Absence Of Sterol Research Articles

A C-Terminally Truncated Variant of Neurospora crassa VDAC Assembles Into a Partially Functional Form in the Mitochondrial Outer Membrane and Forms Multimers in vitro.

The voltage-dependent anion-selective channel (VDAC) is a porin in the mitochondrial outer membrane (MOM). Unlike bacterial porins, several mitochondrial β-barrels comprise an odd number of β-strands, as is the case for the 19-β-stranded VDAC. Previously, a variant of a VDAC from Neurospora crassa, VDAC-ΔC, lacking the predicted 19th β-strand, was found to form gated, anion-selective channels in artificial membranes. In vivo, the two C-terminal β-strands (β18 and β19) in VDAC form a β-hairpin necessary for import from the cytoplasm into mitochondria and the β-signal required for assembly in the mitochondrial outer membrane resides in β19. The current study demonstrated that the putative 18-stranded β-barrel formed by VDAC-ΔC can be imported and assembled in the MOM in vivo and can also partially rescue the phenotype associated with the deletion of VDAC from a strain of N. crassa. Furthermore, when expressed and purified from Escherichia coli, VDAC-ΔC can be folded into a β-strand-rich form in decyl-maltoside. Size exclusion chromatography (SEC) alone or combined with multi-angle light scattering (SEC-MALS) and analytical ultracentrifugation revealed that, unlike full-length VDACs, VDAC-ΔC can self-organize into dimers and higher order oligomers in the absence of sterol.

Influence of amphotericin B on the thermodynamic properties and surface morphology of saturated phospholipid monolayer with different polar head at the air-water interface

Amphotericin B is not only a widely used antifungal drug, but also a potential novel immunotherapy anticancer drug. But it has great side effects on human body due to its pore-forming activity on cell’s membrane. Some researchers have found that AmB can form pores on the phospholipid monolayer in the absence of sterol. However, the effect of different polar heads of phospholipids on the pore-forming activity of AmB on the membrane is rarely reported. In this work, the influence of amphotericin B on the thermodynamic properties and surface morphology of saturated phospholipid monolayer with different polar head at the air-water interface have been studied. The results suggested that the intermolecular force between AmB and DPPC or DPPE molecules was attractive but the force between AmB and DPPG or DPPS molecules was repulsive. No matter what kind of phospholipid’s polar head, AmB can enhance the disorder of the AmB/phospholipid mixed monolayer. But at 30 mN/m, this effect of AmB from strong to weak was in the presence of PS head, PG head, PC head and PE head. And the drug can induce the phase state of the phospholipid monolayer to change from LC phase to LC-LE phase at 30 mN/m. Differently, the shape and distribution of LC phase domains in the mixed monolayer film with non-charged head group were different from that with negatively charged head group, which may be due to the intermolecular force between AmB and different phospholipid. The results are helpful to understand the influence of AmB on the domains without sterols on cell membrane, to lay the foundation for the interaction between AmB and phospholipid-sterol binary model membranes in the further study and to provide a basis for understanding the interaction between AmB and liposome materials.

Amphotericin B/Sterol Co-loaded PEG-Phospholipid Micelles: Effects of Sterols on Aggregation State and Hemolytic Activity of Amphotericin B

To elucidate the effect of sterols on the aggregation of amphotericin B (AmB) in PEG-phospholipid micelles and its consequences on the hemolytic activity of AmB. AmB-incorporated PEG-phospholipid micelles co-loaded with ergosterol, cholesterol, or 7-dehydrocholesterol were prepared at 4:1:1 and 20:5:1 ratios of polymer-to-sterol-to-AmB. The aggregation state of AmB was elucidated by UV-vis spectroscopy. AmB/sterol co-loaded PEG-phospholipid micelles were incubated with red blood cells and the hemolytic activity of AmB assessed by measurement of free hemoglobin. AmB in PEG-phospholipid micelles stayed mostly in a deaggregated state in the absence of sterol or with cholesterol, but aggregated in the presence of ergosterol or 7-dehydrocholesterol. The fraction of aggregated AmB in PEG-phospholipid micelles was lower at the 20:5:1 ratio. In an aggregated state or in the absence of sterol, AmB caused rapid and complete hemolysis. In contrast, deaggregated AmB co-loaded with cholesterol caused slower and incomplete hemolysis, especially at a 20:5:1 ratio. The aggregation state of AmB in PEG-phospholipid micelles was sterol dependant. AmB/cholesterol co-loaded PEG-phospholipid micelles caused low in vitro hemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region.

