Polymer-induced flip-flop in biomembranes

This Account describes the ability of amphiphilic polymers (e.g., EO/PO/EO block copolymers) and polycations [e.g., quaternized poly(4-vinylpyridine)] to accelerate translocation from the inside leaflet to the outside leaflet ("flip-flop") within vesicle bilayer membranes. Driving forces and mechanisms of flip-flop catalyzed by the nonionic and cationic polymers are different. The nonionics are bound to the biological membrane via incorporation of their hydrophobic blocks into the inner part of the lipid bilayer occupied by the hydrocarbon chains. The resulting scrambling of lipid molecules is favored by the overall hydrophobicity of the copolymer and the volume of its hydrophobic block. External binding of the cationic polymers, on the other hand, is driven by electrostatic interactions between the positively charged polymer units and the negatively charged lipid headgroups within the outside leaflet. Electrostatic binding favors both the flip-flop of anionic lipid from the inner to outer leaflet and the formation of anionic domains in the outer leaflet. When it is considered that less than 1% of the liposome surface is occupied by certain bound polymers, their effect upon membrane dynamics, as will be described herein, is considerable. A distinct correlation has been found between the "flippase" activity of the polymers and their ability to mediate drug permeation through biomembranes.     

Wood blocks as a carrier for Saccharomyces Cerevisiae used in the production of ethanol and fructose

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood in order to produce very enriched fructose syrup from synthetic glucose-fructose mixtures, through the selective fermentation of glucose. The kinetics of growth and ethanol production rates were studied. Several tests to assess the influence of substrate and product concentration on the production rates were carried out and appropriate rate equations were proposed as a design basis for continuous immobilized reactors. The ethanol production rate and cell growth rate were found to be inhibited linearly by both substrate and product concentrations. A maximum ethanol productivity of 21.9 g 1⁻¹ h⁻¹ was attained from a feed containing 10% (by weight) glucose and 10% (by weight) fructose. The ethanol concentration was 29.6 g 1⁻¹, the glucose conversion was 78% and a fructose yield of 99% was obtained. This resulted in a final fructose:glucose ratio of 2.7. At lower ethanol productivity levels the fructose:glucose ratio increased, as did the ethanol concentration in the effluent. The ethanol productivities obtained in this study were 33%–132% higher than those obtained in a previous study using the same system, under similar conditions, with the cells immobilized in alginate beads.     

Cholesterol as synthetic building blocks for artificial lipids with characteristic physical,chemical and biological properties

Cholesterol is one of the most widely distributed natural materials and has a unique chemical structure such as a steroid skeleton. Many types of chemical transformations of cholesterol functional groups have been developed. There is an interest in the derivatization of cholesterol and to introduce alkyl branched fatty acids into the molecule. These have found applications in the formulation of cosmetics and toiletries over the past few decades. An extraordinary interesting case is related to cholesteryl esters and their use in gene therapy delivery systems. These results can be attributed to their potential for forming cell‐mimic membranes, because cholesterol is the most important building block of living cell membranes. In terms of organic synthesis, cholesterol is a strategically useful material. A typical case is remote functionalization by chemical reactions or by biocatalysis. In the future, cholesterol should be considered as a key compound, a building block for the construction of artificial lipid‐like membranes by self‐assembly. Also, as cholesterol is one of the members of the fat and oil family, fat and oil chemists should study and develop cholesterol chemistry even further.     

Cholesterol metabolism in the baboon

Studies on the transport of exogenous and endogenous cholesterol in the baboon have been carried out using¹⁴C-acetate,¹⁴C-mevalonic acid, or³H-cholesterol. The results suggest the following characteristics of cholesterol metabolism in the baboon: the serum α- and β-lipoproteins show no preference in the transport of exogenous or endogenous cholesterol; the half-life (t_1/2) of disappearance of cholesterol from the whole animal varied between 31 and 50 days; the daily rate of whole body cholesterol biosynthesis in the baboon (47 mg/kg/day) is comparable with that rate observed in the human being (18–41 mg/kg/day); after administration of¹⁴C-acetate,¹⁴C-mevalonic acid, or³H-cholesterol to baboons, newly synthesized cholesterol esters appear to be incorporated to a greater extent into the serum α-lipoproteins.     

