Membrane Sterol Content Research Articles

Structural Role of Plasma Membrane Sterols in Osmotic Stress Tolerance of Yeast Saccharomyces cerevisiae.

Yeast S. cerevisiae has been shown to suppress a sterol biosynthesis as a response to hyperosmotic stress. In the case of sodium stress, the failure to suppress biosynthesis leads to an increase in cytosolic sodium. The major yeast sterol, ergosterol, is known to regulate functioning of plasma membrane proteins. Therefore, it has been suggested that the suppression of its biosynthesis is needed to adjust the activity of the plasma membrane sodium pumps and channels. However, as the sterol concentration is in the range of thirty to forty percent of total plasma membrane lipids, it is believed that its primary biological role is not regulatory but structural. Here we studied how lowering the sterol content affects the response of a lipid bilayer to an osmotic stress. In accordance with previous observations, we found that a decrease of the sterol fraction increases a water permeability of the liposomal membranes. Yet, we also found that sterol-free giant unilamellar vesicles reduced their volume during transient application of the hyperosmotic stress to a greater extent than the sterol-rich ones. Furthermore, our data suggest that lowering the sterol content in yeast cells allows the shrinkage to prevent the osmotic pressure-induced plasma membrane rupture. We also found that mutant yeast cells with the elevated level of sterol accumulated propidium iodide when exposed to mild hyperosmotic conditions followed by hypoosmotic stress. It is likely that the decrease in a plasma membrane sterol content stimulates a drop in cell volume under hyperosmotic stress, which is beneficial in the case of a subsequent hypo-osmotic one.

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.

Ergosterol Turnover in Yeast: An Interplay between Biosynthesis and Transport.

Sterols are important components of biological membranes that determine the physicochemical properties of lipid bilayer and regulate the functioning of membrane proteins. Being insoluble in water, sterols cannot diffuse between the membrane compartments separated by an aqueous phase. For this reason, distribution of sterols across cellular membranes is rather uneven. Membrane-to-membrane transport of sterols occurs mainly in a non-vesicular fashion and is provided by Lam and Osh proteins. In this review, we discuss the consequences of impairments in sterol biosynthesis and transport mostly relying on the studies performed on the model organism Saccharomyces cerevisiae. Despite the fact that molecular mechanisms underlying the functioning of Lam and Osh proteins are well established, the biological roles of these proteins are still unclear, because deletions of corresponding genes do not affect yeast phenotype. At the same time, disruptions in the biosynthesis of ergosterol, the major sterol of S. cerevisiae, lead to either cell death or reduced stress resistance. However, under certain conditions (e.g., mild salt or thermal stresses), a decrease in the ergosterol levels causes an increase in cell resistance. This suggests that the cells possess a mechanism facilitating rapid adjustment of the plasma membrane sterol content. We argue that the biological role of Lam proteins is, in particular, fast optimization of sterol composition of cell membranes.

Yeasts from Scarlet ibises (Eudocimus ruber): A focus on monitoring the antifungal susceptibility of Candida famata and closely related species.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species.

Synthesis and Live-Cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport.

Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.

Observation on the ultrastructure morphology of HeLa cells treated with ethanol: Statistical analysis

ABSTRACTIt is estimated that 5.9% of all human deaths are attributable to alcohol consumption and that the harmful use of ethanol ranks among the top five risk factors for causing disease, disability, and death worldwide. Ethanol is known to disrupt phospholipid packing and promote membrane hemifusion at lipid bilayers. With the exception of mitochondria involved in hormone synthesis, the sterol content of mitochondrial membranes is low. As membranes that are low in cholesterol have increased membrane fluidity and are the most easily disordered by ethanol, we hypothesize that mitochondria are sensitive targets for ethanol damage. HeLa cells were exposed to 50 mM ethanol and the direct effects of ethanol on cellular ultrastructure were examined utilizing transmission electron microscopy. Our ultramicroscopic analysis revealed that cells exposed to ethanol harbor fewer incidence of apoptotic morphology; however, significant alterations to mitochondria and to nuclei occurred. We observed statistical increases in the amount of irregular cells and cells with multiple nuclei, nuclei harboring indentations, and nuclei with multiple nucleolus-like bodies. Indeed, our analysis revealed that mitochondrial damage is the most extensive type of cellular damage. Rupturing of cristae was the most prominent damage followed by mitochondrial swelling. Ethanol exposure also resulted in increased amounts of mitochondrial rupturing, organelles with linked membranes, and mitochondria localizing to indentations of nuclear membranes. We theorize that these alterations could contribute to cellular defects in oxidative phosphorylation and, by extension, the inability to generate regular levels of cellular adenosine triphosphate.

