Varying Concentrations Of Cholesterol Research Articles

Enhanced sensitivity in Staphylococcus aureus detection: Unveiling the impact of lipid composition on the performance of carboxyfluorescein (CF)-Loaded liposome-based assay

Liposomes have emerged as versatile nanocarriers, finding applications not only in drug delivery but also in pathogen detection and diagnostics. This study aimed to enhance the sensitivity of liposomes to Staphylococcus aureus by investigating the impact of lipid composition on liposomes loaded with 5(6)-carboxyfluorescein (CF). Liposomes were fabricated using various concentrations of cholesterol (10–40 mol%) combined with saturated phospholipids. Dynamic light scattering results revealed that higher cholesterol concentrations led to reduced liposome size, CF release (%), and entrapment efficiency (%). Liposome sensitivity towards S. aureus was evaluated by using CF-loaded liposomes with and without aptamer insertion. Liposomes with a higher cholesterol content (40 mol%) exhibited a strong ability to detect low bacterial concentrations down to 5 × 102 CFU/mL without relying solely on specific receptor-ligand recognition. However, functionalizing the liposome with an aptamer further improved the specificity and sensitivity of S. aureus detection at even lower concentrations, down to 80 CFU/mL, in the wide range of 80–107 CFU/mL. This study highlights the potential for optimizing the lipid composition of liposomes to improve their sensitivity for pathogen detection, particularly when combined with aptamer-based strategies.

Optical detection of total cholesterol based on a dye-displacement method

In the present study, the dye methylene blue (MB) was entrapped in an agarose gel and used as a sensing probe for the detection of total cholesterol. When methylene blue-entrapped agarose cubes were added to the cholesterol solution, methylene blue was displaced by cholesterol and released into the solution. A calibration curve was prepared by plotting the rate of release of methylene blue at 664 nm against varying cholesterol concentrations. A linear response was observed in the concentration range of 1 to 5 mM (40 mg/dL to 200 mg/dL) which covers normal and elevated cholesterol levels in humans. Optical detection of cholesterol using this dye-replacement method is simple, economical, and non-toxic. Characterisation of the system was carried out by FT-IR spectroscopy and cyclic voltammetry. The optical method was validated to determine total cholesterol in serum samples with reasonable accuracy.

Evaluation of Nanochitosan-zein Coating Containing Free and Nano-encapsulated Mentha pulegium L. Hydroalcoholic Extract on Quality Attributes of Persian Shrimp (Fenneropenaeus persian)

ABSTRACT The present study was performed to fabricate a new biodegradable coating containing free, and nano encapsulated Mentha pulegium L. hydroalcoholic extract (MPE) with nano-chitosan (NCs)-zein (Zn) base to assess the biological, chemical, and sensory characteristics of shrimp samples during storage in the refrigerator for 12 days. Various concentrations of cholesterol: soy-lecithin (0:60, 10:50, 20:40, and 30:30) were used to prepare nanoliposomes. The chemical and biological properties results showed that the NCs-Zn-N (nano) MPE group had the highest effect during storage. The pH, peroxide values, thiobarbituric acid reactive substances, total volatile base nitrogen, total viable counts, TPC, and lactic acid bacteria were 7.6, 1.3 meq. kg−1, 0.17 MDA.kg−1, 30 mg.100 g−1 7.9 CFU.g−1, 8.2 CFU.g−1, and 6.4 CFU.g−1, respectively, for NCs-Zn-NMPE on the 12th day. Based on the results, NCs-Zn coating with MPE-loaded nanoliposomes can be used as a safe and suitable coating to increase the shelf life of shrimp.