Etching effects of ethanol on multi-walled carbon nanotubes

The morphology and microstructure of multi-walled carbon nanotubes (MWCNTs) were modified using ethanol as a mild gas reactant. The etching by OH radicals and deposition of C radicals on the carbon nanotubes were considered to be responsible for the modification of the MWCNT structures and the formation of new carbon nanostructures. The effects of etching and deposition on the MWCNTs were confirmed by using methanol as another gas reactant; this molecule has a higher ratio of hydroxyl radicals to carbon atoms than ethanol. In addition, water vapor, containing no carbon atoms in the molecule, was also applied to etch the MWCNTs as a weak oxidant which resulted in stronger etching effects on the MWCNTs than methanol and ethanol.     

Effect of Dietary Cholesterol and Cholesterol Oxides on Blood Cholesterol,Lipids, and the Development of Atherosclerosis in Rabbits

Two studies were conducted to determine the effects of dietary cholesterol (CHO) and cholesterol oxides (COPs) on the development of atherosclerosis and the changes in fatty acid and blood characteristics in rabbits. In the first study, forty male New Zealand white rabbits were divided into 5 groups and fed commercial rabbit chow with no added CHO or COPs, 1 g CHO, 0.9 g CHO + 0.1 g COPs, 0.8 g CHO + 0.2 g COPs, or 0.5 g CHO + 0.5 g COPs per kg diet. In the second study, 24 male New Zealand White rabbits were divided into 3 groups and fed a diet containing 2 g CHO, 1.6 g CHO + 0.4 g COPs, or 1.2 g CHO + 0.8 g COPs per kg diet. All diets induced atherosclerotic lesions in the rabbits’ ascending thoracic aorta. The serum CHO and triglyceride levels (p < 0.05) increased significantly with the increased levels of CHO in the diets. Dietary CHO or COPs did not influence high-density lipoprotein CHO levels. The ratio of saturated fatty acid to unsaturated fatty acid increased as the level of dietary CHO and COPs increased.     

Cholesterol turnover in the central nervous system

Immature rat brains were examined for a metabolite of cholesterol which could conceivably form in the central nervous system as a result of cholesterol turnover. A basic assumption was made that an acidic product would be formed, preferentially by oxidative degradation of the cholesterol side chain. Chemical fractionation of brain tissue and thin-layer chromatography of an appropriate acidic fraction indicated the presence of a monohydroxy steroidal acid(s) which remains to be positively identified. Preliminary tracer experiments did not clarify the origin of the unidentified metabolite.     

In vitro and in vivo effects of ethanol on the formation of endoperoxide metabolites in rat platelets

Preincubation of rat platelet-rich plasma (PRP) with ethanol resulted in dose-dependent inhibition of the formation of endoperoxide metabolites (EPM) when the PRP was aggregated by collagen suspension. The inhibition was manifested at concentrations normally attainable in blood of rats or humans by tolerable amounts of ethanol ingestion. Paradoxically, chronic ingestionof ethanol caused enhanced synthesis of EPM in platelets, indicating that the inhibitory effect of ethanol would be temporary, and that it can be reversed as soon as ethanol is eliminated. The level of arachidonic acid in platelet phospholipids of rats fed the ethanol diet was not different from that of the control, indicating that availability of immediate precursor acid would not be a factor for the enhanced synthesis of EPM in the ethanol group. This result suggested that platelets from rats subjected to chronic ethanol ingestion become hyperactive in synthesizing EPM through an unknown mechanism. When citrated whole blood was incubated in the presence of collagen suspension, amounts of EPM synthesized in the ethanol group were not different from those of the control group, but this was due to significant reduction of platelet counts in the ethanol group. whether the effect of ethanol on other tissues would be similar to that on platelets is unknown. It is tempting to speculate that some of the pathological changes resulting from alcoholism might be mediated through the effect of ethanol on EPM formation.     

