Specific binding of amyloid-beta-protein to IMR-32 neuroblastoma cell membrane.

In flow cytometry using two detecting methods, we have found that amyloid-beta-protein(1-40) [Abeta(1-40)] has high affinity to IMR-32 neuroblastoma cell membrane when it is aggregated to form beta-sheet conformation, whereas random coil small Abeta-species has low affinity. The difference in the binding ability to the cell membranes well accounts for the cytotoxicity of Abeta(1-40); namely, aggregated beta-sheet Abeta(1-40) gives cytotoxicity higher than random coil Abeta(1-40). Specific binding between Abeta(1-40) and ganglioside GM1 of the raft-like domain in lipid membrane is suggested from a surface plasmon resonance (SPR) experiment.     

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.     

Amyloid beta-peptide1-40 increases neuronal membrane fluidity: role of cholesterol and brain region

There is increasing evidence of an interaction between cholesterol dynamics and Alzheimer's disease (AD), and amyloid beta-peptide may play an important role in this interaction. Abeta destabilizes brain membranes and this action of Abeta may be dependent on the amount of membrane cholesterol. We tested this hypothesis by examining effects of Abeta1-40 on the annular fluidity (i.e., lipid environment adjacent to proteins) and bulk fluidity of rat synaptic plasma membranes (SPM) of the cerebral cortex, cerebellum, and hippocampus using the fluorescent probe pyrene and energy transfer. Amounts of cholesterol and phospholipid of SPM from each brain region were determined. SPM of the cerebellum were significantly more fluid as compared with SPM of the cerebral cortex and hippocampus. Abeta significantly increased (P < or = 0.01) annular and bulk fluidity in SPM of cerebral cortex and hippocampus. In contrast, Abeta had no effect on annular fluidity and bulk fluidity of SPM of cerebellum. The amounts of cholesterol in SPM of cerebral cortex and hippocampus were significantly higher (P < or = 0.05) than amount of cholesterol in SPM of cerebellum. There was significantly less (P < or = 0.05) total phospholipid in cerebellar SPM as compared with SPM of cerebral cortex. Neuronal membranes enriched in cholesterol may promote accumulation of Abeta by hydrophobic interaction, and such an interpretation is consistent with recent studies showing that soluble Abeta can act as a seed for fibrillogenesis in the presence of cholesterol.     

Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer's disease amyloid-beta peptide

The lipid membrane has been shown to mediate the fibrillogenesis and toxicity of Alzheimer's disease (AD) amyloid-beta (Abeta) peptide. Electrostatic interactions between Abeta40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Abeta into lipid monolayers, where Abeta exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To elucidate the molecular-scale structural details of Abeta-membrane association, we have used complementary X-ray and neutron scattering techniques (grazing-incidence X-ray diffraction, X-ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Abeta with lipid monolayers composed of either the anionic lipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or the cationic lipid 1,2-dipalmitoyl 3-trimethylammonium propane (DPTAP) at the air-buffer interface. We found that the anionic lipid DPPG uniquely induced crystalline ordering of Abeta at the membrane surface that closely mimicked the beta-sheet structure in fibrils, revealing an intriguing templated ordering effect of DPPG on Abeta. Furthermore, incubating Abeta with lipid vesicles containing the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Abeta at the membrane surface seeded fibril formation. This study provides a detailed molecular-scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Abeta in vivo. Our results implicate that the adsorption of Abeta to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated-aggregation and drive the pathogenesis of AD.     

Structure of the nucleosome core particle at 8 A resolution

The x-ray crystallographic structure of the nucleosome core particle has been determined using 8 A resolution diffraction data. The particle has a mean diameter of 106 A and a maximum thickness of 65 A in the superhelical axis direction. The longest chord through the histone core measures 85 A and is in a non-axial direction. The 1.87 turn superhelix consists of B-DNA with about 78 base pairs or 7.6 helical repeats per superhelical turn. The mean DNA helical repeat contains 10.2 +/- 0.05 base pairs and spans 35 A, slightly more than standard B-DNA. The superhelix varies several Angstroms in radius and pitch, and has three distinct domains of curvature (with radii of curvature of 60, 45 and 51 A). These regions are separated by localized sharper bends +/- 10 and +/- 40 base pairs from the center of the particle, resulting in an overall radius of curvature about 43 A. Compression of superhelical DNA grooves on the inner surface and expansion on the outer surface can be seen throughout the DNA electron density. This density has been fit with a double helical ribbon model providing groove width estimates of 12 +/- 1 A inside vs. 19 +/- 1 A outside for the major groove, and 8 +/- 1 A inside vs. 13 +/- 1 A outside for the minor groove. The histone core is primarily contained within the bounds defined by the superhelical DNA, contacting the DNA where the phosphate backbone faces in toward the core. Possible extensions of density between the gyres have been located, but these are below the significance level of the electron density map. In cross-section, a tripartite organization of the histone octamer is apparent, with the tetramer occupying the central region and the dimers at the extremes. Several extensions of histone density are present which form contacts between nucleosomes in the crystal, perhaps representing flexible or "tail" histone regions. The radius of gyration of the histone portion of the electron density is calculated to be 30.4 A (in reasonable agreement with solution scattering values), and the histone core volume in the map is 93% of its theoretical volume.     