Effect of the cholesterol on electroporation of planar lipid bilayer

Electroporation threshold depends on the membrane composition, with cholesterol being one of its key components already studied in the past, but the results were inconclusive. The aim of our study was to determine behaviour of planar lipid bilayers with varying cholesterol concentrations under electric field. This would give us a better insight into cholesterol effect on membrane properties during electroporation process, since cholesterol is one of the major components of biological membranes and plays a crucial role in membrane organisation, dynamics, and function. Planar lipid bilayers were prepared from phosphatidylcholine lipids with 0, 20, 30, 50 and 80 mol% cholesterol. Capacitance was measured using the discharge method. Results show no statistical difference of cBLM between the cholesterol concentrations. Breakdown voltage Ubr of planar lipid bilayers was measured by means of linear rising voltage with seven different slopes. Obtained results were fitted to a strength-duration curve, where parameter Ubrmin represents minimal breakdown voltage, and parameter τRC represents the inclination of the strength-duration curve. Adding cholesterol to planar lipid bilayer gradually increased its Ubrmin until 50 mol% cholesterol concentration. Afterwards at 80 mol% Ubrmin does not further increase, in fact it reduces by 20% of the Ubrmin at 50 mol% cholesterol concentration.

Molecular Study of Ultrasound-Triggered Release of Fluorescein from Liposomes.

Several investigations have suggested that ultrasound triggers the release of drugs encapsulated into liposomes at acoustic pressures low enough to avoid cavitation or high hyperthermia. However, the mechanism leading to this triggered release as well as the adequate composition of the liposome membrane remains unknown. Here, we investigate the ultrasound-triggered release of fluorescein disodium salt encapsulated into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-distearoylphosphatidyl-ethanolamine (DSPC) lipids with various concentrations of cholesterol (from 0 to 44 mol %). The passive release of encapsulated fluorescein was first characterized. It was observed to be higher when the membrane is in a fluid phase and increased with temperature but decreased upon addition of cholesterol. Next, the release of fluorescein was measured at different acoustic frequencies (0.8, 1.1, and 3.3 MHz) and peak-to-peak pressures (0, 2, 2.5, 5, and 8 MPa). Measurements were performed at temperatures where DOPC and DSPC liposomes were, respectively, in the fluid or gel phase. We found that the release rate did not depend on the ultrasound frequency. For DOPC liposomes, the ultrasound-triggered release of fluorescein decreased with increasing concentration of cholesterol in liposomes, while the behavior was more complex for DSPC liposomes. Overall, the triggered release from DSPC liposomes was up to ten times less than DOPC liposomes. Molecular dynamics simulations performed on a pure DOPC membrane showed that a membrane experiences, under a directional pressure of ±2.4 MPa, various changes in properties such as the area per lipid (APL). An increase in the APL was notably observed when the simulation box was laterally stretched or perpendicularly compressed, which was accompanied by an increase in the number of water molecules crossing the membrane. This suggests that ultrasound most probably enhances the diffusion of encapsulated molecules at small acoustic pressures by increasing the distance between lipids.

Membrane Binding of Flaviviral Nonstructural Protein 1: Comparative Simulations of ZIKA and Dengue Virus NS1 Proteins in Pope Bilayers

The rate of flavivirus infections has increased dramatically in the last few years, in particular infections due to the Dengue virus (DENV) and Zika virus (ZIKV). Hence better inhibition strategies against viral targets are required. One such target in the flaviviral family is the non-structural protein 1 (NS1). Upon infection, the NS1 dimers accumulate in ER lumen and transforms into a hexameric lipoprotein particle under severe disease conditions. Though the presence of dimers and hexamers have been proven, the membrane binding and lipoparticle formation process remains unexplored. This study investigates the membrane binding property of ZIKV and DENV NS1 proteins through all-atom molecular dynamics simulations. Since the NS1 dimers are the most prevalent in the lumen side of ER, the POPE membranes were modelled with varying cholesterol concentrations (0%, 20% and 40%). The 3.6 μs MD simulations clearly showed that ZIKV NS1 inserts deeper into the membrane than DENV NS1. In particular, the β-roll, wing domain and intertwined loop of ZIKV NS1 form the major membrane interacting regions. In this study, two conformations namely the stable membrane bound and solvent exposed conformations were observed for the previously uncharacterised intertwined loop. As ZIKV NS1 inserts more, it shows more number of interacting cholesterol molecules around than that found for DENV NS1. The variation in the number of basic/aromatic amino acids between ZIKV and DENV NS1 might influence their membrane insertion property and this has been quantified in our study. Altogether, our study forms the basis for understanding the membrane binding properties of flaviviral NS1 proteins and the functional characterization of the interacting regions.