Phospholipid monolayer coated microfabricated electrodes to model the interaction of molecules with biomembranes

The hanging mercury (Hg) drop electrode (HMDE) has a classical application as a tool to study adsorption and desorption processes of surface organic films due to its: (a) atomically smooth surface and, (b) hydrophobicity at its potential of zero charge. In this study we report on a replacement of the HMDE for studying supported organic layers in the form of platinum (Pt) working electrodes fabricated using lithography techniques on which a thin film of Hg is electrodeposited. These wafer-based Pt/Hg electrodes are characterised and compared to the HMDE using rapid cyclic voltammetry (RCV) and show similar capacitance-potential profiles while being far more mechanically stable and consuming considerably less Hg over their lifetime of several months. The electrodes have been used to support self-assembled phospholipid monolayers which are dynamic surface coatings with unique dielectric properties. The issue of surface contamination has been solved by regenerating the electrode surface prior to phospholipid coating by application of extreme cathodic potentials more negative than −2.6 V (vs. Ag/AgCl). The phospholipid coated electrodes presented in this paper mimic one half of a phospholipid bilayer and exhibit interactions with the biomembrane active drug molecules chlorpromazine, and quinidine. The magnitudes of these interactions have been assessed by recording changes in the capacitance-potential profiles in real time using RCV at 40 V s⁻¹ over potential ranges >1 V. A method for electrode coating with phospholipids with the electrodes fitted in a flow cell device has been developed. This has enabled sequential rapid cleaning/coating/interaction cycles for the purposes of drug screening and/or on-line monitoring for molecules of interest.     

Vectorial insertion of bacteriorhodopsin for directed orientation assays in various polymeric biomembranes

Here we demonstrate the rapid reconstitution and preserved functionality of vectorially-inserted bacteriorhodopsin from Halobacterium Halobium in ABA block copolymer thin films. We have utilized a triblock copolymeric membrane that simulates biological systems as a matrix to enable protein refolding to observe the activity of bacteriorhodopsin in purple membrane form as well as in individual molecules. In addition to observing protein-polymer compatibility properties using polymers of varying lengths (4 vs. 8.4 nm in height) and compositions (UV-cross-linkable methacrylate or acrylate ended), we have observed that vectorial insertion of the proteins, and hence, the directionality of proton pumping is dependent upon the pH of the medium to manipulate the asymmetric charge density of the polymer membrane during reconstitution.     

A procedure for the simultaneous determination of lipid and protein in biomembranes and other biological samples

Very small sample sizes frequently become the limiting factor in biochemical and biomembrane studies in which routine quantification of protein and bulk lipids are required. The procedure described here allows the simultaneous determination of protein and lipid without initial, multiple aliquots. The method is based on the quantitative precipitation of proteins from a defined hexane/isopropanol mixture. The liquid phase resulting after decanting and concentrating to dryness can then be used to assay the lipid content directly. Quantitative assay of protein can be achieved after resuspension of the pelleted material by addition of sodium dodecyl sulfate (0.1%) and deoxycholate (1%). The method is also applicable to other types of lipid- and protein-containing samples with a broad range of protein/lipid ratios and lipid compositions, as they occur, for example, in serum lipoproteins.     

Cholesterol synthesis in the pathogenesis of lithocholic acid-induced cholestasis

Lithocholic acid (LCA)-induced intrahepatic cholestasis is associated with increased de novo synthesis of hepatic cholesterol and augmented cholesterol content of the liver cell plasma membrane fraction enriched in bile canalicular complexes (BCM). To determine whether inhibition of cholesterol synthesis could prevent LCA-induced cholestasis, adult male Wistar rats were treated daily with the hypocholesterolemic agents, clofibrate (250 mg/kg) or mevinolin (25 mg/kg), for one, two or four days. After bile duct cannulation and bile collection for one hr, the animals were injected intravenously with 120 μmoles/kg of LCA or its carrier (albumin). Cholesterol synthesis was measured in liver homogenates, and its contribution to the BCM was estimated. LCA reduced bile flow by 51%, 35% and 25% after clofibrate pretreatment for one, two and four days, respectively, and by 51%, 30% and 42% in mevinolin-pretreated animals after one, two and four days. In control animals, cholesterol synthesis and the contribution of newly synthesized cholesterol in the BCM were increased after LCA injection. However, despite that cholesterol synthesis and the contribution of newly synthesized cholesterol in the BCM were reduced in drug-pretreated rats, LCA injection caused a relative increase in these parameters of a magnitude similar to that observed in controls. Thus, the ability of LCA injection to augment de novo cholesterol synthesis and its transport to the BCM may be an important pathogenetic step in the development of cholestasis.     

