Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Polyene--sterol interaction and selective toxicity

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.     

Susceptibilities of phospholipid vesicles containing different sterols to amphotericin B-loaded lysophosphatidylcholine micelles

To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.     

A selective cholesterol-dependent induction of H+/OH- currents in phospholipid vesicles by amphotericin B

The effect of amphotericin B on the proton/hydroxide permeability of small unilamellar vesicles has been investigated by using potential-dependent paramagnetic probes. Amphotericin B at 1-10 molecules/vesicle causes a modest 4-8-fold increase in the background H+/OH- permeability of egg phosphatidylcholine (egg PC) vesicles. However, in the presence of cholesterol, amphotericin B promotes a dramatic increase in the H+/OH- permeability of more than 2 orders of magnitude. Surprisingly, this is not observed in vesicle membranes containing ergosterol. In membranes composed of 5-15 mol% ergosterol, amphotericin B is even less effective at promoting H+/OH- currents than in pure egg PC vesicles. The K+ current promoted by amphotericin B in vesicles formed from egg PC and from egg PC plus cholesterol or ergosterol was measured. No significant sterol dependence was found for the K+ current. These results strongly suggest that different mechanisms, or amphotericin B/sterol complexes, are responsible for the induction of H+/OH- and K+ currents. These results have important implications for understanding the therapeutic and toxic effects of amphotericin B.     

Investigation of the role of micellar phospholipid in the preferential uptake of cholesterol over sitosterol by dispersed rat jejunal villus cells

The uptake of radioactive cholesterol and sitosterol by rat jejunal villus cells was examined using mixed micellar solutions containing sodium taurocholate, equimolar mixtures of the two sterols, and a variety of phospholipid types. The addition of phospholipid to the incubation solutions reduced the cellular absorption of both sterols and gave rise to uptake kinetics that were linear with time. In the presence of egg yolk phospholipid, uptake of the sterols by villus cells occurred with a modest preference for cholesterol over sitosterol. The ratio of accumulated cholesterol/sitosterol increased from 1.0 initially to 1.23 +/- 0.04 (n = 18) after a 30-min incubation at 37 degrees C. The selectivity displayed in the villus cells increased significantly as egg phosphatidylethanolamine was added to the egg phosphatidylcholine (PC) preparation in micellar solution. It was markedly decreased when dipalmitoyl PC or the primarily saturated egg yolk sphingomyelin were incorporated into the micelles. In every case examined, phospholipid was taken up by the cells concurrently with the sterols. The selectivity between cholesterol and sitosterol was maintained when the donor species were multilamellar vesicles composed of egg PC and the sterols, but not when the donor particles were albumin-stabilized sterol dispersions or taurocholate solutions in the absence of PC. The results show that the selective absorption of cholesterol over the plant sterol occurs only in the presence of unsaturated phospholipid. The phospholipid may act by influencing the permeability of the cellular membranes to the two sterols or the rate of sterol desorption from the phospholipid-containing micellar or liposomal carriers.(ABSTRACT TRUNCATED AT 250 WORDS)     

How interaction of perfringolysin O with membranes is controlled by sterol structure, lipid structure, and physiological low pH: insights into the origin of perfringolysin O-lipid raft interaction

Perfringolysin O (PFO) is a sterol-dependent, pore-forming cytolysin. To understand the molecular basis of PFO membrane interaction, we studied its dependence upon sterol and lipid structure and aqueous environment. PFO interacted with diverse sterols, although binding was affected by double bond location in the sterol rings, sterol side chain structure, and sterol polar group structure. Importantly, a sterol structure promoting formation of ordered membrane domains (lipid rafts) was not critical for interaction. PFO membrane interaction was also affected by phospholipid acyl chain structure, being inversely related to tight acyl chain packing with cholesterol. Experiments using the pre-pore Y181A mutant demonstrated that sterol binding strength and specificity was not affected by whether PFO forms a transmembrane beta-barrel. Combined, these observations are consistent with a model in which the strength and specificity of sterol interaction arises from both sterol interactions with domain 4 and sterol chemical activity within membranes. The lipid raft-binding portions of sterol bound to PFO may remain largely exposed to the lipid bilayer. These results place important constraints upon the origin of PFO raft affinity. Additional experiments demonstrated that the structure of membrane-inserted PFO at low and neutral pH was similar as judged by the effect of phospholipid and sterol structure upon PFO properties and membrane interaction. However, low pH enhanced PFO membrane binding, oligomerization, and pore formation. In lipid vesicles mimicking the exofacial (outer) membrane leaflet, PFO-membrane binding was maximal at pH 5.5-6. This is consistent with the hypothesis that PFO function involves acidic vacuoles.     

Steric effects at C-20 and C-24 on the metabolism of sterols by Tetrahymena pyriformis

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.     

Phospholipid studies of marine organisms: 26. Interactions of some marine sterols with 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) in model membranes.

