Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip-Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine.

Although lateral and inter-leaflet lipid-lipid interactions in cell membranes play roles in maintaining asymmetric lipid bilayers, the molecular basis of these interactions is largely unknown. Here, we established a method to determine the distribution ratio of phospholipids between the outer and inner leaflets of asymmetric large unilamellar vesicles (aLUVs). The trimethylammonium group, (CH3)3N+, in the choline headgroup of N-palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state 1H NMR (MAS-ss-1H NMR). PSM and DOPC have the same headgroup structure, but one phospholipid was selectively observed by deuterating the trimethylammonium group of the other phospholipid. The addition of Pr3+ to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH3)3N+ group in the outer leaflet from that in the inner leaflet, which allowed estimation of the inter-leaflet distribution ratio of the unlabeled lipid in aLUVs. Using this method, we evaluated the translocation of PSM and DOPC between the outer and inner leaflets of the cholesterol-containing aLUVs, with PSM and DOPC mostly distributed in the outer and inner leaflets, respectively, immediately after aLUV preparation; their flip and flop rates were approximately 2.7 and 6.4 × 10-6 s-1, respectively. During the passive symmetrization of aLUVs, the lipid translocation rate was decreased due to changes in the membrane order, probably through the formation of the registered liquid-ordered domains. Comparison of the result with that of symmetric LUVs revealed that lipid asymmetry may not significantly affect the lipid translocation rates, while the lateral lipid-lipid interaction may be a dominant factor in lipid translocation under these conditions. These findings highlight the importance of considering the effects of lateral lipid interactions within the same leaflet on lipid flip-flop rates when evaluating the asymmetry of phospholipids in the cell membrane.

Behavior of Triterpenoid Saponin Ginsenoside Rh2 in Ordered and Disordered Phases in Model Membranes Consisting of Sphingomyelin, Phosphatidylcholine, and Cholesterol.

The ginsenoside Rh2 (Rh2) is a saponin of medicinal ginseng, and it has attracted much attention for its pharmacological activities. In this study, we investigated the interaction of Rh2 with biological membranes using model membranes. We examined the effects of various lipids on the membrane-disrupting activity of Rh2 and found that cholesterol and sphingomyelin (SM) had no significant effect. Furthermore, the effects of Rh2 on acyl chain packing (DPH anisotropy) and water molecule permeability (GP340 values) did not differ significantly between bilayers containing SM and saturated phosphatidylcholine. These results suggest that the formation of the liquid-ordered (Lo) phase affects the behavior of Rh2 in the membrane rather than a specific interaction of Rh2 with a particular lipid. We investigated the effects of Rh2 on the Lo and liquid-disordered (Ld) phases using surface tension measurements and fluorescence experiments. In the surface tension-area isotherms, we compared the monolayers of the Ld and Lo lipid compositions and found that Rh2 is abundantly bound to both monolayers, with the amount being greater in the Ld phase than in the Lo phase. In addition, the hydration state of the bilayers, mainly consisting of the Lo or Ld phase, showed that Rh2 tends to bind to the surface of the bilayer in both phases. At higher concentrations, Rh2 tends to bind more abundantly to the relatively shallow interior of the Ld phase than the Lo phase. The phase-dependent membrane behavior of Rh2 is probably due to the phase-selective affinity and binding mode of Rh2.

Simultaneous electrochemical detection of ozone and free chlorine with a boron-doped diamond electrode.

O3 and free chlorine play significant roles in disinfection and organic degradation. There are numerous reports about their mixed-use, yet detection of the residual concentrations is not easily accomplished, whilst the interactions between them remain unclear. Herein, we develop a detection method using a boron-doped diamond (BDD) electrode to achieve the simultaneous determination of O3 and free chlorine, which benefits from the unique property of the wide potential window of BDD electrodes. It is indicated that O3 can always be accurately determined at 0.35 V vs. Ag/AgCl in an acidic solution (pH = 4-5), whether or not free chlorine is present in the solution, whereas free chlorine can be precisely monitored at -0.78 V vs. Ag/AgCl only after the O3 is completely depleted. Furthermore, in a basic solution (pH = 9-10), the reduction peak of O3 at 0.57 V vs. Ag/AgCl promptly disappears accompanied by a decrease in the peak current of free chlorine at 1.41 V. All the phenomena observed in the acidic and basic solutions are concurrently confirmed in a quasi-neutral solution. Based on these complementary measurements, a mechanism is proposed, in which the O3 reduction results in partial oxidation of the BDD surface, hindering the reduction of free chlorine in the acidic mixture; whereas O3 reacts quickly with free chlorine in the basic solution, which causes the co-consumption of both of them. It is hoped these results will give us a guide as to how better utilize mixtures and more precisely simultaneously determine O3 and free chlorine in the mixture.

Structures of the Largest Amphidinol Homologues from the Dinoflagellate Amphidinium carterae and Structure-Activity Relationships.

Amphidinols are polyketide metabolites produced by marine dinoflagellates and are chiefly composed of a long linear chain with polyol groups and polyolefins. Two new homologues, amphidinols 20 (AM20, 1) and 21 (AM21, 2), were isolated from Amphidinium carterae collected in Korea. Their structures were elucidated by detailed NMR analyses as amphidinol 6-type compounds with remarkably long polyol chains. Amphidinol 21 (2) has the longest linear structure among the amphidinol homologues reported so far. The congeners, particularly amphidinol 21 (2), showed weaker activity in hemolysis and antifungal assays compared to known amphidinols.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain.

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes.

Centerband-only analysis of rotor-unsynchronized spin echo for measurement of lipid (31) P chemical shift anisotropy.

Structural diversity and molecular flexibility of phospholipids are essential for biological membranes to play key roles in numerous cellular processes. Uncovering the behavior of individual lipids in membrane dynamics is crucial for understanding the molecular mechanisms underlying biological functions of cell membranes. In this paper, we introduce a simple method to investigate dynamics of lipid molecules in multi-component systems by measuring the (31) P chemical shift anisotropy (CSA) under magic angle spinning (MAS) conditions. For achieving both signal separation and CSA determination, we utilized a centerband-only analysis of rotor-unsynchronized spin echo (COARSE). This analysis is based on the curve fitting of periodic modulation of centerband intensity along the interpulse delay time in rotor-unsynchronized spin-echo experiments. The utility of COARSE was examined by using phospholipid vesicles, a three-component lipid raft model system, and archaeal purple membranes. We found that the apparent advantages of this method are high resolution and high sensitivity given by the moderate MAS speed and the one-dimensional acquisition with short spin-echo delays. COARSE provides an alternative method for CSA measurement that is effective in the investigation of lipid polymorphologies.

Convergent synthesis and biological activity of the WXYZA'B'C' ring system of maitotoxin.

The WXYZA'B'C' ring system ( 1) of maitotoxin (MTX) was synthesized in a convergent manner via successive coupling of the W, Z, and C' ring fragments through construction of the XY and A'B' ring systems. The synthetic segment 1 blocked the hemolytic activity elicited by MTX.