Plasma Membrane Sterol Research Articles

Potential Anemia as a Risk Factor for Angular Cheilitis and Pseudomembranous Candidiasis: Case Report

Angular cheilitis and pseudomembranous candidiasis are oral infections caused by Candida albicans. Angular cheilitis patients also experience oral candidiasis because the Candida reservoir is intraoral. Iron deficiency anemia was closely related to this infection. A 50-year-old female patient complaint of pain in the commissure and tongue 3 weeks ago. Extraoral examination showed anemic conjunctiva and fissure erythema with crusting in bilateral commissures. Intraoral examination showed atrophy of papillae with a yellowish-white plaque that can be scraped off and leaves erythema on the tongue. A complete blood examination was performed and showed a decrease in hemoglobin, hematocrit, erythrocytes, MCV, MCH, and MCHC. The patient was diagnosed angular cheilitis and pseudomembranous candidiasis with iron deficiency anemia. The therapy was miconazole 2% cream, nystatin oral suspension 100.000 IU/ml, and iron supplements. The lesions healed after 2 weeks of therapy and the patient was consulted to an internist. The patient has Angular cheilitis and pseudomembranous candidiasis triggered by iron deficiency anemia. Iron deficiency anemia causes a decrease in salivary transferrin protein which is fungistatic so that Candida grows excessively. Angular Cheilitis therapy is miconazole cream which inhibit the synthesis of ergosterol and nystatin for candidiasis therapy which works by binding to Candida plasma membrane sterols, causing leakage and then death. Iron deficiency anemia therapy is iron supplements; however, drug therapy alone is less effective, and consultation with an internist is required for further therapy. Anemia has big potential to be a risk factor for angular cheilitis and pseudomembranous candidiasis so comprehensive treatment is needed.

Comparison of Changes in the Content of Plasma Membrane and Tonoplast Sterols under Oxidative and Osmotic Stress

Comparison of Changes in the Content of Plasma Membrane and Tonoplast Sterols under Oxidative and Osmotic Stress

The Antileishmanial Activity of Eugenol Associated with Lipid Storage Reduction Rather Than Membrane Properties Alterations.

Leishmaniasis is a neglected tropical disease that still infects thousands of people per year throughout the world. The occurrence of resistance against major treatments for this disease causes a healthcare burden in low-income countries. Eugenol is a phenylpropanoid that has shown in vitro antileishmanial activity against Leishmania mexicana mexicana (Lmm) promastigotes with an IC50 of 2.72 µg/mL and a high selectivity index. Its specific mechanism of action has yet to be studied. We prepared large unilamellar vesicles (LUVs), mimicking Lmm membranes, and observed that eugenol induced an increase in membrane permeability and a decrease in membrane fluidity at concentrations much higher than IC50. The effect of eugenol was similar to the current therapeutic antibiotic, amphotericin B, although the latter was effective at lower concentrations than eugenol. However, unlike amphotericin B, eugenol also affected the permeability of LUVs without sterol. Its effect on the membrane fluidity of Lmm showed that at high concentrations (≥22.5× IC50), eugenol increased membrane fluidity by 20-30%, while no effect was observed at lower concentrations. Furthermore, at concentrations below 10× IC50, a decrease in metabolic activity associated with the maintenance of membrane integrity revealed a leishmaniostatic effect after 24 h of incubation with Lmm promastigotes. While acidocalcisomes distribution and abundance revealed by Trypanosoma brucei vacuolar H+ pyrophosphatase (TbVP1) immunolabeling was not modified by eugenol, a dose-dependent decrease of lipid droplets assessed by the Nile Red assay was observed. We hereby demonstrate that the antileishmanial activity of eugenol might not directly involve plasma membrane sterols such as ergosterol, but rather target the lipid storage of Lmm.

Comparison of Changes in the Content of Plasma Membrane and Tonoplast Sterols under Oxidative and Osmotic Stress

Comparison of Changes in the Content of Plasma Membrane and Tonoplast Sterols under Oxidative and Osmotic Stress

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.