Molecular Convergence of Bacterial and Eukaryotic Surface Order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Sterol effect on interaction between amphidinol 3 and liposomal membrane as evidenced by surface plasmon resonance

The affinity of amphidinol 3 (AM3) to phospholipid membranes in the presence and absence of sterol was examined by surface plasmon resonance (SPR) experiments. The results showed that AM3 has 1000 and 5300 times higher affinity for cholesterol- and ergosterol-containing liposomes, respectively, than those without sterol. The two-state reaction model well reproduced the sensor grams, which indicated that the interaction is composed of two steps, which correspond to binding to the membrane and internalization to form stable complexes.

Effects of ergosterol on the structure and activity ofNeurosporamitochondrial porin in liposomes

Mitochondrial porins (also known as voltage-dependent anion-selective channels (VDACs)) regulate and contribute to cellular metabolism. These proteins copurify with sterols, and some purified forms of the protein require sterol for insertion into planar artificial membranes. Recently, interactions between detergent-solubilized mitochondrial porins and sterols have been detected by NMR and spectroscopic methods, but the effects of sterols on pore function remained to be assessed. Therefore, in this work, a freeze-thaw technique was used to introduce recombinant Neurospora porin into liposomes containing, or lacking, the native fungal sterol ergosterol. In both types of liposomes, insertion of the protein converts it to a protease-resistant state and low levels of dimeric and trimeric forms are observed. There are only minor differences between the secondary structural components of the protein in the presence or absence of sterol. Ergosterol in proteoliposomes alters their osmotic responses to sucrose, possibly due to increased membrane rigidity or interactions with the protein that were not revealed by the methods used in this study. The presence of ergosterol is associated with an increased change in conformation and loss of function of liposome-embedded porin at high temperature. Taken with other evidence for direct interactions of sterols with porins, these results support a link between these two molecules in mitochondrial membrane activity.

Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts)

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 ∼ C6:0 ∼ C8:0 < no ceramide < C12:0 < C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 ∼ C6:0 < C2:0 < C12:0 ∼ no ceramide < C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.

Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus.

Cells expressing the hemagglutinin (HA) of influenza virus were fused to planar phospholipid bilayer membranes to evaluate the effects of sterols and sphingolipids in the target bilayer membranes on properties of fusion pores. Typically, in the absence of sterol, flickering pores are observed, followed by a successful pore (i.e., a pore that fully opens). The incorporation of cholesterol into the lipid bilayer had a marked effect: it greatly decreased the number of flickers, and the first pore formed was usually successful. Similar effects were produced by the sterols epicholesterol and 5beta-cholestanol. In contrast, the sterols cholesteryl acetate, coprostanol, and stanolone did not affect pore flickering, and a successful pore was observed to follow the typical number of flickers. 5alpha-cholestanol gave intermediate results. From these results, it follows that the 3-OH of cholesterol is essential to reduce flickering, but it does not matter if the 3-OH is in an alpha or beta configuration. The double bond is also not critical for the actions of cholesterol nor is the fact that it is a flat molecule. The sphingolipids sphingomyelin, lactosyl cerebroside, and glucosyl cerebroside tended to inhibit full pore enlargement, prolonging the stage of pore flickering. If a sphingolipid and a sterol that strongly interact were both included in the planar membrane, the pattern of flickering was the same as if neither had been included in the bilayer. However, if a sphingolipid and sterol that do not interact with each other were included in the bilayer, the reduced flickering characteristic of the sterol was observed.

Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length.

Fourier-transform infrared-spectroscopic and fluorescence measurements have been combined to examine the effect of cholesterol on the intermixing of short-chain dilauroyl phosphatidylcholine (DLPC) and its bromo-substituted derivative (12BrPC) with longer-chain (C16- or C18-) phosphatidylcholines (PCs) in hydrated lipid bilayers. Infrared spectroscopy of mixtures combining protonated DLPC or 12BrPC with chain-perdeuterated dipalmitoyl PC reveals that cholesterol at lower concentrations in the bilayer modifies the resolved thermal melting profiles for both phospholipid components and, at high bilayer concentrations, produces a convergence of the thermal transitions for the two PC species. Fluorescence-quenching measurements using a short-chain fluorescent PC (1-dodecanoyl-2-[8-[N-indolyl]octanoyl] PC) in ternary mixtures combining 12BrPC, dipalmitoyl or distearoyl PC, and cholesterol confirm that very high cholesterol levels (50 mol %) abolish the lateral segregation of the PC components at 25 degrees C, a temperature where the phospholipids extensively phase-separate in the absence of sterol. By contrast, under these same conditions cholesterol at lower concentrations in the bilayer is found to enhance the tendency of the PC components to exhibit lateral segregation. We show that these seemingly contradictory effects of cholesterol can be readily explained in the light of a ternary phase diagram that is fully consistent with out current understanding of the nature of cholesterol-phospholipid interactions in binary mixtures.

Osmotic stress sensitizes sterol-free phospholipid bilayers to the action of Amphotericin B

We have tested the ability of Amphotericin B to form ion channels/defects in osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H + exchange. We found that sterol-free LUV exhibit greatly increased sensitivity to AmB channel formation in the soluble oligomer state (> 0.5 μM) under modestly hypoosmotic conditions (< 100 ΔmosM). These vesicles are completely insensitive under isoosmotic conditions. The related antibiotics, Amphotericin B methyl ester and Nystatin showed almost no activity under hypoosmotic conditions in the absence of sterol. This difference may be attributable to differences in solution oligomeric states. Experiments with KCl and CaCl 2, internal buffers demonstrate that these sterol-free AmB membrane disruptions are highly selective for monovalent cations (K +) over anions (Cl −), ruling out massive lysis or unselective membrane defects caused by osmotic pressure. Thus, AmB seems to be acting as a ‘molecular harpoon’, an express ion coined to describe substances which can selectively target osmotically stressed, strained or highly curved membranes. These results may provide a rationale for AmB's reported anti-HIV activity and reported activity against sterol-free small unilamellar vesicles (highly curved membranes) as well as the reduced activity of liposomal drug delivery systems toward cholesterol-containing and sterol-free membranes (fewer soluble oligomers).

Cholesterol modulation of lipid intermixing in phospholipid and glycosphingolipid mixtures. Evaluation using fluorescent lipid probes and brominated lipid quenchers

Carbazole- and indole-labeled phospholipids have been used to monitor the homo- or heterogeneity of lipid mixing in several types of lipid bilayers combining a brominated and a nonbrominated lipid with varying amounts of cholesterol. Experimental quenching curves (relating the normalized probe fluorescence intensity to the mole fraction of brominated lipid) show a characteristic smooth, monophasic form for homogeneous liquid-crystalline lipid mixtures. However, for mixtures exhibiting lipid lateral segregation, such curves show marked perturbations in form over the region of composition where segregation occurs. Using this approach, it is found that high mole fractions of cholesterol (40-50 mol %) promote the formation of apparently homogeneous solutions in mixtures of disaturated and monounsaturated phosphatidylcholines (PCs) that exhibit extensive thermotropic phase separations in the absence of sterol. At only slightly lower levels of cholesterol, however, these systems exhibit inhomogeneous lipid mixing over a wide range of relative proportions of the two PC components. Mixtures of cerebroside and monounsaturated PCs, even at high bilayer cholesterol contents, exhibit significant inhomogeneity in lipid mixing over a wide range of cerebroside/PC ratios. Phase-separating PC/PC and PC/cerebroside mixtures can readily form long-lived metastable solutions when the level of the higher-melting component in the liquid-crystalline phase exceeds its equilibrium solubility by as much as 20-30 mol %; this tendency is significantly increased by cholesterol. Cholesterol shows no significant ability to enhance lipid intermixing in a third type of phase-separating lipid system, combining a monounsaturated PC with a monounsaturated phosphatidic acid--calcium complex. Experiments using cleavable phospholipid conjugates, linking a fluorescent lipid to a brominated lipid, suggest that each fluorescent molecule probes a local lipid domain comprising approximately less than 40-50 nearby acyl chains.

Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement

The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg-yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios ( R) were necessary ( R > 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R > 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R > 0.1 for EPC vesicles, R > 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations ( R < 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel.

Lecithin-dependent Phytosterol Utilization by Larvae of Culex pipiens (Diptera: Culicidae)

Without sterol in synthetic dietary media Culex pipiens L. could not develop beyond the second instar. With cholesterol as the only dietary lipid, good development to the adult stage occurred, whereas with ergosterol or stigmasterol, development was no better than in the absence of sterol, and with all other phytosterols tested development was variously restricted. When the basic diet incorporated a lipid supplement containing the arachidonic acid needed for newly emerged adults to fly and survive, larval/pupal development and adult viability with ergosterol, stigmasterol, and most phytosterols were as good as with cholesterol. Besides arachidonic acid, the lipid supplement contained an antioxidant, ascorbyl palmitate, and synthetic dipalmitoyl lecithin as a dispersing agent; but with lecithin alone supplementing the basic diet, good development to the adult stage was facilitated with ergosterol, stigmasterol, and other phytosterols, although adults lacking arachidonic acid were not viable. On the basis of diets supplemented with lecithin, we concluded that ergosterol, stigmasterol, sitosterol, 24-methylenecholesterol, fucosterol, desmosterol, and perhaps 7-dehydrocholesterol could support growth and development as well, or almost so, as cholesterol. With cholestanol, development to the fourth instar was good, but nearly all individuals failed to metamorphose. With lathosterol or 22- trans -cholestadienol, development was markedly inferior compared with cholesterol and the well utilized phytosterols, and survival through metamorphosis was severely reduced. These results are discussed in relation to current views on the metabolism of sterols by phytophagous/omnivorous insects.

Isolation and characterization of filipin-resistant LM cell variants not auxotrophic for sterol.

A series of LM cell variants resistant to filipin, but not auxotrophic for sterol, was isolated by plating mutagenized, filipin-treated cells on soft agar medium containing no sterol. Cloned variants were assayed for growth in the presence and absence of sterol or unsaturated fatty acid. Filipin-resistant clones whose growth rate was unaffected by the addition of sterol to the medium were further analyzed. Variants were cultured in minimal medium, and plasma membranes prepared from these cultures were analyzed for sterol content, phospholipid head group composition, and unsaturated fatty acyl content. All variants examined showed a decrease in membrane sterol in conjunction with an increase in unsaturated fatty acyl chains in the membrane phospholipids. In addition, several variants exhibited alterations in phospholipid head group composition, including changes in the phosphatidylcholine/phosphatidylethanolamine ratio, or a decrease in sphingomyelin content. These observations imply that several different metabolic lesions can give rise to decreased plasma membrane sterol content (and hence to filipin resistance). The range of phospholipid alterations observed in these sterol prototrophs emphasizes the complex interrelationship between membrane sterol and phospholipid structure.

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction.

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.

Selective membrane toxicity of the polyene antibiotics: studies on lecithin membrane models (liposomes).