Two types of syringomycin E channels in sphingomyelin-containing bilayers.

The influence of dipole modifiers on the characteristics of single syringomycin E (SRE) channels in bilayers comprising DOPS, DOPE, sphingolipids (sphingomyelin, N-stearoyl-phytosphingosine or N-stearoyl-sphinganine) and sterols (cholesterol or ergosterol) was studied. The effects of dipole modifiers on SRE channel amplitudes were dependent upon the sphingolipid type and were not affected by the membrane sterol content. A decrease in the dipole potential of phytosphingosine- and sphinganine-containing bilayers, which was induced by the adsorption of phloretin, led to a reduction in conductance; however, an increase in this potential, which occurred upon the addition of RH 421, led to an enhancement in the conductance of SRE channels. Two channel populations, one of which is sensitive while the other is insensitive to modifiers, were found in sphingomyelin-containing bilayers. This indicates that SRE channels are distributed in lipid domains with different dipole potentials.

Membrane Permeability Induced by Stereo and Retro Analogs of Histatin 5

Candida species combine to be the major cause of fungal infections in humans. These infections are becoming increasingly difficult to treat due to resistance to current common drug therapies or due to the toxicity of other therapeutic compounds. Antimicrobial peptides, short peptides of less than 100 amino acid residues, are known to kill bacterial and fungal pathogens, with the potential to be a more reliable means of treatment for Candida infections than the drugs currently available. Humans naturally produce the 24 amino acid cationic histatin 5 peptide. Variants of a 16 amino acid peptide derived from histatin 5 were chemically synthesized to delineate the structural determinants of their antifungal activity. In-vitro acridine orange leakage assays were used to evaluate the activity of histatin 5 derivatives with various synthetic membrane bilayers (liposomes) composed of differing lipid and sterol compositions. The variants of histatin 5 were synthesized with alternative stereochemistry and polarity, and then evaluated with liposomes comprised of either Soy PhosphatidylCholine (Soy PC), Soy PC + cholesterol or Soy PC + ergosterol. The results indicate that neither the D or L amino acid stereochemistry nor the N- to C- terminal polarity of the amino acid chain is influential in the induced membrane permeability of these peptides. In addition, these in-vitro studies imply the existence of a factor or a combination of factors aside from strictly membrane sterol content that determines the specificity of these antimicrobial peptides for fungal cells in-vivo. Importantly, the results of this study imply that the relative spatial position of the amino acids in histatin 5 is critical for the antifungal activity.

Triclosan Antagonizes Fluconazole Activity against Candida albicans

Triclosan is a broad-spectrum antimicrobial compound commonly used in oral hygiene products. Investigation of its activity against Candida albicans showed that triclosan was fungicidal at concentrations of 16 mg/L. However, at subinhibitory concentrations (0.5-2 mg/L), triclosan antagonized the activity of fluconazole. Although triclosan induced CDR1 expression in C. albicans, antagonism was still observed in cdr1Δ and cdr2Δ strains. Triclosan did not affect fluconazole uptake or alter total membrane sterol content, but did induce the expression of FAS1 and FAS2, indicating that its mode of action may involve inhibition of fatty acid synthesis, as it does in prokaryotes. However, FAS2 mutants did not exhibit increased susceptibility to triclosan, and overexpression of both FAS1 and FAS2 alleles did not alter triclosan susceptibility. Unexpectedly, the antagonistic effect was specific for C. albicans under hypha-inducing conditions and was absent in the non-filamentous efg1Δ strain. This antagonism may be due to the membranotropic activity of triclosan and the unique composition of hyphal membranes.

Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania

The present study was designed to elucidate the mechanism of tafenoquine uptake in Leishmania and its sterol dependence. Because tafenoquine is a fluorescent compound, spectrofluorimetric analysis allowed us to monitor its uptake by Leishmania promastigotes and intracellular amastigotes, and to evaluate the effect of temperature, energy and H+ gradient on drug entry. The influence of sterols on tafenoquine uptake in Leishmania parasites was determined in experiments using sterol-depleting agents such as methyl-β-cyclodextrin or cholesterol oxidase. Tafenoquine exhibited fast entry kinetics into Leishmania in an energy-independent, but pH- and temperature-dependent, non-saturable process. Furthermore, sterol depletion decreased tafenoquine uptake. These findings suggest that Leishmania takes up tafenoquine by a diffusion process and that decreases in membrane sterol content may induce a decrease in drug uptake.

Lysosomal Membrane Protein Composition, Acidic pH and Sterol Content are Regulated via a Light‐Dependent Pathway in Metazoan Cells

In metazoans, lysosomes are characterized by a unique tubular morphology, acidic pH, and specific membrane protein (LAMP) and lipid (cholesterol) composition as well as a soluble protein (hydrolases) composition. Here we show that perturbation to the eye-color gene, light, results in impaired lysosomal acidification, sterol accumulation, altered endosomal morphology as well as compromised lysosomal degradation. We find that Drosophila homologue of Vps41, Light, regulates the fusion of a specific subset of biosynthetic carriers containing characteristic endolysosomal membrane proteins, LAMP1, V0-ATPase and the cholesterol transport protein, NPC1, with the endolysosomal system, and is then required for the morphological progression of the multivesicular endosome. Inhibition of Light results in accumulation of biosynthetic transport intermediates that contain these membrane cargoes, whereas under similar conditions, endosomal delivery of soluble hydrolases, previously shown to be mediated by Dor, the Drosophila homologue of Vps18, is not affected. Unlike Dor, Light is recruited to endosomes in a PI3P-sensitive fashion wherein it facilitates fusion of these biosynthetic cargoes with the endosomes. Depletion of the mammalian counterpart of Light, hVps41, in a human cell line also inhibits delivery of hLAMP to endosomes, suggesting an evolutionarily conserved pathway in metazoa.

Sterol and Diacylglycerol Acyltransferase Deficiency Triggers Fatty Acid-mediated Cell Death

Deletion of the acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic beta-cell dysfunction, diabetes, and the metabolic syndrome.

Cold adaptation in the yeast Saccharomyces cerevisiae

Although the ability of the yeast Saccharomyces cerevisiae to adapt to environmental stresses has been widely studied, relatively little is know about adaptation to cold temperatures. Previous studies by Loertscher and co‐workers have shown that proteins required for endoplasmic reticulum‐associated degradation (ERAD), such as Ubc7, are also required for cold adaptation and may work by affecting membrane sterol composition. Using both solid and liquid growth media, a panel of deletion mutants lacking endoplasmic reticulum‐related proteins was tested for sensitivity to cold temperatures. Deletion mutant orm2Δ exhibited notable sensitivity to cold when grown on either solid or liquid medium as well as impaired growth at normal temperatures after being grown in cold temperatures for an extended period of time. Orm2 is an ER‐localized protein and may be involved in the cellular response to unfolded proteins. We hypothesize that Orm2 may have a role similar to Ubc7 in helping cells adapt to cold temperature through direct or indirect modification of membrane sterol content. Examining the membrane sterol content of orm2Δ mutants as compared to wild‐type will help determine the role of Orm2 in cold adaptation.

A membrane defect in the pathogenesis of the Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome (SLOS) is an often lethal birth defect resulting from mutations in the gene responsible for the synthesis of the enzyme 3beta-hydroxy-steroid-Delta7-reductase, which catalyzes the reduction of the double bond at carbon 7 on 7-dehydrocholesterol (7-DHC) to form unesterified cholesterol. We hypothesize that the deficiency in cholesterol biosynthesis and subsequent accumulation of 7-DHC in the cell membrane leads to defective composition, organization, dynamics, and function of the cell membrane. Using skin fibroblasts obtained from SLOS patients, we demonstrate that the SLOS membrane has increased 7-DHC and reduced cholesterol content and abnormal membrane fluidity. X-ray diffraction analyses of synthetic membranes prepared to mimic SLOS membranes revealed atypical membrane organization. In addition, calcium permeability is markedly augmented, whereas membrane-bound Na+/K+ATPase activity, folate uptake, inositol-1,4,5-trisphosphate signaling, and cell proliferation rates are markedly suppressed. These data indicate that the disturbance in membrane sterol content in SLOS, likely at the level of membrane caveolae, directly contributes to the widespread tissue abnormalities in this disease.