A combination of plasma membrane sterol biosynthesis and autophagy is required for shade-induced hypocotyl elongation

Plant growth ultimately depends on fixed carbon, thus the available light for photosynthesis. Due to canopy light absorption properties, vegetative shade combines low blue (LB) light and a low red to far-red ratio (LRFR). In shade-avoiding plants, these two conditions independently trigger growth adaptations to enhance light access. However, how these conditions, differing in light quality and quantity, similarly promote hypocotyl growth remains unknown. Using RNA sequencing we show that these two features of shade trigger different transcriptional reprogramming. LB induces starvation responses, suggesting a switch to a catabolic state. Accordingly, LB promotes autophagy. In contrast, LRFR induced anabolism including expression of sterol biosynthesis genes in hypocotyls in a manner dependent on PHYTOCHROME-INTERACTING FACTORs (PIFs). Genetic analyses show that the combination of sterol biosynthesis and autophagy is essential for hypocotyl growth promotion in vegetative shade. We propose that vegetative shade enhances hypocotyl growth by combining autophagy-mediated recycling and promotion of specific lipid biosynthetic processes.

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.

Regulation of Plasma Membrane Sterol Homeostasis by Nonvesicular Lipid Transport.

Sterol contributes to the structural integrity of cellular membranes and plays an important role in the regulation of cell signaling in eukaryotes. It is either produced in the endoplasmic reticulum or taken up from the extracellular environment. In most eukaryotic cells, however, the majority of sterol is enriched in the plasma membrane. Thus, the transport of sterol between the plasma membrane and other organelles, including the endoplasmic reticulum, is crucial for maintaining sterol homeostasis. While vesicular transport that relies on membrane budding and fusion reactions plays an important role in bulk sterol transport, this mode of transport is slow and non-selective. Growing evidence suggests a critical role of nonvesicular transport mediated by evolutionarily conserved families of lipid transfer proteins in more rapid and selective delivery of sterol. Some lipid transfer proteins act primarily at the sites of contacts formed between the endoplasmic reticulum and other organelles or the plasma membrane without membrane fusion. In this review, we describe the similarities and differences of sterol biosynthesis and uptake in mammals and yeast and discuss the role of their lipid transfer proteins in maintaining plasma membrane sterol homeostasis.

The Capture of a Disabled Proteasome Identifies Erg25 as a Substrate for Endoplasmic Reticulum Associated Degradation

Studies in the yeast Saccharomyces cerevisiae have helped define mechanisms underlying the activity of the ubiquitin-proteasome system (UPS), uncover the proteasome assembly pathway, and link the UPS to the maintenance of cellular homeostasis. However, the spectrum of UPS substrates is incompletely defined, even though multiple techniques-including MS-have been used. Therefore, we developed a substrate trapping proteomics workflow to identify previously unknown UPS substrates. We first generated a yeast strain with an epitope tagged proteasome subunit to which a proteasome inhibitor could be applied. Parallel experiments utilized inhibitor insensitive strains or strains lacking the tagged subunit. After affinity isolation, enriched proteins were resolved, in-gel digested, and analyzed by high resolution liquid chromatography-tandem MS. A total of 149 proteasome partners were identified, including all 33 proteasome subunits. When we next compared data between inhibitor sensitive and resistant cells, 27 proteasome partners were significantly enriched. Among these proteins were known UPS substrates and proteins that escort ubiquitinated substrates to the proteasome. We also detected Erg25 as a high-confidence partner. Erg25 is a methyl oxidase that converts dimethylzymosterol to zymosterol, a precursor of the plasma membrane sterol, ergosterol. Because Erg25 is a resident of the endoplasmic reticulum (ER) and had not previously been directly characterized as a UPS substrate, we asked whether Erg25 is a target of the ER associated degradation (ERAD) pathway, which most commonly mediates proteasome-dependent destruction of aberrant proteins. As anticipated, Erg25 was ubiquitinated and associated with stalled proteasomes. Further, Erg25 degradation depended on ERAD-associated ubiquitin ligases and was regulated by sterol synthesis. These data expand the cohort of lipid biosynthetic enzymes targeted for ERAD, highlight the role of the UPS in maintaining ER function, and provide a novel tool to uncover other UPS substrates via manipulations of our engineered strain.

