Incubation of fibroblasts derived from patients with type-C Niemann-Pick disease with low density lipoprotein results in excessive intracellular accumulation of unesterified cholesterol. Cytochemical techniques revealed that this abnormal cholesterol accumulation is associated not only with a massive storage of cholesterol in lysosomes but also with a premature cholesterol enrichment of the Golgi complex. Cholesterol appeared also in the Golgi complex of some normal fibroblasts after 24 hr of low density lipoprotein loading. These findings indicate that components of the Golgi complex play a role in the intracellular translocation of exogenously derived cholesterol and that disruptions of the cholesterol transport pathway at the Golgi may, in part, be responsible for the deficiency in cholesterol utilization in type-C Niemann-Pick fibroblasts. Typb

Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant.Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzymelinked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (antilgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-flxing antibodies to cholesterol can be readily obtained.Cholesterol has been widely studied as a fundamental and ubiquitous constituent of cell membranes and lipoproteins (1,2). Despite occasional reports that conjugates of cholesterol can be immunogenic (3)(4)(5), because of its widespread distribution and important biological roles cholesterol has generally been assumed to be a nonimmunogenic or poorly immunogenic molecule. However, since 1977 several laboratories have reported activation of the classical and alternative pathways of complement by cholesterol (6-11). The exact mechanism (or mechanisms) of complement activation by cholesterol has not been determined. In one case human IgG antibodies to cholesterol were implicated in the activation of the classical pathway (9). On the other hand, it has been proposed that nonimmune activation of the alternative pathway of rabbit complement (7) or human complement (8, 9) also occurs. The issue of the immunogenicity of cholesterol is an important one, since antibodies to cholesterol have been reported to protect against induced atherosclerosis in rabbits (4). On the other hand, activation of the classical pathway of complement by cholesterol has been proposed as a possible mechanism in the pathogenesis of atherosclerosis (6,9,11).In previous studies we induced antibodies to the phospholipid constituents of lipid model membranes (liposomes) (reviewed in ref. 12). Production of antibodies to liposomes was achieved by utilizing lipid A as a powerful adjuvant in the liposomal lipid bilayer. In the present work we used lipid A as an adjuvant to induce antibodies to liposomes containing high concentrations of cholesterol. We found that cholesterol is an excellent immunogen and that murine monoclonal antibodies to cholesterol are eas...

Abstract. It has recently been reported that 8S clathrin trimers or "triskelions" form larger 27S oligomers upon dialysis into low ionic strength buffers (Prasad, K., R. E. Lippoldt, H. Edelhoch, and M. S. Lewis, 1986, Biochemistry, 25:5214-5219). Here, deep-etch electron microscopy of the 27S species reveals that they are closed tetrahedra composed of four clathrin triskelions. This was determined by two approaches. First, standard quick-freezing and freeze-etching of unfixed 27S species suspended in 2 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 5.9, yielded unambiguous images of tetrahedra that measured 33 nm on each edge. Second, the technique of freeze-drying molecules on mica (Heuser, J. E., 1983, J. Mol. Biol., 169:155-195) was modified to overcome the low affinity of mica in 2 mM MES, by pretreating the mica with polylysine. Thereafter, 27S species adsorbed avidly to it and collapsed into characteristic configurations containing four globular domains, each linked to the others by three ,',,33-nm struts. The globular domains look like vertices of deep-etched clathrin triskelions and the links, numbering 12 in all, look like four sets of triskelion legs. New light scattering and equilibrium centrifugation data confirm that 27S polymer is four times as massive as one clathrin triskelion. We conclude that in conditions that do not favor the formation of standard clathrin cages, low affinity interactions lead to closed, symmetrical assemblies of four triskelions, each of which assumes a unique puckered, straight-legged configuration to create the edges of a tetrahedron. Tetrahedra are similar in construction to the cubic octomers of clathrin recently found in ammonium sulfate solutions (Sorger, P. K., R. A. Crowther, J. T. Finch, and B. M. E Pearse, 1986, J. Cell Biol., 103:1213-1219) but are still smaller, involving only half as many clathrin triskelions. W P'HEN clathrin-coated vesicles are exposed to 0.5 M Tris pH 7 (13) or 2 M urea (2, 31), their polygonal cages depolymerize into 8S components. These molecules have been named "triskelions" due to their threelegged pinwheel shape (27). Triskelions dissociate further in SDS into three 180-kD and three ~o30-kD polypeptides (14,17,18,22,24,27), suggesting that each of their three "legs" is composed of one heavy and one light chain. Upon dialysis back into physiological ionic strength buffers, especially when the buffers are slightly acidic and when divalent cations are present, triskelions reassemble spontaneously into empty "cages" that approximate the size and shape of the original vesicle coats (1,4,13,28,31,32). Such empty cages sediment at •150-300S (17-21, 25, 27, 28). If the dialysis conditions are altered, two smaller clathrin polymers will form, one sedimenting at ,'-,42S and one at --27S. The 42S form occurs only when the dialysis solution also contains >10% saturated ammonium sulfate. Structural studies have shown that this species is a cube composed of eight triskelia (26). In contrast, the 27S polymer apparently only forms when the ...

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The mechanisms of cell volume regulation in molluscs have been examined in detail in only a few species. While generalizations must be made with some caution until additional species have been tested, the following mechanism has emerged. When stressed with a decreased external salinity the cells of osmoconforming bivalves swell for a period of time and then recover toward the original cell volume. The volume recovery is effected by an efflux of intracellular free amino acids which act as osmotic solute. While the drop in external osmolality initiates the efliux, the duration of the efflux (restoration of normal amino acid permeability) relies on the activity of a Ca2+ -Mg2+ requiring ATPase.In addition, cell volume regulation and the amino acid efflux are accompanied by an alteration in the number of membrane associated particles per unit of membrane area. While the relationship between the ATPase activity, membrane associated particles and amino acid permeability is not yet clear, there is good evidence that the mechanism of cell volume control resides with the cell membrane in these animals.

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