Distribution Of Sterols Research Articles

Archaeol – a biomarker for foregut fermentation in modern and ancient herbivorous mammals?

A pilot study was conducted to investigate the hydroxylated lipid content of faeces from a range of herbivorous animals with either foregut or hindgut fermenting digestive systems. Assessment of the sterol distributions derived from the faeces revealed that, whilst there were differences in the relative concentrations of individual compounds between species, there was no overall characteristic that could be used to differentiate between foregut and hindgut fermenters. However, the concentration of archaeol in each of the modern faeces varied between 5 and 49 μg g−1dry wt for the foregut fermenters, whilst archaeol was not detected in faeces from hindgut fermenters. Based on these results, it is proposed that archaeol might be a useful proxy for methanogenesis in foregut fermenting digestive systems and, further, that the presence of archaeol may be used to infer a foregut digestive origin for coprolites from ancient herbivores. Consistent with this proposal, analysis of a sub-fossil ovi-caprid coprolite yielded detectable quantities of archaeol.

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-beta-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 degrees C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 degrees C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 degrees C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.

Distribution of sterols in sediment cores from martel inlet, Admiralty Bay, King George Island, Antarctica

In the present study, sterols organic markers were applied to identify the sources of organic matter in Admiralty Bay. For this purpose, sediment samples were extracted using a Soxhlet system, clean up with column chromatography and injected into a gas chromatograph. Measurable levels of all sterols analyzed confirm the multiplicity of sources of sedimentary organic matter. In BTN and STH,the most abundant sterol was the colest-5-en-3β-ol (cholesterol) with 2.24 μg.g–1 and 4.12 μg.g–1, respectively, while in FER, itwas the 24-metil-colest-5-en-3β-ol (campesterol) with 1.83 μg.g–1. The saturated sterols have smaller concentrations in relation to parental unsaturated, which may indicate a low rate of bacterial and hydrogenation processes. A generic profile of the vertical distribution representing all 15 sterols studied was obtained by Principal Component Analysis (PCA). In three cores, the vertical distribution pattern of organic material presented the smallest values in the bottomdepth layers, reflecting the organic matter already immobilized while in the upper layers values showed a gradual increase towards the top, representing the recentdeposition of organic matter.

Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p

BackgroundGUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes.ResultsWe show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane.ConclusionsThis work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.

A comprehensive study of sterols in marine diatoms (Bacillariophyta): Implications for their use as tracers for diatom productivity

Diatoms are one of the most important organisms contributing to aquatic primary productivity and their sterols are frequently used as markers for their presence and abundance. In this study, the sterol composition of >100 diatom cultures was analyzed and its distribution was compared to the diatom phylogeny to identify typical diatom biomarkers. Forty‐four sterols were detected, 11 of them being commonly present as major sterols (contributing >10% to the total sterols). 24‐Methylcholesta‐5,24(28)‐dien‐3β‐ol is the most common sterol in diatoms, being present in 67% of all cultures analyzed, followed by the Δ5 sterols, cholest‐5‐en‐3β‐ol (cholesterol), 24‐methylcholest‐5‐en‐3β‐ol, and 24‐ethylcholest‐5‐en‐3β‐ol. 24‐Methylcholesta‐5,22E‐dien‐3β‐ol, previously suggested to be specific for diatoms, was only the fifth most common sterol; this sterol was absent in some of the major diatom groups, and high relative concentrations seem to be restricted to pennate diatoms. No sterols are restricted to specific phylogenetic groups of diatoms. Cluster analyses, however, do reveal distinct sterol distributions: Thalassiosirales typically contain high relative abundances of 24‐methylcholesta‐5,24(28)‐dien‐3β‐ol, high relative abundances of cholesta‐5,22E‐dien‐3β‐ol are typical for Cymatosirales, high relative abundances of 24‐ethylcholesta‐5,22E‐dien‐3β‐ol are characteristic for related Amphora, Amphiprora, and Entomoneis species, and a combination of high relative abundances of 24‐methylcholest‐5‐en‐3β‐ol, 24‐methylcholesta‐5,24(28)‐dien‐3β‐ol, and 24‐ethylcholest‐5‐en‐3β‐ol is typical for Attheya species. High contributions of 24‐methylcholesta‐5,22E‐dien‐3β‐ol (>50% of all sterols) seem to be restricted to pennate diatoms. None of the major sterols found in diatoms can be used as an unambiguous diatom biomarker because all of them have been reported as common sterols in other algal groups.