Genomic adaptations of the green alga Dunaliella salina to life under high salinity

Life in high salinity environments poses challenges to cells in a variety of ways: maintenance of ion homeostasis and nutrient acquisition, often while concomitantly enduring saturating irradiances. Dunaliella salina has an exceptional ability to thrive even in saturated brine solutions. This ability has made it a model organism for studying responses to abiotic stress factors. Here we describe the occurrence of unique gene families, expansion of gene families, or gene losses that might be linked to osmoadaptive strategies. We discovered multiple unique genes coding for several of the homologous superfamily of the Ser-Thr-rich glycosyl-phosphatidyl-inositol-anchored membrane family and of the glycolipid 2-alpha-mannosyltransferase family, suggesting that such components on the cell surface are essential to life in high salt. Gene expansion was found in families that participate in sensing of abiotic stress and signal transduction in plants. One example is the patched family of the Sonic Hedgehog receptor proteins, supporting a previous hypothesis that plasma membrane sterols are important for sensing changes in salinities in D. salina. We also investigated genome-based capabilities regarding glycerol metabolism and present an extensive map for core carbon metabolism. We postulate that a second broader glycerol cycle exists that also connects to photorespiration, thus extending the previously described glycerol cycle. Further genome-based analysis of isoprenoid and carotenoid metabolism revealed duplications of genes for 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and phytoene synthase (PSY), with the second gene copy of each enzyme being clustered together. Moreover, we identified two genes predicted to code for a prokaryotic-type phytoene desaturase (CRTI), indicating that D. salina may have eukaryotic and prokaryotic elements comprising its carotenoid biosynthesis pathways. In brief, our genomic data provide the basis for further gene discoveries regarding sensing abiotic stress, the metabolism of this halophilic alga, and its potential in biotechnological applications.

Antifungal Activity of the Natural Coumarin Scopoletin Against Planktonic Cells and Biofilms From a Multidrug-Resistant Candida tropicalis Strain.

Candida tropicalis is one the most relevant biofilm-forming fungal species increasingly associated with invasive mucosal candidiasis worldwide. The amplified antifungal resistance supports the necessity for more effective and less toxic treatment, including the use of plant-derived natural products. Scopoletin, a natural coumarin, has shown antifungal properties against plant yeast pathogens. However, the antifungal activity of this coumarin against clinically relevant fungal species such as C. tropicalis remains to be established. Here, we investigated the potential antifungal properties and mechanisms of action of scopoletin against a multidrug-resistant C. tropicalis strain (ATCC 28707). First, scopoletin was isolated by high-performance liquid chromatography from Mitracarpus frigidus, a plant species (family Rubiaceae) distributed throughout South America. Next, scopoletin was tested on C. tropicalis cultivated for 48h in both planktonic and biofilm forms. Fungal planktonic growth inhibition was analyzed by evaluating minimal inhibitory concentration (MIC), time-kill kinetics and cell density whereas the mechanisms of action were investigated with nucleotide leakage, efflux pumps and sorbitol and ergosterol bioassays. Finally, the scopoletin ability to affect C. tropicalis biofilms was evaluated through spectrophotometric and whole slide imaging approaches. In all procedures, fluconazole was used as a positive control. MIC values for scopoletin and fluconazole were 50 and 250 μg/L respectively, thus demonstrating a fungistatic activity for scopoletin. Scopoletin induced a significant decrease of C. tropicalis growth curves and cell density (91.7% reduction) compared to the growth control. Its action was related to the fungal cell wall, affecting plasma membrane sterols. When associated with fluconazole, scopoletin led to inhibition of efflux pumps at the plasma membrane. Moreover, scopoletin not only inhibited the growth rate of preformed biofilms (68.2% inhibition at MIC value) but also significantly decreased the extent of biofilms growing on the surface of coverslips, preventing the formation of elongated fungal forms. Our data demonstrate, for the first time, that scopoletin act as an effective antifungal phytocompound against a multidrug-resistant strain of C. tropicalis with properties that affect both planktonic and biofilm forms of this pathogen. Thus, the present findings support additional studies for antifungal drug development based on plant isolated-scopoletin to treat candidiasis caused by C. tropicalis.

The Role of LAM Genes in the Pheromone-Induced Cell Death of S. cerevisiae Yeast.

