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Antibody light chain (LC) aggregation in vivo leads to the systemic deposition of Ig light chain domains in the form of either amyloid fibrils (AL-amyloidosis) or amorphous deposits, light-chain deposition disease (LCDD), in mainly cardiac or renal tissue and is a pathological condition that is often fatal. Molecular factors that may contribute to the propensity of LCs to aggregate in vivo, such as the protein primary structure or local environment, are intensive areas of study. Herein, we show that the aggregation of a human antibody kappa-(kappa-MJM) and lambda-(lambda-L155) light chain (1 mg/mL) can be accelerated in vitro when they are incubated under physiologically relevant conditions, PBS, pH 7.4 and 37 degrees C, in the presence of a panel of biologically relevant lipid-derived aldehydes, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), glyoxal (GLY), atheronal-A (KA), and atheronal-B (ALD). Thioflavin-T (ThT) and Congo Red (CR) binding assays coupled with turbidity studies reveal that this aldehyde-induced aggregation can be associated with alteration of protein secondary structure to an increased beta-sheet conformation. We observed that the nature of the conformational change is primarily dependent upon the lipidic aldehyde studied, not the protein sequence. Thus, the cholesterol 5,6-secosterols, KA and ALD, cause an amorphous-type aggregation which is ThT and CR negative for both the kappa-MJM and lambda-L155 light chains, whereas 4-HNE, MDA, and GLY induce aggregates that bind both ThT and CR. TEM analysis revealed that amyloid fibrils were formed during the 4-HNE-mediated aggregation of kappa-MJM and lambda-L155 light chains, whereas ALD-induced aggregates of these LCs where amorphous in nature. Kinetic profiles of LC aggregation reveal clear differences between the aldehydes, KA and ALD, causing a classic nucleated polymerization-type aggregation, with a lag phase (of approximately 150 h) followed by a growth phase that plateaus, whereas 4-HNE, MDA, and GLY trigger a seeded-type aggregation process that has no lag phase. In-depth studies of the 4-HNE-accelerated aggregation of kappa-MJM and lambda-L155 reveal a clear aldehyde concentration dependence and a process that can be inhibited by the naturally occurring osmolyte trimethylamine N-oxide (TMAO). Given these data, we feel our recently discovered paradigm of inflammatory aldehyde-induced protein misfolding may now extend to LC aggregation.

The proatherogenic properties of the cholesterol 5,6-secosterols (atheronal-A and atheronal-B), recently discovered in atherosclerotic arteries, have been investigated in terms of their effects on monocyte/macrophage function. A fluorescent analogue of atheronal-B (1) (50 microM), when cultured in either aqueous buffer (PBS) or in media containing fetal calf serum (10%), is rapidly taken-up into cultured macrophage (J774.1 or RAW 264.7) cells and accumulates at perinuclear sites (within 1 h). Co-incubation of macrophage cells (J774.1) with atheronal-A (25 microM) and atheronal-B (25 microM) when complexed with low-density lipoprotein (LDL) (100 microg/mL) leads to a significant upregulation of scavenger receptor class A (approximately 3-fold increase relative to LDL alone, p < 0.05) but not CD36, showing that cultured macrophages respond to LDL-complexed atheronals in a manner highly analogous to acetylated LDL rather than oxidized LDL. Both atheronal-A and atheronal-B in solution exhibit a dose-dependent (0-25 microM) induction of chemotaxis of cultured macrophages (p < 0.001). When complexed with LDL (100 microg/mL), atheronal-A (but not atheronal-B) induces a dose-dependent (0-25 microM, p < 0.05) upregulation of the cell-surface adhesion molecule endothelial (E)-selectin on vascular endothelial cells (HUVECs). LDL (100 microg/mL) complexed atheronal-B (25 microM) but not atheronal-A induces cultured human monocytes (THP-1) to differentiate into macrophage cell lineage. When these in vitro data are taken together with the already known effects of cholesterol 5,6-secosterols on foam cell formation and macrophage cytotoxicity, the atheronals possess biological effects that if translated to an in vivo setting could lead to the recruitment, entrapment, dysfunction, and ultimate destruction of macrophages, with the major leukocyte player in inflammatory artery disease. As such, the atheronal molecules may be a new association, in the already complex inter-relationship, between inflammation, cholesterol oxidation, the tissue macrophage, and atherosclerosis.

When phospholipase C is added to a suspension of large unilamellar vesicles of egg phosphatidylcholine, maximal rates of hydrolysis occur only after a latency period. No lag period is seen when the substrate is in the form of small (sonicated) vesicles, or of short-chain phosphatidylcholine monomers. For a given vesicle concentration, the lag time may vary as a function of Ca2+, enzyme concentration, or temperature, but activation occurs at a fixed molar fraction of diacylglycerol produced. Lag times decrease gradually with vesicle size, and also with the amount of diacylglycerol present in the bilayers when it is mixed with phospholipid prior to enzyme addition. Parallel recordings of enzyme activity and suspension turbidity reveal that in all cases the latency period ends concomitantly with the start of a process of vesicle aggregation. Both the lag time and the amount of diacylglycerol formed before activation decrease with vesicle concentration, suggesting that enzyme activation is somehow related to vesicle aggregation. The latency period of phospholipase C may be explained in terms of a hypothesis according to which (a) full enzyme activity requires the presence of membrane surface irregularities or defects, (b) the diacylglycerol generated in the lag phase produces some kind of phase separation, with the formation of diacylglycerol-rich "patches" or domains, (c) vesicles aggregate through contacts between those patches, and (d) aggregation causes (and/or increases, and/or stabilizes) the surface inhomogeneities that allow fast enzyme activity. These data and suggestions may be relevant to the process of model membrane fusion promoted by phospholipase C.

