The interaction of the brominated flame retardant: tetrabromobisphenol A with phospholipid membranes

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy [partition coefficient (Log K(p))=5.7+/-0.7]. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 microM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.     

Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak

TBBPA (tetrabromobisphenol A) is currently the most widely used type of BFR (brominated flame retardant) employed to reduce the combustibility of a large variety of electronic and other manufactured products. Recent studies have indicated that BFRs, including TBBPA, are bio-accumulating within animal and humans. BFRs including TBBPA have also been shown to be cytotoxic and potentially endocrine-disrupting to a variety of cells in culture. Furthermore, TBBPA has specifically been shown to cause disruption of Ca2+ homoeostasis within cells, which may be the underlying cause of its cytotoxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform-specific inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) (apparent K(i) 0.46-2.3 microM), thus we propose that TBBPA inhibition of SERCA contributes in some degree to Ca2+ signalling disruption. TBBPA binds directly to the SERCA without the need to partition into the phospholipid bilayer. From activity results and Ca2+-induced conformational results, it appears that the major effect of TBBPA is to decrease the SERCA affinity for Ca2+ (increasing the K(d) from approx. 1 microM to 30 microM in the presence of 10 microM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the SERCA and this quenching can be reversed by BHQ [2,5-di-(t-butyl)-1,4-hydroquinone] and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the SERCA.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Linking membrane physical properties and low temperature tolerance in arthropods

Maintenance of membrane fluidity is of crucial importance in ectotherms experiencing thermal changes. This maintenance has in ectotherms most often been indicated using indirect measures of biochemical changes of phospholipid membranes, which is then assumed to modulate the physico-chemical properties of the membrane. Here, we measure bending rigidity characterizing the membrane flexibility of re-constituted membrane vesicles to provide a more direct link between membrane physical characteristics and low temperature tolerance. Bending rigidity of lipid bilayers was measured in vitro using Giant Unilamellar Vesicles formed from phospholipid extracts of the springtail, Folsomia candida. The bending rigidity of these membranes decreased when exposed to 0.4 vol% ethanol (0.23 mM/L). Springtails exposed to ethanol for 24 h significantly increased their cold shock tolerance. Thus, by chemically inducing decreased membrane rigidity, we have shown a direct link between the physico-chemical properties of the membranes and the capacity to tolerate low temperature in a chill-susceptible arthropod.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

Influence of Phospholipid Species on Membrane Fluidity: A Meta-analysis for a Novel Phospholipid Fluidity Index

Generalized membrane lipid composition determinants of fluidity have been widely investigated, including phospholipid/cholesterol ratio and unsaturation index. Individual phospholipids differ in their physical characteristics, including their interaction with cholesterol and level of unsaturation, emphasizing the importance of examining their individual influence on membrane fluidity. Thus, the purpose of this study was to examine the dominant phospholipids of biological membranes (phosphatidylcholine, PC; phosphatidylethanolamine, PE; sphingomyelin, SM) through a meta-analysis to assess the validity of an inclusive phospholipid fluidity index (PFI = PC/(PE + SM)) as a determinant for membrane fluidity (expressed as polarization of fluorescent probe 1,6 diphenyl-1,3,5-hexatriene) in comparison to previous phospholipid ratios (PC/PE and PC/SM). The results demonstrate that all indices significantly predicted membrane fluidity at 25°C (based on 10–13 data points). In contrast, only PFI approached significance when predicting membrane fluidity at 37°C (P = 0.10 based on five points). As a result, PFI appears to be the only phospholipid index close to significantly predicting membrane fluidity at mammalian physiological temperature. Because this meta-analysis only assessed studies using mammalian membranes, future work should experimentally assess the validity of the PFI utilizing membranes from mammals and a variety of other species and tissues at their respective physiological temperatures.     

Teratogenic effects of tetrabromobisphenol A on Xenopus tropicalis embryos

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant and a known thyroid disruptor. We reported exposing Xenopus tropicalis embryos (NF10) to 0.01, 0.1 or 1 mg/L of TBBPA with or without 70 µg/L triiodothyronine (T₃). Compared with the controls, 1 mg/L of TBBPA significantly reduced the body length of embryos after 24, 36 and 48 h of exposure. Embryos treated with TBBPA showed multiple malformations, including: abnormal eyes, skin hypopigmentation, enlarged proctodaeum, narrow fins and pericardial edemas. The effect of abnormal eyes manifested itself in the loss of pigmentation, reduction in size, or absence of external eyes. The degree of eye malformations was quantified with the index of eye malformations (IEM) with 0 being normal and 3 being severe. In the 1 mg/L TBBPA treatment groups, the incidence of total malformations (ITM) was 68–93%, and IEM was 0.8–0.9. T₃ showed no teratogenic effects on embryos, but it significantly enhanced TBBPA-induced teratogenic effects. In the T₃ + 1 mg/L TBBPA treatment groups, ITM was 91–99%, and IEM was 1.8–1.9. Histological observations showed that the retinas were generally smaller, and the lenses were underdeveloped or even absent. These results indicate that TBBPA at relatively high concentration has teratogenic effects on X. tropicalis embryos. The results also suggest that thyroid hormone signaling might be involved in TBBPA induced-teratogenicity.     

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

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

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.     

Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.     