Lam1-4 proteins perform non-vesicular transport of sterols from the plasma membrane to the endoplasmic reticulum. Disruption of their function leads to an increase in the content of sterols in the plasma membrane. In mammals, homologs of Lam proteins are responsible for the internalization of plasma cholesterol. The biological role of Lam proteins in yeast remains unclear, since the strains lacking individual LAM genes do not display any pronounced phenotype. Deletion of LAM1 (YSP1) gene inhibits the regulated death of Saccharomyces cerevisiae yeast cells induced by the mating pheromone. Here, we investigated whether LAM2 also plays a role in the cell death induced by the excess of mating pheromone and assessed genetic interactions between LAM2 and genes responsible for ergosterol biosynthesis. We have shown that LAM2 deletion partially prevents pheromone-induced death of yeast cells of the laboratory strain W303, while deletions of three other LAM genes - LAM1, LAM3, and LAM4 - does not provide any additional rescuing effect. The UPC2-1 mutation in the transcription factor UPC2 gene, which leads to the excessive accumulation of sterols in the cell, promotes cell survival in the presence of the pheromone and shows additivity with the LAM2 deletion. On the contrary, LAM2 deletion stimulates pheromone-induced cell death in the laboratory strain BY4741. We have found that the deletion of ergosterol biosynthesis genes ERG2 and ERG6 reduces the effect of LAM2 deletion. Deletion of LAM2 in the Δerg4 strain lacking the gene of the last step of ergosterol biosynthesis, significantly increased the proportion of dead cells and decreased the growth rate of the yeast suspension culture even in the absence of the pheromone. We suggest that the absence of the effect of LAM2 deletion in the Δerg6 and Δerg2 strains indicates the inability of Lam2p to transport some ergosterol biosynthesis intermediates, such as lanosterol. Taken together, our data suggest that the role of Lam proteins in the regulated death of yeast cells caused by the mating pheromone is due to their effect on the plasma membrane sterol composition.

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

Cholesterol: The Plasma Membrane’s Constituent that Chooses Sides

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.

TOR complex 2-regulated protein kinase Ypk1 controls sterol distribution by inhibiting StARkin domain-containing proteins located at plasma membrane-endoplasmic reticulum contact sites.

In our proteome-wide screen, Ysp2 (also known as Lam2/Ltc4) was identified as a likely physiologically relevant target of the TOR complex 2 (TORC2)–dependent protein kinase Ypk1 in the yeast Saccharomyces cerevisiae. Ysp2 was subsequently shown to be one of a new family of sterol-binding proteins located at plasma membrane (PM)–endoplasmic reticulum (ER) contact sites. Here we document that Ysp2 and its paralogue Lam4/Ltc3 are authentic Ypk1 substrates in vivo and show using genetic and biochemical criteria that Ypk1-mediated phosphorylation inhibits the ability of these proteins to promote retrograde transport of sterols from the PM to the ER. Furthermore, we provide evidence that a change in PM sterol homeostasis promotes cell survival under membrane-perturbing conditions known to activate TORC2-Ypk1 signaling. These observations define the underlying molecular basis of a new regulatory mechanism for cellular response to plasma membrane stress.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

CgMED3 Changes Membrane Sterol Composition To Help Candida glabrata Tolerate Low-pH Stress.

Candida glabrata is a promising microorganism for organic acid production. The present study aimed to investigate the role of C. glabrata Mediator complex subunit 3 (CgMed3p) in protecting C. glabrata under low-pH conditions. To this end, genes CgMED3A and CgMED3B were deleted, resulting in the double-deletion Cgmed3ABΔ strain. The final biomass and cell viability levels of Cgmed3ABΔ decreased by 64.5% and 35.8%, respectively, compared to the wild-type strain results at pH 2.0. In addition, lack of CgMed3ABp resulted in selective repression of a subset of genes in the lipid biosynthesis and metabolism pathways. Furthermore, C18:1, lanosterol, zymosterol, fecosterol, and ergosterol were 13.2%, 80.4%, 40.4%, 78.1%, and 70.4% less abundant, respectively, in the Cgmed3ABΔ strain. In contrast, the concentration of squalene increased by about 44.6-fold. As a result, membrane integrity, rigidity, and H+-ATPase activity in the Cgmed3ABΔ strain were reduced by 62.7%, 13.0%, and 50.3%, respectively. In contrast, overexpression of CgMED3AB increased the levels of C18:0, C18:1, and ergosterol by 113.2%, 5.9%, and 26.4%, respectively. Moreover, compared to the wild-type results, dry cell weight and pyruvate production increased, irrespective of pH buffering. These results suggest that CgMED3AB regulates membrane composition, which in turn enables cells to tolerate low-pH stress. We propose that regulation of CgMed3ABp may provide a novel strategy for enhancing low-pH tolerance and increasing organic acid production by C. glabrataIMPORTANCE The objective of this study was to investigate the role of Candida glabrata Mediator complex subunit 3 (CgMed3ABp) and its regulation of gene expression at low pH in C. glabrata We found that CgMed3ABp was critical for cellular survival and pyruvate production during low-pH stress. Measures of the levels of plasma membrane fatty acids and sterol composition indicated that CgMed3ABp could play an important role in regulating homeostasis in C. glabrata We propose that controlling membrane lipid composition may enhance the robustness of C. glabrata for the production of organic acids.

Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells

We compared the potency of four derivatives of the antimicrobial peptide halictine-2 against six Candida species. Observed activity was peptide and species specific. Halictines rapidly permeabilized cell membranes and caused the leakage of cytosolic components. Their killing potential was enhanced by the commercial antimicrobial agent octenidine dihydrochloride. The effect on C. glabrata cells did not depend on the activity of Cdr pumps, but was influenced by their lipid composition. The pre-treatment of cells with myriocin, an inhibitor of sphingolipid synthesis, enhanced the peptides' activity, whereas pre-treatment with terbinafine and fluconazole, inhibitors of sterol synthesis, significantly weakened their efficacy. The killing efficacy of peptides increased in combination with amphotericin B. Thus the mode of action of halictines is likely to depend on the plasma-membrane sterols, which might explain the observed differences among the tested Candida species.

Influence of difenoconazole on lipid metabolism in marine medaka (Oryzias melastigma).

Difenoconazole (DFZ) is a triazole fungicide that inhibits the biosynthesis of sterols in cell membranes and is widely used in agriculture for effectively treating fungal infections. However, there are few studies available addressing the effects of DFZ on lipid metabolism in marine fishes. The present study was conducted to investigate the effects of DFZ on lipid metabolism in marine medaka (Oryzias melastigma). After exposure to 1, 10, 100 and 1000ng/L DFZ for 180days, an increase in condition factor (CF), total lipids and polyunsaturated fatty acids (PUFA) contents accompanied with a decrease in saturated fatty acids was observed in the muscle of DFZ-exposed fish. The expression of peroxisome proliferator-activated receptor γ as well as retinoid X receptors in the muscle was up-regulated, which would be responsible for the lipid accumulation in the muscle. The elevation of Δ6-desaturase (FADS2) and Δ9-desaturase (SCD) mRNA levels in the muscle and liver might result in the increase of PUFA content. The increased CF index and total lipid amounts indicated that DFZ exposure could affect the health of fish. ∑SFA (sum of saturated fatty acids) and DHA (docosahexaenoic acid; 22:6n-3) concentrations decreased, and the levels of ∑PUFA and ∑n-6PUFA increased in the muscle, which suggested that DFZ exposure could change lipid metabolism and profiles in fish.

Effects of Sterol Structure and Sterol Ability to form Ordered Membrane Domains upon Cellular Endocytosis

Ordered membrane domains (lipid rafts) have been hypothesized to participate in many cellular processes, including post-golgi trafficking, endocytosis, membrane protein sorting, cell viability and pathogen infection. In many cases, the evidence consists of an effect of the partial removal or sequestering of cholesterol from plasma membranes, based on the assumption that this disrupts formation of lipid domains. However, partial cholesterol removal does not establish that membrane domains were disrupted, and does not rule out a direct effect of cholesterol not related to domain formation. Thus, to investigate the role of the sterol structure and sterol raft-forming ability upon endocytosis, we used a sterol-substitution strategy. To do this, cyclodextrin-catalyzed lipid exchange was used to replace cholesterol with each of 16 sterols having various raft-forming abilities and variable structures. Endocytosis of two different proteins was studied - clustered placental alkaline phosphatase (PLAP)-endocytosis (clathrin-independent) and transferrin endocytosis (clathrin-mediated). Both types of endocytosis were affected similarly by sterol type. The ability to support lipid raft formation was found to be necessary for endocytosis. However, it was not sufficient for endocytosis, because sterols/steroids in which the 3β-OH group of cholesterol was modified did not support endocytosis. The loss of endocytosis could be reversed by a second substitution step that replenished cholesterol, and although some sterols that do not support ordered domain formation altered cell shape, cell shape changes were not found to be closely linked to the loss of endocytosis. This study shows the sterol-substitution method using diverse sterols is a useful approach to study the potential role of rafts and sterols in plasma membrane on cellular functions.