Role of Sterol Superlattice in Free Radical-Induced Sterol Oxidation in Lipid Membranes

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.

Role of the sterol superlattice in the partitioning of the antifungal drug nystatin into lipid membranes.

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.

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.

Head-specific mannose-ligand receptor expression in human spermatozoa is dependent on capacitation-associated membrane cholesterol loss.

D-Mannose binding lectins appear on the human sperm head following in-vitro incubation under capacitating conditions. Surface expression of lectin is dependent on reduction of the sterol content of sperm membranes. Mannose-specific lectin distribution over the head differs in acrosome-intact and acrosome-reacted spermatozoa. Sugar competition experiments show that mannose is the only saccharide competitive with D-mannosylated albumin binding and that free mannose induces an acrosome reaction in capacitated spermatozoa. A total of 183 men with normozoospermic semen parameters were screened for the ability of their spermatozoa to bind fluorescein isothiocyanate (FITC)-labelled mannosylated albumins, before and after incubation in vitro. The spermatozoa from 176 men 'responded' to incubation by exhibiting time-dependent increases in head-directed mannosylated albumin binding, accompanied by increases in the percentage of spermatozoa showing spontaneous acrosome loss. Motile spermatozoa from the remaining seven men failed to express mannose-lectin binding activity after 18 h of incubation and only low percentages of their spermatozoa showed spontaneous acrosome loss. These seven men were classified as 'non-responders'. The relative amounts of non-esterified cholesterol in the sperm membranes of the responder and non-responder males were analysed by gas-liquid chromatography. Responder spermatozoa showed decreases in free cholesterol content whereas non-responder spermatozoa exhibited either no decrease or increases in relative free cholesterol per cell. Fresh swim-up spermatozoa contain sub-plasma membrane stores of mannose lectins which are revealed by FITC-mannosylated albumin labelling before and after removal of the plasma membrane by vortexing. In contrast, the mannose-lectin binding activity of capacitated spermatozoa is entirely limited to the sperm surface. Western blots of proteins isolated from sperm plasma membranes after capacitation revealed two molecular species reactive with polyclonal antibodies against human macrophage mannose receptors. A model is proposed for the molecular mechanism whereby mannose lectins are transposed from sub-plasma membrane sites to an integral membrane position as a consequence of the loss of cholesterol from the sperm membrane.

Sulfate metabolism in Entamoeba histolytica

Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing [ 35S]sulfate as a marker. Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1,1 mM) total uptake was 1.5 μmol h −1 (5 × 10 7 cells) −1. The fate of sulfate within the cells was investigated by thin-layer chromatography. Major metabolites (together >3 μmol (5 × 10 7 cells) −1) were monoethyl sulfate and 3-cholesteryl sulfate; both those products were released into the growth medium. As minor components we identified the activated sulfate derivatives, adenosine-5′-phosphosulfate and 3′-phosphoadenosine-5′-phosphosulfate. In addition, up to 10% of the sulfate taken up was incorporated into high-molecular weight material (possibly proteoglycans). We propose that sulfurylation of cholesterol may play a role in controlling membrane sterol content.

Flow‐cytometric investigation of sterol content and profliferation activity of yeast

AbstractTo understand and control the dynamics of microbial growth and metabolism, a theoretical background based on segregated models is necessary, and therefore flow cytometry is the suitable measuring method.It is shown that additional information to the usual mean‐value information, and to the well‐known cytometric methods for determining DNA content and size/shape measurement by scattered light, like the distribution of membrane sterol content of baker's yeast, leads to a more detailed knowledge of growth dynamics.We have found, that a low content of membrane sterols is a characteristic of cells with suppressed proliferation activity, but necessary for high fermentation activity. Also, a high content of membrane sterols seems to enhance the viability and survival capability of harvested and dryed baker's yeast cells. Therefore, we recommend harvesting cells at a point of medium content of membrane sterols.