Characterization of the ternary mixture of sphingomyelin, POPC, and cholesterol: support for an inhomogeneous lipid distribution at high temperatures

A ternary lipid mixture of palmitoyl-oleoyl-phosphatidylcholine (POPC), palmitoyl-erythro-sphingosylphosphorylcholine (PSM), and cholesterol at a mixing ratio of 37.5:37.5:25 mol/mol/mol was characterized using fluorescence microscopy, ²H NMR, and electron paramagnetic resonance spectroscopy. The synthetic PSM provides an excellent molecule for studying the molecular properties of raft phases. It shows a narrow phase transition at a temperature of 311 K and is commercially available with a perdeuterated sn-2 chain. Fluorescence microscopy shows that large inhomogeneities in the mixed membranes are observed in the coexistence region of liquid-ordered and liquid-disordered lipid phases. Above 310 K, no optically detectable phase separation was shown. Upon decrease in temperature, a redistribution of the cholesterol into large liquid-ordered PSM/cholesterol domains and depletion of cholesterol from liquid-disordered POPC domains was observed by ²H NMR and electron paramagnetic resonance experiments. However, there is no complete segregation of the cholesterol into the liquid-ordered phase and also POPC-rich domains contain the sterol in the phase coexistence region. We further compared order parameters and packing properties of deuterated PSM or POPC in the raft mixture at 313 K, i.e., in the liquid crystalline phase state. PSM shows significantly larger ²H NMR order parameters in the raft phase than POPC. This can be explained by an inhomogeneous interaction of cholesterol between the lipid species and the mutual influence of the phospholipids on each other. These observations point toward an inhomogeneous distribution of the lipids also in the liquid crystalline phase at 313 K. From the prerequisite that order parameters are identical in a completely homogeneously mixed membrane, we can determine a minimal microdomain size of 45-70 nm in PSM/POPC/cholesterol mixtures above the main phase transition of all lipids.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Sterols impart significant changes to the biophysical properties of lipid bilayers. In this regard the impact of cholesterol on membrane organization and dynamics is particularly well documented and serves for comparison with other sterols. However, the factors underlying the molecular evolution of cholesterol remain enigmatic. To this end, cholesterol attenuates membrane perturbation by the so-called antimicrobial peptides (AMPs), produced ubiquitously by eukaryotic cells to combat bacterial infections by compromising the permeability barrier function of the microbial target membranes. In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Interestingly, sterol concentration dependent effects on the membrane association of these peptides were observed. At X_Sterol = 0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol > lanosterol ≥ ergosterol, and cholesterol > lanosterol > ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cholesterol may have derived from the ability to protect the AMP-secreting host cell from the membrane damaging action of the antimicrobial peptides.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

The Structure of the C-Terminal Domain of the Pro-Apoptotic Protein Bak and Its Interaction with Model Membranes

Bak is a pro-apoptotic protein widely distributed in different cell types that is associated with the mitochondrial outer membrane, apparently through a C-terminal hydrophobic domain. We used infrared spectroscopy to study the secondary structure of a synthetic peptide (⁺₃HN-¹⁸⁸ILNVLVVLGVVLLGQFVVRRFFKS²¹¹-COO^-) with the same sequence as the C-terminal domain of Bak. The spectrum of this peptide in D₂O buffer shows an amide I′ band with a maximum at 1636 cm⁻¹, which clearly indicates the predominance of an extended β-structure in aqueous solvent. However, the peptide incorporated in multilamellar dimyristoylphosphatidylcholine (DMPC) membranes shows a different amide I′ band spectrum, with a maximum at 1658 cm⁻¹, indicating a predominantly α-helical structure induced by its interaction with the membrane. It was observed that through differential scanning calorimetry the transition of the phospholipid model membrane was broadened in the presence of the peptide. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) in fluid DMPC vesicles showed that increasing concentrations of the peptide produced increased polarization values, which is compatible with the peptide being inserted into the membrane. High concentrations of the peptide considerably broaden the phase transition of DMPC multilamellar vesicles, and DPH polarization increased, especially at temperatures above the T_c transition temperature of the pure phospholipid. The addition of peptide destabilized unilamellar vesicles and released encapsulated carboxyfluorescein. These results indicate that this domain is able to insert itself into membranes, where it adopts an α-helical structure and considerably perturbs the physical properties of the membrane.     

Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l_o-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φ_o, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φ_o was related to the assumed partition coefficient k_p of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k_p = 0 (probe completely excluded from liquid-ordered phase), Φ_o = 0.16 and with k_p = 0.2, Φ_o = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.     

The influenced of PAC, zeolite, and Moringa oleifera as biofouling reducer (BFR) on hybrid membrane bioreactor of palm oil mill effluent (POME)

The main objective of this work was to determine the effectiveness of various biofouling reducers (BFRs) to operational condition in hybrid membrane bioreactor (MBR) of palm oil mill effluent (POME). A series of tests involving three bench scale (100 L) hybrid MBR were operated at sludge retention times (SRTs) of 30 days with biofouling reducer (BFR). Three different biofouling reducers (BFRs) were powdered actived carbon (PAC), zeolite (Ze), and Moringa oleifera (Mo) with doses of 4, 8 and 12 g L⁻¹ respectively were used. Short-term filtration trials and critical flux tests were conducted. Results showed that, all BFRs successfully removed soluble microbial products (SMP), for PAC, Ze, and Mo at 58%, 42%, and 48%, respectively. At their optimum dosages, PAC provided above 70% reductions and 85% in fouling rates during the short-term filtration and critical flux tests.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997