Cholesterol metabolism in gnotobiotic gerbils

Germfree gerbils were associated with a murine-derived hexaflora which produced only minor changes in the primary bile acid pattern of rats. These hexaflora-associated gerbils had relatively small ceca (4% of body weight) and reproduced well. Although serum cholesterol levels of both conventional and hexaflora-associated gerbils increased in response to dietary cholesterol, the hexaflora-associated gerbil showed a greater elevation in serum cholesterol than the conventional gerbil when maintained on a diet containing 0.1% cholesterol. This increase in serum cholesterol manifested itself almost totally in the very low density lipoprotein and low density lipoprotein fractions. The fecal bile acids of the hexaflora-associated gerbil were largely deconjugated, but very little further modification of either cholic or chenodeoxycholic acid had taken place. The data suggest that in the absence of elements of the intestinal microflora that can express a bile acid-modifying potential, and particularly a 7-α-dehydroxylating capacity, catabolism of cholesterol to bile acids is reduced, and cholesterol accumulates in the very low density and low density serum lipoprotein fractions.     

The application of the laser induced proton pulse for studying electroneutral ion exchange across biomembranes

An electroneutral ion exchange across a membrane cannot generate an electric signal. Yet on a molecular level the reaction consists of steps where surface charges are formed and neutralised. Thus with proper synchronisation and time resolution the electrosilence should be broken.

Using the laser induced proton pulse as synchronizing perturbation, we monitored the capacitance current associated with H⁺---Na⁺ exchange by monensin impregnated in a phospholipid membrane.

A global kinetic analysis, based on numerical solution of coupled non linear differential rate equations, reconstructed the observed current and the charge dynamics on each side of the membrane.     

HDL Cholesterol Efflux and Serum Cholesterol Loading Capacity Alterations Associate to Macrophage Cholesterol Accumulation in FH Patients with Achilles Tendon Xanthoma

Achilles tendon xanthoma (ATX) formation involves macrophage cholesterol accumulation within the tendon, similar to that occurring in atheroma. Macrophage cholesterol homeostasis depends on serum lipoprotein functions, namely the high-density lipoprotein (HDL) capacity to promote cell cholesterol efflux (cholesterol efflux capacity, CEC) and the serum cholesterol loading capacity (CLC). We explored the HDL-CEC and serum CLC, comparing 16 FH patients with ATX to 29 FH patients without ATX. HDL-CEC through the main efflux mechanisms mediated by the transporters ATP binding cassette G1 (ABCG1) and A1 (ABCA1) and the aqueous diffusion (AD) process was determined by a cell-based radioisotopic technique and serum CLC fluorimetrically. Between the two groups, no significant differences were found in terms of plasma lipid profile. A trend toward reduction of cholesterol efflux via AD and a significant increase in ABCA1-mediated HDL-CEC (+18.6%) was observed in ATX compared to no ATX patients. In ATX-presenting patients, ABCG1-mediated HDL-CEC was lower (−11%) and serum CLC was higher (+14%) compared to patients without ATX. Considering all the patients together, ABCG1 HDL-CEC and serum CLC correlated with ATX thickness inversely (p = 0.013) and directly (p < 0.0001), respectively. In conclusion, lipoprotein dysfunctions seem to be involved in ATX physiopathology and progression in FH patients.     

Thermally induced conformational and structural disordering in polyethylene crystal studied by near-infrared spectroscopy