Amyloid-beta peptide assembly: a critical step in fibrillogenesis and membrane disruption

Identifying the mechanisms responsible for the assembly of proteins into higher-order structures is fundamental to structural biology and understanding specific disease pathways. The amyloid-beta (Abeta) peptide is illustrative in this regard as fibrillar deposits of Abeta are characteristic of Alzheimer's disease. Because Abeta includes portions of the extracellular and transmembrane domains of the amyloid precursor protein, it is crucial to understand how this peptide interacts with cell membranes and specifically the role of membrane structure and composition on Abeta assembly and cytotoxicity. We describe the results of a combined circular dichroism spectroscopy, electron microscopy, and in situ tapping mode atomic force microscopy (TMAFM) study of the interaction of soluble monomeric Abeta with planar bilayers of total brain lipid extract. In situ extended-duration TMAFM provided evidence of membrane disruption via fibril growth of initially monomeric Abeta1-40 peptide within the total brain lipid bilayers. In contrast, the truncated Abeta1-28 peptide, which lacks the anchoring transmembrane domain found in Abeta1-40, self-associates within the lipid headgroups but does not undergo fibrillogenesis. These observations suggest that the fibrillogenic properties of Abeta peptide are in part a consequence of membrane composition, peptide sequence, and mode of assembly within the membrane.     

Rapid and opposite effects of cortisol and estradiol on human erythrocyte Na+,K+-ATPase activity: relationship to steroid intercalation into the cell membrane.

We determined whether two naturally occurring steroids, cortisol and 17beta-estradiol (E2), can rapidly modulate the activity of an important membrane protein, human erythrocyte (RBC) Na+,K+-ATPase, an enzyme that does not bind either hormone directly. We also determined the membrane binding locations for cortisol and E2 and their effects on membrane molecular structure and fluidity. Direct application of both steroids to intact human RBC significantly altered maximum ouabain-sensitive 86Rb uptake within 5 min: Cortisol decreased it by 24%, whereas E2 increased it by 18%. As determined by small angle x-ray diffraction, these steroids occupied distinct time-averaged binding locations in the RBC membrane, cortisol localizing near the bilayer surface, 14-29 A from the bilayer center, and E2 localizing deep within the hydrocarbon core, 0-7 A from the bilayer center. Neither steroid significantly changed overall bilayer width or membrane fluidity. These data suggest that cell membrane protein function can be altered rapidly and differentially by naturally occurring steroids. This effect did not appear to be related to the different binding locations of the steroids in the membrane or to their influence on membrane fluidity.     

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction.

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of cholesterol and monosialoganglioside (GM1) on the release and aggregation of amyloid beta-peptide from liposomes prepared from brain membrane-like lipids

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid beta peptide (Abeta)-(1-40) and Abeta-(1-42), we have examined Abeta peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing Abeta peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four Abeta-containing liposomes were investigated: CCM-lipid; liposomes with no GM1 (GM1-free lipid); those with no cholesterol (Ch-free lipid); liposomes with neither cholesterol nor GM1 (Ch-GM1-free lipid). In CCM liposomes, Abeta was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, Abeta was first released to yield a fibril structure about the membrane surface, then the membrane became disrupted resulting in the formation of small vesicles. In Ch-free lipid, a fibril structure with a phospholipid membrane-like shadow formed, but this differed from the well defined fibril structure seen for CCM-lipid. In Ch-GM1-free lipid, no fibril structure formed, possibly because of membrane solubilization by Abeta. The absence of fibril structure was noted at physiological extracellular pH (7.4) and also at liposomal/endosomal pH (5.5). Our results suggest a possible role for both Ch and GM1 in the membrane release of Abeta from brain lipid bilayers.     

