Membrane Sterol Research Articles

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome.

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A (13)C MAS NMR Study.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state 13C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol 13C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.

Contribution of Accelerated Degradation to Feedback Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase and Cholesterol Metabolism in the Liver

Accumulation of sterols in endoplasmic reticulum membranes stimulates the ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which catalyzes a rate-limiting step in synthesis of cholesterol. This ubiquitination marks HMGCR for proteasome-mediated degradation and constitutes one of several mechanisms for feedback control of cholesterol synthesis. Mechanisms for sterol-accelerated ubiquitination and degradation of HMGCR have been elucidated through the study of cultured mammalian cells. However, the extent to which these reactions modulate HMGCR and contribute to control of cholesterol metabolism in whole animals is unknown. Here, we examine transgenic mice expressing in the liver the membrane domain of HMGCR (HMGCR (TM1-8)), a region necessary and sufficient for sterol-accelerated degradation, and knock-in mice in which endogenous HMGCR harbors mutations that prevent sterol-induced ubiquitination. Characterization of transgenic mice revealed that HMGCR (TM1-8) is appropriately regulated in the liver of mice fed a high cholesterol diet or chow diet supplemented with the HMGCR inhibitor lovastatin. Ubiquitination-resistant HMGCR protein accumulates in the liver and other tissues disproportionately to its mRNA, indicating that sterol-accelerated degradation significantly contributes to feedback regulation of HMGCR in vivo Results of these studies demonstrate that HMGCR is subjected to sterol-accelerated degradation in the liver through mechanisms similar to those established in cultured cells. Moreover, these studies designate sterol-accelerated degradation of HMGCR as a potential therapeutic target for prevention of atherosclerosis and associated cardiovascular disease.

Rhizosphere associated bacteria trigger accumulation of terpenes in leaves of Vitis vinifera L. cv. Malbec that protect cells against reactive oxygen species

It has been proposed that plant growth promoting rhizobacteria (PGPR) stimulate plant growth and development by inducing the biosynthesis of secondary metabolites, like terpenes, which reduce stress incidence. Three bacteria previously isolated from grapevine roots and adjacent soil (Microbacterium imperiale Rz19M10, Kocuria erythromyxa Rt5M10 and Terribacillus saccharophilus Rt17M10) were tested as PGPR. After 30 days since root inoculation of in vitro grown Vitis vinifera cv. Malbec plants, the monoterpenes α-pinene, terpinolene and 4-carene, and the sesquiterpene nerolidol were detected only in bacterized-plant leaves. Also, the concentrations of the diterpenes α and γ-tocopherol, and the sterols sitosterol and lupeol were significantly enhanced compared to controls. The leaf extracts of bacterized plants showed photoprotective properties since they decreased the oxygen consumption (that is photo-oxidation) of the amino acid tryptophan in a sensitized solution, thus indicating an increment of the antioxidant capacity of the tissues. In addition, experiments with α-pinene and nerolidol standards showed the capability to intercept reactive oxygen species in the sensitized solution. Moreover, bacterized plants infected with the pathogen Botrytis cinerea showed a reduction in the lesion diameter compared with non-bacterized plants. The results suggest that M. imperiale, K. erythromyxa and mainly T. saccharophilus are able to induce a systemic response that trigger increases on monoterpenes, sesquiterpenes, tocopherols and membrane sterols. These compounds enhance the antioxidant capacity in leaf tissues that may help grapevine to cope with stresses.

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.

DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

BackgroundPlant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome.ResultsBy the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence.ConclusionsRAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high-efficiency AMT, possibly by avoiding congestion. The involvement of the cell wall synthesis regulator SMI1 remains to be elucidated.

Cholesterol-mediated Degradation of 7-Dehydrocholesterol Reductase Switches the Balance from Cholesterol to Vitamin D Synthesis

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis.

Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer.