Preparing and assessing the physiochemical properties of curcumin niosomes and evaluating their cytotoxicity in 3T3 and MCF-7 cell lines.

Objective:Application of vesicular drug delivery systems has made major progress in pharmaceutical science and technology. Niosomal drug delivery is potentially efficient to improve the pharmacokinetic and pharmacological properties of many compounds. Curcumin (CUR) has several documented anticancer activities; however, it has a low bioavailability that necessitates the development of efficient delivery systems. Accordingly, different niosomal preparations were prepared and evaluated in the present study to find a suitable delivery system.Materials and Methods:Span and Tween 20, 40, 60, and 80 were employed with various concentrations of cholesterol for studying the ability to form curcumin-loaded niosomes. Multiple characterization techniques including visual evaluation, particle size analysis, stability, encapsulation efficiency (EE), and release profile were studied. Cytotoxicity of curcumin niosomes on MCF-7 and 3T3 cell lines was determined using MTT assay. Results:Visual and particle size analysis indicated the formation of seven niosomal formulations in the micron size range, while the formulation consisted of Tween 40/cholesterol (50/50 M%) with 0.05% w/v CUR had an average diameter of 475 nm. The latter formulation was selected and it had EE of 78.5%. The CUR release profile showed 18.7% release over a period of 300 min. The MTT results showed that CUR incorporation significantly increased the cytotoxicity of niosomes and the extent of toxicity was higher in MCF-7 cells. Conclusion:In this study, a simple niosomal formulation was developed for CUR loading with favorable physicochemical properties. The presented niosomal curcumin had also considerable effects in cell toxicity studies, which can be suggested for future anticancer studies.

Exploring membrane viscosity at the headgroup region utilizing a hemicyanine-based fluorescent probe

Membrane viscosity is an important determinant for several cellular functions. In spite of having vast literature on the interfacial and acyl chain region viscosity of the membrane, there is hardly any report on the membrane viscosity at the headgroup region. In this present work, we have exploited the photophysical properties of a hemicyanine dye covalently linked with a C16 alkyl chain (HC16) to explore the membrane viscosity at the headgroup region. The chromophoric group of HC16 locates at the headgroup region due to its overall positive charge. Methanol-glycerol mixture of known composition and viscosity were used to develop the standard curve of apparent rotational correlation time against viscosity, and utilized this standard curve to evaluate viscosity of dimyristoylphosphatidylcholine (DMPC) membrane as a function of temperature. The result exhibited that there is a 4-fold change in headgroup viscosity of the DMPC membrane between gel and fluid phase; with a sharp change during the phase transition. Moreover, we have monitored the effect of cholesterol on the viscosity of the headgroup region of a biologically relevant lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), keeping in mind the role of cholesterol on membrane organization, dynamics, and cellular functions. Interestingly, the headgroup viscosity of POPC membrane does not show a significant change in presence of various concentrations of cholesterol, though literature showed remarkable change in interfacial and acyl chain region viscosity in presence of similar concentrations of cholesterol.

Investigating the Nanocomposite Thin Films of Hematite α-Fe2O3 and Nafion for Cholesterol Biosensing Applications

Nanocomposite materials are multi-phase materials, usually solids, which have two or more component materials having different chemical and physical properties. When blended together, a newer material is formed with distinctive properties which make them an eligible candidate for many important applications. In the present study, thin films of nafion (polymer) and hematite or α-Fe2O3 (nanoparticles) nanocomposite is fabricated on indium tin oxide (ITO) coated glass substrates, due to its enhanced ionic conductivity, for cholesterol biosensor applications. Scanning electron microscopy and Atomic force microscopy revealed the formation of nanorod structured α-Fe2O3 in the films. The cyclic voltammetry (CV) studies of nafion-α-Fe2O3/ITO revealed the redox properties of the nanocomposites. The sensing studies were performed on nafion-α-Fe2O3/CHOx/ITO bioelectrode using differential pulse voltammetry (DPV) at various concentrations of cholesterol. The enzyme immobilization leaded to the selective detection of cholesterol with a sensitivity of 64.93 × 10−2 μA (mg/dl)−1 cm−2. The enzyme substrate interaction (Michaelis–Menten) constant Km, was obtained to be 19 mg/dl.