β-淀粉肽(1-40)对海马神经元高电压激活钙电流的作用及银杏内酯B对该作用的影响

应用全细胞膜片钳技术探讨β-淀粉肽(1-40)(β-amyloid peptide1-40,Aβ1-40)对新鲜分离的大鼠海马CA1区锥体神经元高电压依赖性钙通道电流(high voltage-activated calcium channel current,IHVA)的作用并观察银杏内酯B(ginkgolideB,GB)对该作用的影响.利用细胞外灌流或者电极内液给药的方法,比较加药前后电流幅度的变化以判断药物是否发挥作用.细胞外给予老化处理的Aβ1-40可以浓度依赖性地增强IHVA的幅度,Aβ1-40的浓度为0.01～30 μmol/L时可分别使IHVA幅度增加(5.43±3.01)%(n=8,P＞0.05)、(10.49±4.13)%(n=11,P＞0.05)、(40.69±8.01)%(n=16,P＜0.01)、(58.32±4.85)%(n=12,P＜0.01)和(75.45±5.81)%(n=6,P＜0.01);新鲜配制的Aβ1-40对IHVA几乎没有影响(n=5,P＞0.05).L-型钙通道阻断剂nifedipine可以抵消Aβ1-40对IHVA的增强作用.Aβ1-40(1.0μmol/L)对IHVA的增强作用可以被cAMP的类似物8-Br-cAMP和腺苷酸环化酶(adenylyl cyclase,AC)的激动剂forskolin增强[分别为(66.19±5.74)%,P＜0.05和(73.21±6.90)%,P＜0.05],被蛋白激酶A(protein kinaseA,PKA)的抑制剂H-89减弱[(20.08±2.18)%,P＜0.05].GB可有效地减弱Aβ1-40对IHVA的增强作用.以上结果表明Aβ1-40可通过AC-cAMP-PKA增强IHVA引起胞内钙超载,这可能是其产生神经毒性作用的机制之一.GB可通过抑制Aβ1-40引起的异常钙离子内流对神经元起一定保护作用.     

Apolipoprotein E enhances uptake of soluble but not aggregated amyloid-beta protein into synaptic terminals

The cellular mechanism by which apolipoprotein E (apoE) affects the pathogenesis of Alzheimer's disease (AD) is not understood. We have examined the effect of apolipoprotein E on the internalization of exogenous amyloid-beta 1-40 (Abeta40) into a rat brain crude synaptosomal preparation. Abeta40 peptide in soluble (within 1 h of dilution in buffer) or aggregated (aged 4 days before dilution in buffer) form was pre-incubated with lipidated apoE then added to synaptosomes; intraterminal amyloid-beta labeling was quantified using flow cytometry following immunolabeling with the anti-Abeta (10G4) antibody. The number of Abeta-positive synaptosomes was increased ( approximately 50%) by treatment with a soluble Abeta/apoE mixture compared with treatment with soluble Abeta40 alone. However, when the Abeta was aggregated, less sodium dodecyl sulfate (SDS)-stable Abeta/apoE complex was formed and the addition of apoE decreased the number of Abeta-positive terminals. The addition of the lipoprotein-receptor related protein (LRP) antagonist receptor-associated protein (RAP) inhibited the apoE-induced increase in synaptosomal Abeta, and controls treated with trypsin and heparinase confirm intraterminal localization of the majority of the soluble Abeta. The apoE-mediated increase in Abeta labeling was confirmed in intact cells by immunocytochemistry of dorsal root ganglion (DRG) neurons. These results suggest that complex formation with apoE enhances internalization of soluble Abeta uptake into terminals.     

