VariVis: a visualisation toolkit for variation databases

Background  

With the completion of the Human Genome Project and recent advancements in mutation detection technologies, the volume of data available on genetic variations has risen considerably. These data are stored in online variation databases and provide important clues to the cause of diseases and potential side effects or resistance to drugs. However, the data presentation techniques employed by most of these databases make them difficult to use and understand.     

The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.     

Membrane cholesterol content modulates activation of volume-regulated anion current in bovine endothelial cells

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-beta-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-beta-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening.     

Sensitivity of volume-regulated anion current to cholesterol structural analogues

Depletion of membrane cholesterol and substitution of endogenous cholesterol with its structural analogues was used to analyze the mechanism by which cholesterol regulates volume-regulated anion current (VRAC) in endothelial cells. Depletion of membrane cholesterol enhanced the development of VRAC activated in a swelling-independent way by dialyzing the cells either with GTPgammaS or with low ionic strength solution. Using MbetaCD-sterol complexes, 50-80% of endogenous cholesterol was substituted with a specific analogue, as verified by gas-liquid chromatography. The effects of cholesterol depletion were reversed by the substitution of endogenous cholesterol with its chiral analogue, epicholesterol, or with a plant sterol, beta-sitosterol, two analogues that mimic the effect of cholesterol on the physical properties of the membrane bilayer. Alternatively, when cholesterol was substituted with coprostanol that has only minimal effect on the membrane physical properties it resulted in VRAC enhancement, similar to cholesterol depletion. In summary, our data show that these channels do not discriminate between the two chiral analogues of cholesterol, as well as between the two cholesterols and beta-sitosterol, but discriminate between cholesterol and coprostanol. These observations suggest that endothelial VRAC is regulated by the physical properties of the membrane.     

Pitavastatin increases ABCA1-mediated lipid efflux from Fu5AH rat hepatoma cells

ATP binding cassette A1 (ABCA1) is responsible in vivo for the formation of HDL by promoting the lipidation of apoprotein A-I (apoA-I) via cholesterol and phospholipid efflux from the liver. Treatment of patients with statins produces an increase in HDL plasma level, but the underlying mechanism is not completely understood. In this work we investigated the ability of pitavastatin to modulate ABCA1-mediated efflux from Fu5AH rat hepatoma cells, that here we demonstrate to express functional ABCA1 upon treatment with 22OH/cRA. In both basal and ABCA1 expressing cells pitavastatin 0.1-50microM induced a dose-dependent increase in cholesterol efflux to apoA-I; this effect was reversed by mevalonate or geranyl geraniol. A stimulatory effect was also observed on phospholipid efflux. Similar results were obtained with compactin, suggesting a class-related effect of statins. These results indicate a potential mechanism for the improvement in HDL plasma profile observed in patients treated with statins.     

Effects of apolipoprotein A-I on ATP-binding cassette transporter A1-mediated efflux of macrophage phospholipid and cholesterol: formation of nascent high density lipoprotein particles

The mechanism of formation of high density lipoprotein (HDL) particles by the action of ATP-binding cassette transporter A1 (ABCA1) is not defined completely. To address this issue, we monitored efflux to apoA-I of phosphatidylcholine (PC), sphingomyelin (SM), and unesterified (free) cholesterol (FC) from J774 macrophages, in which ABCA1 is up-regulated, and investigated the nature of the particles formed. The various apoA-I/lipid particles appearing in the extracellular medium were separated by gel filtration chromatography. The presence of apoA-I in the extracellular medium led to the simultaneous formation of more than one type of poorly lipidated apoA-I-containing particle: there were 9- and 12-nm diameter particles containing approximately 3:1 and 1:1 phospholipid/FC (mol/mol), respectively, which were present together with 6-nm monomeric apoA-I. Removal of the C-terminal alpha-helix (residues 223-243) of apoA-I reduced phospholipid and FC efflux and prevented formation of the 9- and 12-nm HDL particles; the apoA-I variant formed larger particles that eluted in the void volume. FC loading of the J774 cells also led to the formation of larger apoA-I-containing particles that were highly enriched in FC. Besides creating HDL particles, ABCA1 mediated release of larger (20-450-nm diameter) FC-rich particles that were not involved in HDL formation and that are probably membrane vesicles. These particles contained 1:1 PC/SM in contrast to the HDL particles, which contained 2:1 PC/SM. This is consistent with lipid raft and non-raft plasma membrane domains being involved primarily in ABCA1-mediated vesicle release and nascent HDL formation, respectively.     

