ß-Glucosylation of cholesterol reduces sterol-sphingomyelin interactions.

Cholesteryl-ß-D-glucoside (ChoGlc) is a mammalian glycolipid that is expressed in brain tissue. The effects of glucosylation on the ordering and lipid interactions of cholesterol (Cho) were examined in membranes composed of N-stearoyl sphingomyelin (SSM), which is abundant in the brain, and to investigate the possible molecular mechanism involved in these interactions. Differential scanning calorimetry revealed that ChoGlc was miscible with SSM in a similar extent of Cho. Solid-state 2H NMR of deuterated SSM and fluorescent anisotropy using 1,6-diphenylhexatriene demonstrated that the glucosylation of Cho significantly reduced the effect of the sterol tetracyclic core on the ordering of SSM chains. The orientation of the sterol core was further examined by solid-state NMR analysis of deuterated and fluorinated ChoGlc analogues. ChoGlc had a smaller tilt angle between the long molecular axis (C3-C17) and the membrane normal than Cho in SSM bilayers, and the fluctuations in the tilt angle were largely unaffected by temperature-dependent mobility changes of SSM acyl chains. This orientation of the sterol core of ChoGlc leads to reduce sterol-SSM interactions. The MD simulation results suggested that the Glc moiety perturbs the SSM-sterol interactions, which reduces the umbrella effect of the phosphocholine headgroup because the hydrophilic glucose moiety resides at the same depth as an SSM amide group. These differences between ChoGlc and Cho also weaken the SSM-ChoGlc interactions. Thus, the distribution and localization of Cho and ChoGlc possibly control the stability of sphingomyelin-based domains that transiently occur at specific locations in biological membranes.

Interactions of OSW-1 with Lipid Bilayers in Comparison with Digitonin and Soyasaponin.

OSW-1, a unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane (Kapp = 0.38 µM-1) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage (r50 = 0.47) occurrence. OSW-1 showed slightly lower activity (Kapp = 0.31 µM-1; r50 = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity (Kapp = 0.017 µM-1; r50 = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 µM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 µM, r50 = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 µM, r50 = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.

Antioxidant, Cytotoxicity, and Antiophidian Potential of Alstonia macrophylla Bark.

The objective of this research was to find the possible pharmacognosy of the bark of the Philippine Alstonia macrophylla Wall. ex G.Don (AM). Gas chromatographic-mass spectral (GC-EI-MS) characterization and energy dispersive X-ray spectroscopy (EDX) were performed to detect the bioactive constituents. EDX analysis of AM bark displayed a high content of potassium (3.26%) and calcium (2.96%). Eight constituents were detected in AM crude dichloromethane (DCM) extracts, which consisted of a long-chain unsaturated fatty acid (17:0) and fatty acid esters such as ethyl hexadecanoate and methyl hexadecanoate. Extraction of AM bark using methanol and dimethyl sulfoxide (MeOH/DMSO) solvents resulted in the identification of 17 constituents, principally alkaloids (alstonerine, 34.38%; strictamin, 5.23%; rauvomitin, 4.29%; and brucine, 3.66%) and triterpenoids (γ-sitosterol, 3.85%; lupeol, 3.00%; 24-methylenecycloartanol, 2.81%; campesterol, 2.71%; ß-amyrin, 2.30%; and stigmasterol, 2.13%). MeOH/DMSO samples of AM were used in the selected bioassays. The samples exhibited efficient free radical scavenging activity (IC50 = 0.71 mg/mL) and were noncytotoxic to normal HDFn (IC50 > 100 µg/mL) and neoplastic THP-1 cell lines (IC50 = 67.22 µg/mL) while highly degenerative to MCF-7 (IC50 = 6.34 µg/mL), H69PR (IC50 = 7.05 µg/mL), and HT-29 (IC50 = 9.10 µg/mL). Most interestingly, the AM samples inhibited the northern Philippine Cobra's (Naja philippinensis Taylor) venom (IC50 = 297.27 ± 9.33 µg/mL) through a secretory phospholipase A2 assay.

Theonellamide A, a marine-sponge-derived bicyclic peptide, binds to cholesterol in aqueous DMSO: Solution NMR-based analysis of peptide-sterol interactions using hydroxylated sterol.

Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. The inclusion of cholesterol (Chol) or ergosterol in the phosphatidylcholine membrane is known to significantly enhance the membrane affinity for theonellamide A (TNM-A). We have previously revealed that TNM-A stays in a monomeric form in dimethylsulfoxide (DMSO) solvent systems, whereas the peptide forms oligomers in aqueous media. In this study, we utilized 1H NMR chemical shift changes (Δδ1H) in aqueous DMSO solution to evaluate the TNM-A/sterol interaction. Because Chol does not dissolve well in this solvent, we used 25-hydroxycholesterol (25-HC) instead, which turned out to interact with membrane-bound TNM-A in a very similar way to that of Chol. We determined the dissociation constant, KD, by NMR titration experiments and measured the chemical shift changes of TNM-A induced by 25-HC binding in the DMSO solution. Significant changes were observed for several amino acid residues in a certain area of the molecule. The results from the solution NMR experiments, together with previous findings, suggest that the TNM-Chol complex, where the hydrophobic cavity of TNM probably incorporates Chol, becomes less polar by Chol interaction, resulting in a greater accumulation of the peptide in membrane. The deeper penetration of TNM-A into the membrane interior enhances membrane disruption. We also demonstrated that hydroxylated sterols, such as 25-HC that has higher solubility in most NMR solvents than Chol, act as a versatile substitute for sterol and could be used in 1H NMR-based studies of sterol-binding peptides.

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.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.