Early synergy between Abeta42 and oxidatively damaged membranes in promoting amyloid fibril formation by Abeta40

Oxidative lipid membrane damage is known to promote the misfolding of Abeta42 into pathological beta structure. In fully developed senile plaques of Alzheimer's disease, however, it is the shorter and more soluble amyloid beta protein, Abeta40, that predominates. To investigate the role of oxidative membrane damage in the misfolding of Abeta40, we have examined its interaction with supported lipid monolayer membranes using internal reflection infrared spectroscopy. Oxidatively damaged lipids modestly increased Abeta40 accumulation, with adsorption kinetics and a conformation that are distinct from that of Abeta42. In stark contrast, pretreatment of oxidatively damaged monolayer membranes with Abeta42 vigorously promoted Abeta40 accumulation and misfolding. Pretreatment of saturated or undamaged membranes with Abeta42 had no such effect. Parallel studies of lipid bilayer vesicles using a dye binding assay to detect fibril formation and electron microscopy to examine morphology demonstrated that Abeta42 pretreatment of oxidatively damaged membranes promoted the formation of mature Abeta40 amyloid fibrils. We conclude that oxidative membrane damage and Abeta42 act synergistically at an early stage to promote fibril formation by Abeta40. This synergy could be detected within minutes using internal reflection spectroscopy, whereas a dye-binding assay required several days and much higher protein concentrations to demonstrate this synergy.     

Interaction between amyloid beta-protein aggregates and membranes.

The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.     

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.     

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.     

Hexane dissolved in dioleoyllecithin bilayers has a partial molar volume of approximately zero

Neutron diffraction has been used to measure the amount and distribution of hexane incorporated from the vapor phase into oriented dioleoylphosphatidylcholine bilayers at 66% relative humidity. We reported earlier that hexane at low concentrations is located largely in a zone 10 A wide at the center of the bilayer [White, S. H., King, G. I., & Cain, J. E. (1981) Nature (London) 290, 161-163]. Extending these studies to high hexane concentrations, we find no readily apparent change in the volume of the hydrocarbon region of the bilayer even though more than one hexane molecule per lipid enters the region. The hexane partial molar volume in the bilayer hydrocarbon region is thus approximately zero. Within our statistical confidence limits, the partial molar volume is certainly no greater than one-third the molecular volume of the hexane. Further, analysis of the data suggests that the mass density of the bilayer is considerably less than 1 in the absence of hexane. These findings are in conflict with the assumptions usually made about lipid bilayers and their interaction with nonpolar hydrophobic molecules. In the course of these experiments, we found that standard methods of interpreting diffraction results were not suitable for our purposes. We thus developed several new methods which are summarized in the text and two appendixes. One of these methods allows us to define with precision the width of the hydrocarbon core of the bilayer. The other provides a means of calculating the effects of changes in the absolute scaling of the bilayers with changes in composition without placing the structures on an absolute scattering length density scale.     

Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.     

Structure and Dynamics of Cholesterol-Containing Polyunsaturated Lipid Membranes Studied by Neutron Diffraction and NMR

A direct and quantitative analysis of the internal structure and dynamics of a polyunsaturated lipid bilayer composed of 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0-22:6n3-PC) containing 29 mol% cholesterol was carried out by neutron diffraction, (2)H-NMR and (13)C-MAS NMR. Scattering length distribution functions of cholesterol segments as well as of the sn-1 and sn-2 hydrocarbon chains of 18:0-22:6n3-PC were obtained by conducting experiments with specifically deuterated cholesterol and lipids. Cholesterol orients parallel to the phospholipids, with the A-ring near the lipid glycerol and the terminal methyl groups 3 ? away from the bilayer center. Previously, we reported that the density of polyunsaturated docosahexaenoic acid (DHA, 22:6n3) chains was higher near the lipid-water interface. Addition of cholesterol partially redistributes DHA density from near the lipid-water interface to the center of the hydrocarbon region. Cholesterol raises chain-order parameters of both stearic acid and DHA chains. The fractional order increase for stearic acid methylene carbons C(8)-C(18) is larger, reflecting the redistribution of DHA chain density toward the bilayer center. The correlation times of DHA chain isomerization are short and mostly unperturbed by the presence of cholesterol. The uneven distribution of saturated and polyunsaturated chain densities and the cholesterol-induced balancing of chain distributions may have important implications for the function and integrity of membrane receptors, such as rhodopsin.     

Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.     

Mutant presenilin 2 transgenic mice. A large increase in the levels of Abeta 42 is presumably associated with the low density membrane domain that contains decreased levels of glycerophospholipids and sphingomyelin

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid beta protein (Abeta) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Walter, J., Grünberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313-322). Here, using a new extraction protocol, we quantitated the Abeta40 and Abeta42 levels in the Tris-saline-insoluble fraction. The insoluble Abeta levels were found to be higher than the soluble Abeta levels, and the insoluble Abeta42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble Abeta42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutant PS2 transgenic mice contained remarkably increased levels of Abeta42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of Abeta42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.