An investigation of morphological, microscopic dynamics, fluidity, and physicochemical variations in Cu-decorated metallosomes with cholesterol

The controlled stoichiometry of single-tailed surfactant cetylpyridinium chloride (CPC) in the presence of Cu ions led to a vesicular assembly known as ‘metallosomes’. Unlike liposomes, their membrane properties such as microscopic dynamics, fluidity, and physicochemical variations, have not been explored. Therefore, metallosomes with a water-soluble metallosurfactant (bishexadecylpyridiniumcopper tetrachloride) with varying cholesterol concentrations were synthesized and explored for the induced effect of cholesterol on different membrane properties. Three different metallosomes were synthesized by varying the metallosurfactant to cholesterol molar ratio to 1:0, 1:0.5, and 1:1 and characterized with cryo-TEM, FESEM, DLS, and ζ-potential. For microscopic dynamics studies, quasielastic neutron scattering (QENS), for bilayer molecular arrangement XRD and SAXS, and for physicochemical properties, three fluorescent probes (ANS, Prodan, and Laurdan) were utilized. The Well Diffusion method was employed for exploring the antimicrobial behavior of empty as well as drug-loaded metallosomes. The spherical morphology of synthesized metallosomes was established with different studies and insertion of cholesterol has been found to lead to more stable and homogeneous suspensions. Also, more restricted, less fluid, and dehydration of membrane was observed with cholesterol. The antimicrobial activity as well as releasing profile was observed to be directly influenced in the presence of cholesterol.

Facilitated and Non-Gaussian Diffusion of Cholesterol in Liquid Ordered Phase Bilayers Depends on the Flip-Flop and Spatial Arrangement of Cholesterol.

The diffusion of cholesterol in biological membranes is critical to cellular processes such as the formation of cholesterol-enriched domains. The cholesterol diffusion may be complicated especially when cholesterol flip-flops and/or stays at the membrane center. Understanding the diffusion mechanism of cholesterol at a molecular level should be, therefore, a topic of interest. We perform molecular dynamics simulations up to 100 μs for lipid bilayers with various concentrations of cholesterol. We find that cholesterol diffusion in the liquid ordered phase depends on whether it is within leaflets or at the bilayer center, is non-Gaussian for several microseconds, and is enhanced significantly compared to that of lipids. Cholesterol at the bilayer center diffuses fast, while cholesterol in the hydrocarbon region with upright orientation diffuses relatively slowly. Such position-dependent dynamics of cholesterol leads to facilitated and non-Gaussian diffusion.

Covalently Cross-Linked Cholesterol Oxidase for an Indirect Detection Cholesterol Biosensor

Nearly a third of the population in the United States suffer from high cholesterol. This condition approximately doubles one's risk of cardiovascular disease, which is the leading cause of death in the country. As such, increasingly cost-effective, robust, easy-to-use, and accurate methods of detection of cholesterol are necessary to curtail such health risks. A particularly accurate method for this includes enzymatic electrochemical detection; however, most electrochemical detection methods use enzyme-modified electrodes developed based on the physisorption of enzymes onto the electrodes. This approach does not result in high activity and severely limits the enzyme stability on the electrode. This can be mitigated by immobilizing the enzyme onto carbon nanotubes using molecular tethers. Our biosensor was fabricated using Cholesterol oxidase, from Streptomyces, immobilized on multi-walled carbon nanotubes (MWCNT) with 1-pyrenebutanoic acid, succinimidyl ester (PBSE) as a molecular tethering agent. The biosensor shows a linear detection region ranging from at least 0.03-0.5 mM and high selectivity against common interferents, such as glucose at less than five percent. Fig. 1:(top): Differential Pulse Voltammetry study of Cholesterol oxidase immobilized on MWCNT with PBSE in an oxygen-saturated 0.1 M phosphate buffer (pH 7.4) at varying cholesterol concentrations ranging from 0 to 0.5 mM. (bottom): Standard plot of cholesterol concentration versus amperometric signal (change in peak current). Acknowledgments: The authors extend gratitude to Dr. Nicole S. Sampson of Stony Brook University for the cholesterol oxidase enzyme used herein. * Corresponding Author: rama@uga.edu

Secretory vesicle cholesterol: Correlating lipid domain organization and Ca2 + triggered fusion

Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca2+ sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure–area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal–spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca2+ triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure.