Membrane-active antibiotics are known to selectively target certain pathogens based on cell membrane properties, such as fluidity, lipid ordering, and phase behavior. These are in turn modulated by the composition of a lipid bilayer and in particular by the presence and type of membrane sterols. Amphotericin B (AmB), the golden standard of antifungal treatment, exhibits higher activity toward ergosterol-rich fungal membranes, which permits its use against systemic mycoses; however, the selectivity for fungal membranes is far from satisfactory leading to severe side effects. Despite decades of research, no consensus has emerged on the origin of AmB specificity for fungal cells and its actual mode of action at the molecular level. Previously, it has been proposed that the specific action of AmB is related to differences in its affinity for membranes of different composition. In this work, we investigate this relationship by employing molecular dynamics simulations to compare the free energy of insertion of AmB into three types of membranes: a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol or ergosterol. We analyze the orientation of AmB molecules within the bilayer in order to unambiguously establish their membrane binding mode and relate the orientational freedom to the sterol-dependent tightness of lipid packing. Our results strongly indicate that the membrane insertion of AmB proceeds virtually to completion independent of membrane type, and hence the higher toxicity against fungal membranes may rather result from differences in subsequent oligomerization in the membrane and assembly of monomers into functional transmembrane pores. In particular, the latter could be facilitated by sterol-induced ordering of AmB molecules along the membrane normal, revealed by our free energy profiles. Moreover--in contrast to certain claims--we find no stable binding mode corresponding to the horizontal adsorption of AmB on the membrane surface.

Effects of Sterol Substitution on Influenza Viral Membrane Fusion

Influenza virus membranes are enriched in cholesterol in comparison to the plasma membrane, as cholesterol can comprise as much as 50% of viral envelope lipids. This enrichment in sterol content likely plays an adaptive role for the virus. Prior work has shown that influenza viral fusion rates vary with cholesterol concentration of both viral and target membranes. To understand which cholesterol properties affect influenza fusion, we measured fusion kinetics between viral and liposomal membranes where cholesterol was replaced with different sterols. Influenza fusion rates are surprisingly invariant across different sterols in the target membrane (all tested at the same mol %) and across a fairly broad range of sterols in the viral membrane. Two sterols--lanosterol and 25-hydroxycholesterol—did slow fusion moderately when introduced into the viral membrane, while substitution of cholesterol with polar sterols greatly slowed fusion. No other sterols tested showed a significant perturbation in fusion rates, including ones altering membrane phase behavior. Our findings suggest that the rate-limiting step in influenza viral fusion may be sensitive to membrane bending moduli but does not depend strongly on sterol-mediated changes to liquid-liquid phase coexistence in membranes.

7-Dehydrocholesterol Modifies the Operation of Kv1.3 Channels in T Cells Isolated from Smith-Lemli-Opitz Syndrome Patients

In vitro manipulation of membrane sterol level has an impact on the regulation of ion channels, however, a comprehensive study is lacking to confirm the physiological or pathophysiological significance of these experiment. Smith-Lemli-Opitz syndrome (SLOS) is characterized by a reduced or abolished activity of 7-dehydrocholesterol (7DHC) reductase, which leads to the elevation of the 7DHC in the tissues and blood. We utilized T cells isolated from SLOS patients to address the question if in vivo altered membrane sterol composition impairs the operation of Kv1.3, the predominant voltage-gated ion channel of T cells, and if altered Kv1.3 function is reflected in impaired mitogenic responses of SLOS T lymphocytes.Using whole-cell patch-clamp technique we showed that the activation kinetics of Kv1.3 is slower and the midpoint of the voltage-dependence of steady-state activation is shifted to depolarized potentials in SLOS T cells as compared to age-matched controls. Similar changes in the kinetic and equilibrium parameters of Kv1.3 gating were detected in control T cells loaded with 7DHC using 7DHC/cyclodextrin complex. Upon removal of putative sterol binding sites of Kv1.3 the channel become insensitive to 7DHC loading. Functional assays revealed that modified operation of Kv1.3 in the SLOS T cells is associated with impaired proliferation rate (CFSE method) and a defect in the early steps of Kv1.3¬- and Ca2+-dependent activation process (CD154/CD40L expression) in CD3+ cells.Our conclusion is that the function of Kv1.3 is modified in SLOS via a direct coupling to the 7DHC in the cell membrane. We propose that this ion channel-sterol interaction reveals a molecular mechanism that may contribute to the pathophysiological conditions in SLOS and lead to the most prominent neurological and cardiovascular symptoms via influencing the physiological function of ion channels.