Sterol concentration and distribution in sunflower seeds ( Helianthus annuus L.) during seed development

Sunflower seeds are currently used for edible oil production. Among oil minor compounds, phytosterols are of special interest due to their cholesterol reducing properties. This paper reports studies on their accumulation and distribution in the embryo and hull, and the effects of temperature on phytosterol contents in sunflower seed produced under both conventional and organic field conditions. An optimised method of sterol determination, adapted to studies on small samples of seed, is presented. Seventy-two percent of phytosterols were found in the embryo, 28% in the hull. The periods of phytosterols concentration varied according to sterol category and seed part. Application of these results to improve production of natural sterols for functional food use is discussed.

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The Tm was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.

Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15-32%. When the same quenchers have access to both leaflets, they quench 70-80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60-70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes approximately 40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.

BODIPY‐Cholesterol: A New Tool to Visualize Sterol Trafficking in Living Cells and Organisms

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol-storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY-cholesterol did not disturb zebrafish development and was targeted to sterol-enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time-lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Distribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae.

The yeast O-acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly

Saccharomyces cerevisiae Gup1p is a membrane-bound O-acyltransferase. Previous works involved GUP1 in a wide range of crucial processes for cell preservation and functioning. These include cytoskeleton polarization and secretory/endocytic pathway, GPI-anchor remodelling, wall composition and integrity, and membrane lipids, with a reduction in phospholipids and an increase in acylglycerols. DRM fractions were found in considerably lower amounts in gup1Δ than in wt strain. Additionally, the proteins presumably associated with lipid micro domains, Gas1p and Pma1p, were present in much smaller amounts in the mutant DRMs. Pma1p is also found in minor quantities in the whole cells extracts of the gup1Δ mutant. Accordingly, H +-ATPase activity was reduced in about 40%. Deletion of GUP1 resulted in higher sensibility to specific sphingolipid biosynthesis inhibitors and a notorious resistance to ergosterol biosynthesis inhibitors. Furthermore, the majority of mutant cells displayed an even (less punctuated) sterol distribution. The present work presents improvements to DRMs extraction methodology and filipin-sterol staining, provides evidence supporting that Gup1p is involved in lipid metabolism and shows the direct consequences of its absence on the plasma membrane sphingolipid-sterol-ordered domains integrity/assembly.

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

A laboratory method to evaluate the sensitivity of Colletotrichum musae to postharvest fungicides

Introduction. Ce protocole vise a detecter et evaluer des variations de sensibilite dans des populations de Colletotrichum musae vis-a-vis des fongicides actuellement utilises pour le controle des maladies de conservation des bananes. Le principe, les principaux avantages, le materiel vegetal de depart, le temps necessaire et les resultats attendus de la methode sont presentes. Materiel et methodes. Le materiel de laboratoire necessaire, et le detail des douze etapes du protocole realise pour l'echantillonnage des bananes et le developpement des lesions dues a l'anthracnose, la preparation des boites de milieu gelose (agar), les isolements monospores, les tests de germination et l'analyse des donnees sont decrits. De possibles problemes sont evoques. Nous obtenons (a) pour les produits antimitotiques, le pourcentage de souches resistantes dans la population analysee ; (b) pour des fongicides inhibiteurs de la biosynthese des sterols (SBI), la variation et la distribution de la sensibilite des isolats dans la population analysee en fonction de la reduction de leur croissance radiale, imputable au fongicide. (Resume d'auteur)

Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

Accumulation of excess non-esterified free cholesterol (FC) in macrophages is a key factor in macrophage death during late stages of atheroslerosis. Raising FC content in macrophages has been shown to trigger Rac activation and actin polymerisation and to inhibit cell migration. Here, the plasma membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments. DHE's surface distribution matched exactly large ruffles and membrane protrusions which were pronounced in FC-loaded cells. Plasma membrane blebs, however, formed in FC-loaded J774 cells had a homogenous staining along the membrane bilayer at 20 °C. The results show that even in FC-loaded cells with increased membrane cholesterol content, sterols do not form a separate phase in the plasma membrane.

