Nystatin isolated from Streptomyces is a polyene antibiotic that is frequently used in the treatment and prophylaxis of fungal infections. Here, the fractional sterol concentration dependencies of the partition coefficient for partitioning of nystatin into ergosterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), cholesterol/DMPC, ergosterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC), and ergosterol/POPC/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylethano lam ine (POPE) multilamellar vesicles have been determined fluorometrically at 37 degrees C using approximately 0.3-1.0 mol % sterol concentration increments over a wide concentration range (e.g., 18-54 mol % sterol). This unconventional approach of varying membrane sterol content, in contrast to previous studies using large sterol concentration increments (e.g., 10 mol %), leads to a striking observation. The partition coefficient of nystatin changes dramatically with membrane sterol content in a well-defined alternating manner, displaying a local minimum at or very close to the critical sterol mole fractions (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % sterol) predicted for sterols regularly distributed in either hexagonal or centered rectangular superlattices. In ergosterol/DMPC bilayers, for example, there is a >3-fold increase in nystatin partitioning with a minute change (approximately 1 mol %) in sterol content on either side of the critical sterol mole fraction, 25.0 mol %. These results provide semifunctional evidence supporting the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073]. More importantly, these results reveal a new membrane phenomenon, that is, that nystatin partitioning is affected by the extent of sterol regular distribution in the plane of the membrane. This phenomenon occurs not only in saturated (e.g., DMPC) but also in unsaturated (e.g., POPC) lipid membranes, and persists in the presence of polar headgroup heterogeneity (e.g., POPC/POPE). This membrane property points to a new method for studying the interactions of polyene antibiotics with sterol-containing membranes, and the need to consider the membrane sterol content of the target cells when administering nystatin or other polyene antibiotics.

Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model. Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 degrees C using cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, approximately 800 nm in diameter). In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol %. Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C(r)) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation. The activity variation with cholesterol content was correlated well with the area of regular distribution (A(reg)) in the plane of the membrane as determined by nystatin fluorescence. A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (approximately 168 nm in diameter). These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (approximately 168-800 nm). The present work on COD and the previous study on phospholipase A(2) (sPLA(2)) [Liu and Chong (1999) Biochemistry 38, 3867-3873] suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner. When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C(r). When phospholipid is the substrate, the minimum activity is at C(r), as is the case with sPLA(2). Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.

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We have investigated the spectroscopic evidence for sterol superlattice formation in bilayers composed of dehydroergosterol (DHE) and a homologous series of phosphatidylcholine, namely, C(18:0):C(18:1∆ n )PC with n ) 6, 7, 9, 11, and 13. These phosphatidylcholines (PCs) possess the same number of carbons and the same number of cis double bonds; however, the position of the double bond of these PCs varies. We have examined the fractional concentration dependence of the steady-state fluorescence intensity of DHE in the liquid-crystalline multilamellar vesicles composed of DHE and this series of PCs. The DHE contents in the membranes were varied by ∼0.2 mol % in two concentration regions, 18.7-21.3 and 23.9-26.0 mol % DHE. In these two regions, 20.0 and 25.0 mol % are the critical sterol mole fractions predicted for maximal superlattice formation. A DHE intensity dip appears at each of these two critical sterol mole fractions in the PC bilayers that have the double bond located between C9 and the terminal carbon. However, no distinct DHE intensity dips are observed in these two concentration regions as well as in the region 18.4-27.5 mol % DHE, when the double bond is located at C6-C7 or C7-C8. These data suggest that sterol superlattice can occur in liquid-crystalline state of unsaturated phospholipid bilayers so long as the cis double bonds are not located between C9 and the carboxyl carbon. It appears that appropriate van der Waals contacts between the steroid ring and the C2-C9 segment of the phospholipid acyl chain are required for sterol superlattice formation.

The focus of the current study was a G protein‑coupled estrogen receptor (GPER)/microRNA (miR)‑148a/human leukocyte antigen‑G (HLA‑G) signaling pathway in ovarian endometriosis. Reverse transcription‑quantitative polymerase chain reaction was performed to analyze the changes in miR‑148a expression. A MTT assay, flow cytometry and caspase‑3/9 activity assays were performed to analyze cell proliferation, apoptosis and caspase‑3/9 activity levels, respectively. Protein expression was measured using western blot analysis. In tissue samples from healthy controls, and patients with endometriosis and endometriosis‑associated ovarian cancer, the expression of miR‑148a was lower in in endometriosis and EAOC samples compared with healthy controls. Overexpression of miR‑148a using miR mimics significantly decreased proliferation, promoted apoptosis, increased the Bcl‑2 associated X apoptosis regulator (Bax)/Bcl‑2 apoptosis regulator (Bcl‑2) ratio and caspase3/9 activity, and suppressed HLA‑G protein expression in Hs 832(C).T cells. miR‑148a downregulation using miR inhibitor significantly increased cell viability, inhibited apoptosis, and reduced the Bax/Bcl‑2 ratio and caspase3/9 activity, and induced HLA‑G protein expression in Hs 832(C).T cells. The GPER inhibitor, G15, suppressed GPER protein expression, upregulated miR‑148a expression, decreased cell proliferation, promoted apoptosis, increased the Bax/Bcl‑2 ratio and caspase3 activity, and suppressed HLA‑G protein expression in Hs 832(C).T cells. The findings indicate that GPER/miR‑148a/HLA‑G signaling pathway may mediates the development of ovarian endometriosis and may become a potential therapeutic target for the treatment of endometriosis.

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