric-xylenol orange method (1) characteristics of the ferric-xylenol orange/membrane phosphatidylcholine complex.

The ferric-xylenol orange (FOX) method for measurement of hydroperoxides is based on a technique that employs reduction of peroxides in an acidic condition by Fe2+ and formation of the colored ferric-xylenol orange (XO/Fe3+) product with a peak at 560 nm. The 560 nm absorbance peak of XO/Fe3+ shifts to a 610 nm peak with high absorption intensity in the presence of phosphatidylcholine. This is useful for quantification of peroxides such as phospholipid hydroperoxides. Based on this finding, we recently reported a modified FOX method. We now show by measurements of absorbance, broadening of the electron paramagnetic resonance spectrum, changes in the vesicle size and their zeta potentials, the effects of detergents, and manipulation of the membrane lipid composition that the XO/Fe3+-phosphatidylcholine complex forms only in the presence of intact phosphatidylcholine membranes. The phosphate group on the phospholipid plays a role in this interaction which may involve an electron transfer from the phosphate to the Fe3+. A positively charged quaternary amine on the phosphatidylcholine is also necessary to give a peak absorbance at 610 nm. Our observations are consistent with binding of one XO/Fe3+ complex to about 3 molecules of the egg yolk phosphatidylcholine carrying a zero net charge.

Measurement of lipid hydroperoxides by the ferric-xylenol orange method (2) application to lipoxygenase assay.

The ferric-xylenol orange (FOX) method has been used for quantification of hydroperoxides by measuring the colored ferric-xylenol orange (XO/Fe3+) product with a peak absorbance at 560 nm. We recently reported a modified FOX method, the sensitivity of which was increased in the presence of membranous phosphatidylcholine (PC) by forming a XO/Fe3+-PC complex with a peak absorbance at 610 nm. Lipoxygenases (LOXs) and their metabolites have been implicated in a wide range of disease states. We applied our newly developed FOX method to the assay of human 15-lipoxygenase-2 (15-LOX-2) and soybean lipoxygenase (SLOX) as typical animal and plant lipoxygenases, respectively. The amounts of 15-S-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (15-HPETE) produced by 15-LOX-2 measured by UV-absorption at 237 nm attributed to the conjugated diene, coincided with the results of our FOX method measuring absorbance at 610 nm. The 15-HPETE production time courses measured by the two methods also correlated well. SLOX rapidly oxidized unesterified linoleic acids (LA) and slowly esterified fatty acids in egg yolk PC (EYPC). Availability of EYPC was increased if the membrane structure was moderately disturbed by MeOH and Triton X-100, but LA oxidation was readily decreased by them. These results indicate that our method is useful for lipoxygenase assay. Furthermore, our method was applicable to assaying the inhibitory effect of 5,8,11,14-eicosatetraynoic acid (ETYA) on SLOX activity using LA and EYPC as substrates. The inhibition dose-dependency of ETYA was almost the same in the LA and EYPC systems, although the enzyme concentrations differed by a factor of 1,000, suggesting that ETYA functioned as a competitive inhibitor. These results indicate that our method may be useful as a screen for the identification of novel inhibitors of lipoxygenases.

Motion picture imaging of a nanometer-thick liquid film dewetting by ellipsometric microscopy with a submicrometer lateral resolution.

We visualized the detwetting of a nanometer-thick unstable liquid film on a nanotextured solid surface with a high lateral spatial resolution. The dewetting was imaged as a motion picture at a submicrometer spatial resolution and a frame rate of 4 frames/s, using ellipsometric microscopy in a vertical objective configuration. The observation revealed that the dewetting process significantly depends on the sign of the disjoining pressure Pi. When Pi is negative, the film rupture due to the spinodal dewetting proceeds to droplet formation in a single step, whereas, when Pi is positive, the film rupture due to the spinodal dewetting stops when the pressure of the thicker region balances with that of the thinner region, and then the heterogeneous grooves are nucleated and grow. The dewetting process dependence on the sign of Pi can be found in systems other than that reported here because the sign of Pi changes at the local maximum of the surface energy.

Dynamics of lipid peroxidation and antioxidion of alpha-tocopherol in membranes.

The dynamics of initiation and inhibition of lipid peroxidation by alpha-tocopherol (alpha-Toc) in membranes were investigated under biological conditions using phosphatidylcholine liposomes. First, I examined how superoxide generated in the bulk water phase is able to induce lipid peroxidation in the inner hydrophobic region of the membrane. Second, I studied the localization of the antioxidant OH group of alpha-Toc in membranes and its lipid radical-trapping dynamics. Third, I investigated how alpha-Toc that is oxidized during radical trapping in membranes is recycled by ascorbic acid (AsA) in the bulk water phase. Finally, I studied the deactivation by alpha-Toc of singlet oxygen ((1)O(2)), which was generated by photoirradiation at the membrane surface, in the hydrophobic membrane inner region, and in bulk water, and measured the (1)O(2) deactivating rate constant of alpha-Toc in membranes considering: the concentration and mobility of alpha-Toc molecule in membranes, especially those of its active OH moiety located at the membrane domains, such as the membrane surface polar zone, inner hydrogen belt, and hydrophobic core, and the dielectric constant reflecting the reactivity of the OH moiety and (1)O(2) in the membrane domains where they interact.