Cholesterol Substitution Increases the Structural Heterogeneity of Caveolae

Caveolin-1 binds cholesterol and caveola formation involves caveolin-1 oligomerization and cholesterol association. The role of cholesterol in caveolae has so far been addressed by methods that compromise membrane integrity and abolish caveolar invaginations. To study the importance of sterol specificity for the structure and function of caveolae, we replaced cholesterol in mammalian cells with its immediate precursor desmosterol by inhibiting 24-dehydrocholesterol reductase. Desmosterol could substitute for cholesterol in maintaining cell growth, membrane integrity, and preserving caveolar invaginations. However, in desmosterol cells the affinity of caveolin-1 for sterol and the stability of caveolin oligomers were decreased. Moreover, caveolar invaginations became more heterogeneous in dimensions and in the number of caveolin-1 molecules per caveola. Despite the altered caveolar structure, caveolar ligand uptake was only moderately inhibited. We found that in desmosterol cells, Src kinase phosphorylated Cav1 at Tyr(14) more avidly than in cholesterol cells. Taken the role of Cav1 Tyr(14) phosphorylation in caveolar endocytosis, this may help to preserve caveolar uptake in desmosterol cells. We conclude that a sterol C24 double bond interferes with caveolin-sterol interaction and perturbs caveolar morphology but facilitates Cav1 Src phosphorylation and allows caveolar endocytosis. More generally, substitution of cholesterol by a structurally closely related sterol provides a method to selectively modify membrane protein-sterol affinity, structure and function of cholesterol-dependent domains without compromising membrane integrity.

Geochemistry of fecal sterols in a contaminated estuary in southeastern Brazil

Domestic sewage contamination in the Iguaçu River, located in the northwestern sector of Guanabara Bay (Rio de Janeiro, Brazil) was investigated by using sterols extracted from suspended particles and sediments. Coprostanone (5β-cholestan-3-one), epicholestanol (5α-cholestan-3α-ol) and coprostanol (5β-cholestan-3β-ol) occurred in high concentration (up to 12.3 μg L −1 and 70.6 μg g −1 in suspended particles and sediments, respectively). In general, fecal sterol concentrations decreased from the river to the bay, associated with increasing distance from the diffuse sewage sources. Seasonal variation also influenced the sterol distribution. The distribution of fecal sterols suggests that system dynamics, as well as pre- and post-depositional processes, must be carefully taken into consideration if sterols are to be used in the evaluation of sewage derived contamination.

Organic matter sources in an enclosed coastal inlet assessed using lipid biomarkers and stable isotopes

The sources of organic matter (OM) in surface sediments from Wilson Inlet, an enclosed and seasonally barred inlet in Western Australia, have been determined using a combination of lipid biomarkers (including fatty acids, sterols, phytol, long chain alcohols, alkyl diols, tetrahymanol and C 32 hopanol) and stable carbon and nitrogen isotope data for bulk OM and δ 13C data for n-alkanols. The organic composition of sediments from the inflowing rivers was used as a proxy for terrestrial inputs of higher plant OM, since these sediments contained only small amounts of carbon from microalgae (mostly diatoms), bacteria and cyanobacteria. The relative proportions of “marine” and “terrestrial” OM in the sediments could be estimated using the stable carbon isotope signature of the bulk organic carbon. The sediments from Wilson Inlet showed similar lipid compositions and isotope values, and the sterol and fatty acid distributions showed the complexity typical of marine ecosystems. Relative contributions from algal (phytoplankton and microphytobenthos), bacterial, terrestrial plants and seagrass sources were estimated using biomarker/organic carbon ratios in the different source terms. Microalgae and bacteria are the most important source of OM within the inlet (over 80% in most sediments) and most of the algal material originates from the water column, except in sandy shallow sediments at the eastern end of the inlet where a contribution from microphytobenthos can be discerned. The distributions, abundances and δ 13C isotope values of long chain n-alkanols proved to be useful in quantifying the contributions from terrestrial plants and the seagrass Ruppia megacarpa. The latter is a relatively small contributor (< 10%) to the OM in the sediments, despite the presence of large seagrass beds around the edges of the inlet and the presumed importance of seagrass as a source of carbon in the foodwebs of the inlet. Nonetheless, seagrass is a significant contributor of the “higher plant” marker 24-ethylcholesterol (sitosterol) in the sediments.

YeastARV1Is Required for Efficient Delivery of an Early GPI Intermediate to the First Mannosyltransferase during GPI Assembly and Controls Lipid Flow from the Endoplasmic Reticulum

Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.