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.

Correlating Sterol Structure with Membrane Solubility Limits and Phase Behavior in Ternary Model Membranes

Evolution selected for an array of structurally similar sterols in cell membranes. While animal cells rely on cholesterol, yeast utilize ergosterol, and plants incorporate a variety of phytosterols including stigmasterol and s-sitosterol. These sterols differ by one or two degrees of unsaturation and/or by an added methyl or ethyl group on their tail; these subtle changes have substantial effects on their membrane solubility. A previous study found the solubility limits of cholesterol, ergosterol, stigmasterol, and s-sitosterol in 1:1 DOPC/DPPC bilayers to be 65-70 mol %, 30-35 mol %, 20-25 mol %, and ∼40 mol % [Stevens, M. M.; Honerkamp-Smith, A. R.; Keller, S. L. Soft Matter, 2010, 6, 5882-5890]. Here we use fluorescence microscopy to correlate the structural features of ergosterol, stigmasterol, s-sitosterol with the miscibility temperature of ternary membranes containing DOPC, DPPC, and one of the aforementioned sterols. We map the full ternary phase diagram of each system and compare it with that of the well-characterized DOPC/DPPC/cholesterol system. By dark-field microscopy we determine upper bounds of the solubility limits of each sterol in DOPC/DPPC membranes, and we also provide qualitative tie lines for each system. Our data reveal that slight changes in sterol structure have strong effects on phase behavior and membrane solubility. Our results provide insight into intermolecular interactions between sterols and PC-lipids. This research was supported by NSF MCB-1402059.

Phase Behavior of Complex Lipid Mixtures: Signatures of Spatial Organization

The spatial organization of phospholipids and sterols in cellular membranes plays an important role in membrane function and cell activities. Both experimental and simulation studies have shown that the spatial distribution of lipids and sterols can be heterogeneous with various morphologies in multi-component bilayer systems. Here we use coarse-grained Molecular Dynamics simulations to study the phase behavior of a quaternary lipid mixture. We obtain quantities such as density correlation functions and composition distribution functions, and we show how these observables can be analyzed to yield insight into the structure of complex lipid mixtures.

Control of Influenza Virus Binding by Target Membrane Composition

Despite the availability of a vaccine for more than six decades, influenza infection remains a major cause of morbidity and mortality and a global health threat. Infectivity depends on viral hemagglutinin recognizing sialic acid in the host cell membrane to initiate endocytosis and deliver the viral RNA. We have investigated how the lipid composition of target membranes modulates viral binding by visualizing the interaction of fluorescently labeled virions with synthetic planar lipid bilayers in a microfluidic device. In comparative studies of membranes supplemented with seven different sterol analogues, ketocholesterol (K-chol) outperformed other sterol variants in enhancing viral capture. Membranes containing 10 mol % K-chol showed a 35% increase in viral binding when compared to 10 mol % cholesterol membranes and a 60% enhancement when compared to sterol-free membranes. FRAP measurements demonstrated that K-chol decreased lipid lateral diffusion constants by a factor of two, confirming that K-chol is differentially altering membrane behavior. Both heightened viral capture and diminished lipid diffusion rates were nonlinearly dependent on K-chol concentration. In addition, at a constant mol % sterol, K-chol-containing membranes showed a much greater sensitivity to GD1A concentration, saturating binding at 4-fold lower mol % GD1A than membranes containing an equimolar amount of cholesterol. These data are compatible with virus-membrane contact involving a multivalent interaction that is highly sensitive to membrane organization and lipid mobility. Therefore, membrane sterol composition modulates influenza virus-receptor binding in a manner closely correlated to nanoscale organization and dynamics. The resulting changes to receptor sensitivity may be important in controlling permissiveness to infection.