The thermotropic behavior of multilamellar vesicles (MLV) composed of different mole fractions of various marine sterols and 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) was examined by differential scanning calorimetry (DSC), and was compared to pure SOPC as well as their mixtures with cholesterol. The marine sterols investigated were capable of interacting with the phospholipid bilayers. Upon addition of marine sterols, the apparent transition temperature (Tm) of SOPC decreased significantly. Desmosterol (cholesta-5,24-dien-3 beta-ol) had the least interaction with SOPC, as reflected by the larger delta H values of its mixtures with the phospholipid. Fucosterol (24-ethylcholesta-5,24(28)-dien-3 beta-ol) showed a non-linear trend as the mole percent of the sterol increased. Mixtures of sutinasterol (24R-24-ethyl-26,26-dimethylcholesta-7,25(27)-dien-3 beta-ol) with SOPC had similar enthalpy values to cholesterol. The shape of the SOPC/marine sterol endotherm and their delta H values were not identical when liposomes prepared by dialysis were compared to MLV.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranes and small unilamellar vesicles

This study examined the kinetics of sterol desorption from monolayer and small unilamellar vesicle membranes to 2-hydroxypropyl-beta-cyclodextrin. The sterols used include cholesterol, dehydroergosterol (ergosta-5,7,9,(11),22-tetraen-3beta-ol) and cholestatrienol (cholesta-5,7,9,(11)-trien-3beta-ol). Desorption rates of dehydroergosterol and cholestatrienol from pure sterol monolayers were faster (3.3-4.6-fold) than the rate measured for cholesterol. In mixed monolayers (sterol: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 30:70 mol%), both dehydroergosterol and cholestatrienol desorbed faster than cholesterol. clearly indicating a difference in interfacial behavior of these sterols. In vesicle membranes desorption of dehydroergosterol was slower than desorption of cholestatrienol, and both rates were markedly affected by the phospholipid composition. Desorption of sterols was slower from sphingomyelin as compared to phosphatidylcholine vesicles. Desorption of fluorescent sterols was also faster from vesicles prepared by ethanol-injection as compared to extruded vesicles. The results of this study suggest that dehydroergosterol and cholestatrienol differ from cholesterol in their membrane behavior, therefore care should be exercised when experimental data derived with these probes are interpreted.     

The functional importance of structural features of ergosterol in yeast.

As an approach to the study of the relationship between the structure of sterols and their capacity to function in the lipid leaflet of membranes, various sterols were examined for their ability to support the growth of anaerobic Saccharomyces cerevisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24beta-methyl-delta22-grouping greater than 24beta-methyl group greater than delta5,7-diene system = delta5-bond approximately or equal to no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic processes removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C-22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol probably acts functionally in the form of its preferred conformer in which C-22 is to the right ("right-handed") in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.     

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibilities of phospholipid membranes containing cholesterol or ergosterol to gramicidin and its derivative incorporated in lysophospholipid micelles

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.     

Steinernema feltiae (= Neoaplectana carpocapsae): effect of sterols and hypolipidemic agents on development

The structure and concentration of sterol in a lipid-defined artificial medium affected the development of the entomogenous nematode, Steinernema feltiae (= Neoaplectana carpocapsae). The nematode grew normally in vitro when the medium was supplemented with delta 5-desalkylsterol (cholesterol) or delta 5-desalkylsteryl ester (cholesterol oleate). The minimum amount of cholesterol in the medium that was necessary to support the development of S. feltiae to the climax population (i.e., dauer stage) was 0.0025%. The nematode also completed its life cycle normally when delta 0- or delta 7-desalkylsterols (cholestanol and lathosterol) were substituted for cholesterol. In contrast, development was inhibited when the medium contained delta 5,7-desalkylsterol (7-dehydrocholesterol); however, the nematode population reached the climax stage, in medium containing this sterol, when cholesterol was also present. S. feltiae was able to utilize delta 5- and delta 0-24 alpha-ethylsterols (sitosterol and sitostanol) as dietary sterols; however, when a delta 22-bond was introduced into the side chain (stigmasterol) the rate of development of the nematode slowed significantly. The growth of the nematode was also retarded when the medium contained delta 5,7,22-24 beta-methylsterol (ergosterol). The nematode population reached the climax stage in medium containing delta 8,24-4,14 alpha-trimethylsterol (lanosterol) only when cholesterol was also present. When S. feltiae was exposed to certain hypolipidemic agents, which are known to lower the level of lipids in human plasma (clofibrate, cholestyramine resin, niacin, and D-thyroxine), all but D-thyroxine affected the growth and development of the nematode in vivo (in Heliothis zea) and/or in vitro. Therefore further studies are warranted to determine how these drugs affect the lipid biochemistry of this nematode.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.     

Substrate specificity of the biotransformation enzymes in a Nocardia erythropolis culture

Substrate specificity of biotransformation enzymes of culture Nocardia erythropolis was studied. Products of transformation of cholesterol and three sterols of microbial origin: ergosterol, ergosta-5,7-dien-3 beta-ol and ergosta-7,22-dien-3 beta-ol was identified with a help of thin-layer chromatography, UV spectrophotometry and mass-spectrometry. It was established, that delta 22-bond in the side chains of sterols and delta 7-bond slows and delta 5-bond makes impossible cleavage of side chains of sterols.