In the absence of sterol, amphotericin B at 5 x 10(-6) M caused maximum marker release from the saturated dipalmitoyl lecithin liposomes, minimum release from the unsaturated dioleoyl lecithin liposomes, and an in-between response from egg lecithin liposomes. Nystatin at 2.5 to 4.0 x 10(-5) M induced appreciable marker release from all three types of sterol-free liposomes. The amphotericin B- and nystatin-induced permeability changes in dipalmitoyl lecithin liposomes were drastically suppressed by the incorporation of cholesterol or stigmasterol (with identical Delta5 sterol nuclei), but were unaffected by the incorporation of ergosterol or 5,7-cholestadien-3beta-ol (with identical Delta5,7 sterol nuclei). The nystatin sensitivity of dioleoyl lecithin liposomes remained low after the incorporation of cholesterol or stigmasterol, but was greatly enhanced by the incorporation of ergosterol or 5,7-cholestadien-3beta-ol. Digitonin, a compound known to interact specifically with membrane sterol, induced marker release from liposomes in proportion to the amount of either cholesterol or ergosterol incorporated; epicholesterol did not sensitize to digitonin. These results lead to the following conclusions: (i) polyene-induced permeability alteration in model membrane systems is effected by the composition of membrane phospholipid fatty acyl chains; (ii) the distribution of double bonds in the sterol nucleus is related to the selective toxicity of the polyenes toward natural sterol-containing membranes; and (iii) polyenes differ in membrane selectivity.

Theeffect of cholesterol and polyene antibiotics on the permeability of the protoplasmic membrane of Pythium PRL 2142.

Intracellular constituents of Pythium PRL 2142 leak from the mycelium when it is placed in distilled water. The effect of cholesterol association with the protoplasmic membrane of this fungus is to decrease leakage of nucleotide, nitrogen, and protein constituents of the mycelium but leakage of carbohydrate is significantly increased.The polyene antibiotics, amphotericin B, nystatin, candidin, and candicidin, whilst ineffective on mycelium grown in the absence of sterol, increase leakage of these intracellular components from sterol-grown mycelium, particularly the carbohydrate and protein components, but do not cause lysis of the organism.

Induction de l'oogénèse par les antibiotiques polyéniques et interaction avec le cholestérol chez un Pythium sp. PRL 2142

Crystals of the polyene antibiotics amphotericin B, candicidin, candidin, and nystatin placed on the hyphae of Pythium sp. PRL 2142 induce the formation of many oogonia. Candidin incorporated into agar plates, at a concentration of 1000 μg/ml, promotes abundant oospore production which can be completely inhibited by 5 μg/ml of cholesterol. Conversely, cholesterol by itself, at 4 g per kilogram dry mycelium stimulates oogonial formation which can be inhibited by the antibiotics listed above. Filipin induces the formation of only one or two oogonia and causes lysis of the mycelium growing in the presence of cholesterol. Amphotericin B increases slightly the mycelial yield of cholesterol-grown mycelium. High concentrations of filipin and amphotericin B increase the dry weight of mycelium growing in the absence of sterol.

Sterol requirements of a ciliate Tetrahymena corlissi Th-X : I. A nutritional analysis of the sterol requirements of T. Corlissi Th-X

1. 1. Tetrahymena corlissi Th-X, a tetrahymenid ciliate, has been cultivated under axenic conditions in a medium of known chemical composition (within limits of contemporary standards of chemical purity) and required a sterol for best growth. 2. 2. It responded to many plant and animal 3ß-OH sterols and their esters, with saturated and unsaturated ring systems and variously-substituted side chains. 3. 3. The activity of potential cholesterol precursors was correlated approximately with their presumed proximity to cholesterol; however, tritiated lophenol was not converted to cholesterol or sterols of comparable polarity but was directly incorporated. 4. 4. dl-α-Glycerophosphate, oleic acid and other oleate-containing compounds, and some synthetic phosphatides, spared the sterol requirement and permitted submaximal growth in the absence of sterol.