Thermally induced structural and conformational changes in polyethylene (PE) samples were explored by using near-infrared (NIR) spectroscopy. The differences in the temperature-dependent structural disordering process among six PE samples were depicted by monitoring the intensities of NIR bands characteristic of orthorhombic crystalline phase. The temperature dependency of bands in the NIR region that have been considered to be due to orthorhombic crystalline lattice was compared to that of a band at 1378 cm⁻¹ due to the methyl symmetric bending mode. The intensity decrease of the band in the mid-infrared (MIR) region seems to sensitively reflect the overall disordering of orthorhombic crystalline structure. As a result of this study, the intensity decrease of the bands in the NIR spectral region was found to proceed at lower temperature than that of the band at 1378 cm⁻¹. This finding suggests the status of orthorhombic crystalline structure probed by the intensity of the band at 1378 cm⁻¹ and that by the “crystalline” bands in the NIR spectral region may not be identical. The NIR spectra were further analyzed by two-dimensional (2D) correlation spectroscopy to provide the in-depth analysis of NIR bands. The 2D correlation spectroscopy has detected the presence of two NIR bands at 4342 and 4290 cm⁻¹ due to orthorhombic crystalline phase and those at 5840 and 5640 cm⁻¹ due to amorphous phase. The hetero-spectral 2D correlation analysis was carried out between the NIR spectral region of 4365-4240 cm⁻¹ and the well-established MIR spectral region for CH₂ wagging deformation region of 1390-1240 cm⁻¹, where bands due to nonplanar conformer are detected. This approach allowed us to determine NIR bands, which behave in a way similar to MIR bands originating from conformational defect sequences that exist in the orthorhombic crystalline lattice, the amorphous domain and the chain fold regions. As a result of the hetero-spectral 2D NIR-MIR correlation spectroscopic studies on the development of conformational defect sequence in three types of PE samples, it was concluded that the intensity of a band at 4265 cm⁻¹ changes in the same manner as the MIR bands at 1368, 1353 and 1308 cm⁻¹ assignable to gtg, gg and gtg′ (kink) conformations. This finding means that the state of conformational disorder in PE crystal can be studied by monitoring the intensity of the NIR band at 4265 cm⁻¹. The use of NIR spectroscopy makes it possible to directly probe the degree in the formation of conformational defect sequences in thick PE products typically produced in industry, which cannot be studied by MIR spectroscopy. This paper thus provides in-depth fundamental understandings on NIR spectra of PE as well as the results of our study regarding structural and conformational changes in PE crystals probed by NIR spectroscopy.     

Effect of Dietary Cholesterol and Fat on Cell Cholesterol Transfer to Postprandial Plasma in Hyperlipidemic Men

Postprandial chylomicrons are potent ultimate acceptors of cell membrane cholesterol and are believed to accelerate reverse cholesterol transport (RCT). We compared the effects of meals rich in polyunsaturated fat (PUFA) and either high (605 mg) or low (151 mg) in cholesterol and a meal rich in dairy fat (DF) in the form of cream on net in vitro transport of red blood cell (RBC) membrane cholesterol to 4 and 6 h postprandial plasma in eight normotriglyceridemic (NTG-H) and eight hypertriglyceridemic (HTG-H) men with mild to moderate hypercholesterolemia. In HTG-H men, cell cholesterol accumulation in 6-h postprandial plasma was significantly (P = 0.02) less after the PUFA-HC meal compared with the other meals. The significant (P < 0.001) increase in cell plus endogenous cholesterol accumulation in the triglyceride-rich lipoprotein (TRL) fraction of 4 h postprandial plasma incubated with RBC was significantly (P = 0.007) higher after the PUFA-HC meal compared with DF meal in HTG-H men. In NTG-H men, cholesterol accumulation in plasma and plasma lipoproteins in the presence and absence of RBC was not significantly affected by the type of meal ingested. These data suggest that addition of large amounts of cholesterol to a PUFA meal may impair diffusion-mediated transport of cell membrane cholesterol to postprandial plasma and that replacing DF with PUFA in a meal increases postprandial lipemia and may potentially increase cholesterol accumulation in atherogenic postprandial TRL in HTG-H men.     

Cholesterol: A Key in the Pathogenesis of Alzheimer's Disease

Herein we highlight recent advances in our understanding of the role of cholesterol in Alzheimer′s disease (AD). It has been proposed that cholesterol could enhance the risk of AD, and the interaction between cholesterol and amyloid‐β peptide 42 (Aβ42) has been studied extensively, yet until recently, the specific interaction mechanisms between them and how this affects Aβ42 aggregation had not yet been fully explored and had remained ambiguous. Vendruscolo and co‐workers addressed these issues in their recent article entitled “Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes” (Habchi et al., Nat. Chem. 2018 , 10, 673). In this article, the authors revealed the mechanism behind cholesterol‐catalyzed Aβ42 aggregation, providing the potential to address the molecular origins of AD, thereby opening a new avenue for effective AD therapy.