This paper presents a study on the membrane fusion activity of a 23-residue synthetic peptide, representing the N-terminus of gp41 of the human immunodeficiency virus type I (HIV-1; LAV1a strain), in a model system involving large unilamellar vesicles (LUV) composed of the negatively charged 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG). The peptide (HIVarg) induced fusion of POPG LUV as evidenced by (i) mixing of membrane lipids, (ii) mixing of aqueous vesicle contents, and (iii) an irreversible increase in vesicle size. Fusion could be induced only in the presence of millimolar concentrations of Ca2+ or Mg2+, needed for induction of vesicle aggregation; the divalent cations by themselves did not induce any fusion. The rate constant of the fusion reaction, as determined by simulation of the process according to a kinetic model, increased dramatically with the peptide-to-lipid molar ratio, indicating that the peptide was the mediator of the process. In the absence of divalent cations, the HIVarg peptide induced leakage of small molecules due to formation of pores in the membrane of single vesicles. Final extents and kinetics of this leakage process could be simulated adequately by model calculations for peptide-to-lipid ratios ranging from 1:25 to 1:750. Experiments, in which the order of peptide and Ca2+ addition to the vesicles was varied, indicated that the peptide is likely to adopt two different structures, one in the absence of Ca2+, primarily supporting leakage by formation of pores in separate vesicles, and one in the presence of Ca2+, primarily supporting fusion. Once a final structure had been established, it persisted even upon addition or removal of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane fusion, in particular the fusion of enveloped viruses, is often measured with an assay based on octadecylrhodamine (R18) fluorescence dequenching. We have studied the association of R18 with membranes and used the R18 assay to measure virus fusion in model systems and in cultured cells. The results were compared with those of an assay based on the decrease in excimer fluorescence of pyrene-labeled phospholipids. For liposomes made from premixed R18 and phosphatidylcholine (PC), R18 fluorescence quenching was proportional to the concentration of the probe up to about 4 mol %. No quenching was found at very low concentrations of R18. However, various artificial and biological membranes labeled by the addition of R18 from an ethanolic solution showed significant quenching at such low R18 concentrations. Thus, some of the R18 was not randomly distributed but likely was associated with the surface of the membranes in the form of highly quenched clusters or micelles. Moreover, in influenza virus membranes, R18 appeared highly quenched at very low concentrations, indicative of the probe interacting with viral proteins. In contrast, pyrene-labeled PC incorporated in either liposomes or reconstituted viral membranes (virosomes) showed an excimer/monomer fluorescence ratio proportional to the concentration of probe. When intracellular membrane fusion was investigated with R18-labeled influenza virus or Semliki Forest virus (SFV), fluorescence dequenching was observed in the absence of fusion, most likely due to spontaneous probe exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

The catalytic activity of phospholipase C induces fusion of pure lipid vesicles. When large unilamellar liposomes composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) are treated with phospholipase C, in the presence of 10 mM Ca2+, two enzyme effects can be distinguished: a fast one (half-time on the order of seconds) consisting mainly of vesicle-vesicle fusion and a slow one (half-time on the order of minutes) representing bulk lipid hydrolysis. The fast fusion process is inhibited by the end-product diacylglycerol, as well as by lysophosphatidylcholine and by low Ca2+ concentrations. The temperature dependence of enzyme activity (phospholipid hydrolysis), vesicle aggregation, and vesicle fusion (mixing of aqueous contents) has been separately studied. Enzyme activity and vesicle aggregation rates increase monotonically with temperature, while an optimum temperature is found for vesicle fusion, depending on liposome composition and assay conditions. The presence of diacylglycerol incorporated to the membrane (up to 10 mol %) does not produce any fusion effect even at temperatures as high as 80 degrees C: in situ diacylglycerol production by the enzyme appears to be required. The data are interpreted in support of a hypothesis according to which a "fusion intermediate" would be required, depending (among others) on bilayer composition, temperature, and Ca2+ concentration, for vesicle fusion to occur.

Large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (50:25:25 mole ratio) were treated with phospholipase C. The early stages of phospholipid cleavage are accompanied by mixing of bilayer lipids (monitored by dequenching of octadecylrhodamine fluorescence) and leakage-free mixing of vesicle contents [measured by using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX)]. These results are interpreted in terms of vesicle fusion induced by the catalytic activity of phospholipase C. The use of sonicated unilamellar vesicles decreases the lag time, but does not modify the amplitude, of the fusion process. The presence of both phosphatidylethanolamine and cholesterol appears to be essential for measurable fusion effects to occur with low levels of phospholipid hydrolysis. Optimal fusion rates are observed with about 10-20 enzyme molecules per large unilamellar vesicle. This system of catalytically induced liposome fusion may be of relevance for the interpretation of physiological membrane fusion processes.