Cholesterol-mediated surfactant dysfunction is mitigated by surfactant protein A

The ability of pulmonary surfactant to reduce surface tension at the alveolar surface is impaired in various lung diseases. Recent animal studies indicate that elevated levels of cholesterol within surfactant may contribute to its inhibition. It was hypothesized that elevated cholesterol levels within surfactant inhibit human surfactant biophysical function and that these effects can be reversed by surfactant protein A (SP-A). The initial experiment examined the function of surfactant from mechanically ventilated trauma patients in the presence and absence of a cholesterol sequestering agent, methyl-β-cyclodextrin. The results demonstrated improved surface activity when cholesterol was sequestered in vitro using a captive bubble surfactometer (CBS). These results were explored further by reconstitution of surfactant with various concentrations of cholesterol with and without SP-A, and testing of the functionality of these samples in vitro with the CBS and in vivo using surfactant depleted rats. Overall, the results consistently demonstrated that surfactant function was inhibited by levels of cholesterol of 10% (w/w phospholipid) but this inhibition was mitigated by the presence of SP-A. It is concluded that cholesterol-induced surfactant inhibition can actively contribute to physiological impairment of the lungs in mechanically ventilated patients and that SP-A levels may be important to maintain surfactant function in the presence of high cholesterol within surfactant.

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-beta-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquid-disordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquid-ordered behavior observed in the various types of liposomes as well as some interesting differences.

Cholesterol oxidase modified gold electrodes as bioanalytical devices

Cholesterol oxidase (ChOx) has been immobilized by direct adsorption on gold electrodes. The resulting ChOx monolayers have been characterized using atomic force microscopy (AFM) under liquid conditions and quartz crystal microbalance (QCM) techniques. The immobilized enzyme retains its catalytic activity, thus spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). The replacement, in the enzymatic reaction, of the natural electron acceptor (O 2) by an artificial mediator has been also evaluated, in particular, hydroxymethylferrocene (HMF), thionin, nile blue and azure A, have been studied as electron acceptors for reduced ChOx. In addition, the influence of the low cholesterol solubility on the experimental conditions using redox mediators was also discussed. Finally, the response of the enzymatic electrode to varying cholesterol concentrations has been obtained by measuring directly the H 2O 2 generated in the enzymatic reaction. Cholesterol can be determined amperometrically at +0.5 V (versus SSCE) with a detection limit of 60 μM and a sensitivity of 0.13 μA mM −1.

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

Lipid Peroxidation Induces Cholesterol Domain Formation in Model Membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.

Manipulation of Cholesterol Levels in Rod Disk Membranes by Methyl-β-cyclodextrin: EFFECTS ON RECEPTOR ACTIVATION

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.

Effects of lipids on n-alkane attenuation in media supporting oil-utilizing microorganisms from the oily Arabian Gulf coasts.

The Arabian Gulf is one of the most extensively oil-polluted areas of the world. The major objectives of this work were to study whether hydrocarbon-utilizing microorganisms indigenous to that area would readily accumulate added lipids, and whether this might affect their hydrocarbon consumption potential. Two prokaryotes, Arthrobacter nicotianae KCC B35 and the unidentified organisms KCC B6, as well as one eukaryote, Candida parapsilosis KCC Y1, were selected for this study. Biomass samples of the test organisms were incubated in an inorganic medium containing various concentrations of cholesterol, stearic acid, triolein or egg-phospholipids. The results revealed that all lipid classes were readily accumulated by the three test organisms. In addition, biomass samples were incubated for 6 h in an inorganic medium containing mixtures of individual lipid classes and either n-octadecane or n-docosane. The cells were removed and the residual alkanes in the medium were quantitatively recovered and analyzed by GLC. The results showed that out of the tested lipid classes only stearic acid exhibited a common stimulatory effect on the consumption of both n-alkanes by all test organisms. Other lipid classes were either inhibitory or had less pronounced effects than stearic acid.