In vitro Inhibition of Leishmania braziliensis Promastigotes Growth by a Fluconazole Microemulsion

Cutaneous leishmaniasis (CL) is the most prevalent form of the disease caused by the parasites Leishmania. However, effective topical treatments for the disease are lacking. Fluconazole (FLZ) is an antifungal agent that inhibits a key enzyme for the production of ergosterol, the main sterol in membranes of fungi and parasites. An in vitro evaluation of antileishmanial activity of a microemulsion intended for topical use, with FLZ as active pharmaceutical ingredient, was performed. The formulation effectively inhibited L. braziliensis promastigotes growth. This study provides baseline data about the efficacy of FLZ microemulsion on L. braziliensis promastigotes. Previous reports had shown poor activity of the drug, so this result highlighted the role of the vehicle. Although further work is needed, this semisolid dosage form could be useful for the local treatment of CL in the New World. It is also interesting to mention the importance of repositioning well-known drugs for new uses to offer treatments more quickly, particularly for this disease that greatly affects public health of poor regions.

Comparison of Membrane Characteristics between Freshly Ejaculated and Cryopreserved Sperm in the Chicken.

Cryopreserved sperm undergoes serious damage which affects its fertilizing ability. Despite progress in understanding the nature of functional deterioration in mammalian sperm, little is known about the mechanism involved in the induction of functional damage in avian sperm. Cellular membranes are considered the primary site of cryodamage to sperm. Membrane rafts are specific membrane regions enriched in sterols, ganglioside GM1, and functional proteins and they play important roles in the regulation of diverse functions exerted in mammalian sperm during fertilization. Several reports investigating cryopreservation-induced membrane changes in mammalian sperm have suggested that cryopreservation induces a compositional alteration of membrane rafts via a loss of membrane sterols, leading to impaired fertilizing ability. Recently, we demonstrated that membrane rafts are present in chicken sperm. Therefore, we investigated a possible mechanism for the induction of functional damage in cryopreserved chicken sperm, with particular attention to cryopreservation-induced compositional changes in membrane rafts. Sterol quantification showed that loss of sterols from sperm membranes occurred following cryopreservation. Biochemical analyses of detergent-insoluble membranes showed that the lipid and protein compositions of membrane rafts were altered dramatically by cryopreservation. To determine the physiological role of these changes, we examined external translocation of phosphatidylserine (PS), representing an early apoptotic change, and found that cryopreservation induced apoptotic changes in chicken sperm. Furthermore, methyl-β-cyclodextrin-induced loss of sterols from the plasma membranes stimulated PS translocation that was not accompanied with caspase-3 activation, which plays an important role downstream of the apoptotic cascade. Based on the results obtained in this study, we discuss a new mechanism for reduction of the fertilizing ability in avian sperm after cryopreservation.

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.

Early sponges and toxic protists: possible sources of cryostane, an age diagnostic biomarker antedating Sturtian Snowball Earth.

