Diffusible subfibrillar aggregates of amyloid proteins are potent neurotoxins and primary suspects in amyloid diseases including Alzheimer's disease. Despite widespread interest, the molecular structures of the amyloid intermediates and the conformational conversions in amyloid misfolding are poorly understood. Here we present a molecular-level examination of sequence-specific secondary structures and supramolecular structures of a neurotoxic amyloid intermediate of the 40-residue β-amyloid (Aβ) peptide involved in Alzheimer's disease. Using solid-state NMR and electron microscopy, we show that, before fibrillization, natively unstructured monomeric Aβ is subject to large conformational changes into a spherical amyloid intermediate of 15–35 nm diameter, which has predominantly parallel β-sheet structures. Structural comparison with Aβ fibrils demonstrates that formation of this β-sheet intermediate I(β) largely defines conformational transitions in amyloid misfolding. Neurotoxicity assays on PC12 cells show that I(β) shows higher toxicity than the fibril, indicating that the β-sheet formation may trigger neurotoxicity.

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans10 cis12 (A), trans9 trans11 (B), cis9 trans11 (C), and cis9 cis11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (−80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.

Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ 9 desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.

We previously showed that sphingomyelin (SM), the second most abundant phospholipid in plasma, inhibits lecithin‐cholesterol acyltransferase (LCAT), the enzyme activity critical for HDL maturation, partly by competing with its substrate, phosphatidylcholine (PC). The physiological relevance of this inhibition, however, has not been investigated. In this study, using plasma from various genetic models of mice with naturally varying ratios of SM/PC, we show that this ratio is inversely correlated with the rate of cholesterol esterification by LCAT. Mice deficient in serine palmitoyltransferase, or SM synthase, both of which have reduced SM/PC ratios, showed significantly higher than normal LCAT activity when assayed with the endogenous substrates. On the other hand, LDL receptor deficient mice, and apo E knockout mice, both of which have high SM/PC ratios, had lower than normal LCAT activities. The enzyme levels in plasma, as assayed with an exogenous substrate, were not significantly different, except for a small decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 cholesteryl esters (CE), and higher percentage of 18:1CE, showing that in vivo, the contribution of LCAT to plasma CE is diminished while that of liver ACAT is enhanced. These results show that plasma SM is a physiological modulator of cholesterol esterification and plasma CE composition.