The period 800-717million years (Ma) ago, in the lead-up to the Sturtian Snowball glaciation, saw an increase in the diversity of eukaryotic microfossils. To afford an independent and complementary view of this evolutionary period, this study presents the distribution of eukaryotic biomarkers from three pre-Sturtian successions across the supercontinent Rodinia: the ca. 780Ma Kanpa Formation of the Western Australian Officer Basin, the ca. 800-740Ma Visingsö Group of Sweden, and the 740Ma Chuar Group in Arizona, USA. The distribution of eukaryotic steranes is remarkably similar in the three successions but distinct from all other known younger and older sterane assemblages. Cholestane was the only conventional structure, while indigenous steranes alkylated in position C-24, such as ergostane, stigmastane, dinosterane and isopropylcholestane, and n-propylcholestane, were not observed. This sterane distribution appears to be age diagnostic for the pre-Sturtian Neoproterozoic. It attests to the distinct evolutionary state of pre-Snowball eukaryotes, pointing to a taxonomic disparity that was still lower than in the Ediacaran (635-541Ma). All three basins also show the presence of a new C28 sterane that was tentatively identified as 26-methylcholestane, here named cryostane. The only known extant organisms that can methylate sterols in the 26-position are demosponges. This assignment is plausible as molecular clocks place the appearance of the earliest animals into the pre-Sturtian Neoproterozoic. The unusual 26-methylsterol may have protected sponges, but also other eukaryotes, against their own membranolytic toxins. Some protists release lytic toxins to deter predators and kill eukaryotic prey. As conventional membrane sterols can be the site of attack for these toxins, sterols with unusual side-chain modification protect the cell. This interpretation of cryostane supports fossil evidence of predation in the Chuar Group and promotes hypotheses about the proliferation of eukaryophagy in the lead-up to the Cryogenian.

Membrane lipid rafts and neurobiology: age-related changes in membrane lipids and loss of neuronal function.

A better understanding of the cellular physiological role that plasma membrane lipids, fatty acids and sterols play in various cellular systems may yield more insight into how cellular and whole organ function is altered during the ageing process. Membrane lipid rafts (MLRs) within the plasma membrane of most cells serve as key organizers of intracellular signalling and tethering points of cytoskeletal components. MLRs are plasmalemmal microdomains enriched in sphingolipids, cholesterol and scaffolding proteins; they serve as a platform for signal transduction, cytoskeletal organization and vesicular trafficking. Within MLRs are the scaffolding and cholesterol binding proteins named caveolin (Cav). Cavs not only organize a multitude of receptors including neurotransmitter receptors (NMDA and AMPA receptors), signalling proteins that regulate the production of cAMP (G protein-coupled receptors, adenylyl cyclases, phosphodiesterases (PDEs)), and receptor tyrosine kinases involved in growth (Trk), but also interact with components that modulate actin and tubulin cytoskeletal dynamics (e.g. RhoGTPases and actin binding proteins). MLRs are essential for the regulation of the physiology of organs such as the brain, and age-related loss of cholesterol from the plasma membrane leads to loss of MLRs, decreased presynaptic vesicle fusion, and changes in neurotransmitter release, all of which contribute to different forms of neurodegeneration. Thus, MLRs provide an active membrane domain that tethers and reorganizes the cytoskeletal machinery necessary for membrane and cellular repair, and genetic interventions that restore MLRs to normal cellular levels may be exploited as potential therapeutic means to reverse the ageing and neurodegenerative processes.

3-H-[1,2]Dithiole as a New Anti-Trypanosoma cruzi Chemotype: Biological and Mechanism of Action Studies.

The current pharmacological Chagas disease treatments, using Nifurtimox or Benznidazole, show limited therapeutic results and are associated with potential side effects, like mutagenicity. Using random screening we have identified new chemotypes that were able to inhibit relevant targets of the Trypanosoma cruzi. We found 3H-[1,2]dithioles with the ability to inhibit Trypanosoma cruzi triosephosphate isomerase (TcTIM). Herein, we studied the structural modifications of this chemotype to analyze the influence of volume, lipophilicity and electronic properties in the anti-T. cruzi activity. Their selectivity to parasites vs. mammalian cells was also examined. To get insights into a possible mechanism of action, the inhibition of the enzymatic activity of TcTIM and cruzipain, using the isolated enzymes, and the inhibition of membrane sterol biosynthesis and excreted metabolites, using the whole parasite, were achieved. We found that this structural framework is interesting for the generation of innovative drugs for the treatment of Chagas disease.