Pulmonary abnormalities due to ABCA1 deficiency in mice

Mice gene targeted for ATP-binding cassette transporter A1 (ABCA1; Abca1(-/-)) have been shown to have low-serum high-density lipoprotein and abnormal lung morphology. We examined alterations in the structure and function of lungs from -/- mice (DBA1/J). Electron microscopy of the diseased mouse lung revealed areas of focal disease confirming previous results (47). Lipid analysis of the lung tissue of -/- mice showed a 1.2- and 1.4-fold elevation in total phospholipid (PL) and saturated phosphatidylcholine, respectively, and a marked 50% enrichment in total cholesterol content predominantly due to a 17.5-fold increase in cholesteryl ester compared with wild type (WT). Lung surfactant in the -/- mice was characterized by alveolar proteinosis (161%), a slight increase in total PL (124%), and a marked increase in free cholesterol (155%) compared with WT. Alveolar macrophages were enriched in cholesterol (4.8-fold) due to elevations in free cholesterol (2.4-fold) and in cholesteryl ester (14.8-fold) compared with WT macrophages. More PL mass was cleared from the alveolar space of -/- mice lungs, measured using intratracheal installation of (3)H-PL liposomes. Compared with WT mice, the Abca1(-/-) mice demonstrated respiratory distress with rapid, shallow breathing. Thus the lungs of mice lacking ABCA1 protein demonstrated abnormal morphology and physiology, with alveolar proteinosis and cholesterol enrichment of tissue, surfactant, and macrophages. The results indicate that the activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function.     

Induction of cellular cholesterol efflux to lipid-free apolipoprotein A-I by cAMP

In the present study apolipoprotein-mediated free cholesterol (FC) efflux was studied in J774 macrophages having normal cholesterol levels using an experimental design in which efflux occurs in the absence of contributions from cholesteryl ester hydrolysis. The results show that cAMP induces both saturable apolipoprotein (apo) A-I-mediated FC efflux and saturable apo A-I cell-surface binding, suggesting a link between these processes. However, the EC50 for efflux was 5-7-fold lower than the Kd for binding in both control and cAMP-stimulated cells. This dissociation between apo A-I binding and FC efflux was also seen in cells treated for 1 h with probucol which completely blocked FC efflux without affecting apo A-I specific binding. Thus, cAMP-stimulated FC efflux involves probucol-sensitive processes distinct from apo A-I binding to its putative cell surface receptor. FC efflux was also dramatically stimulated in elicited mouse peritoneal macrophages, suggesting that cAMP-regulated apolipoprotein-mediated FC efflux may be important in cholesterol homeostasis in normal macrophages. The presence of a cAMP-inducible cell protein that interacts with lipid-free apo A-I was investigated by chemical cross-linking of 125I-apo A-I with J774 cell surface proteins which revealed a Mr 200 kDa component when the cells were treated with cAMP.     

Mechanism of the hepatic lipase induced accumulation of high-density lipoprotein cholesterol by cells in culture

Hepatic lipase can enhance the delivery of high-density lipoprotein (HDL) cholesterol to cells by a process which does not involve apoprotein catabolism. The incorporation of HDL-free (unesterified) cholesterol, phospholipid, and cholesteryl ester by cells has been compared to establish the mechanism of this delivery process. Human HDL was reconstituted with 3H-free cholesterol and [14C]sphingomyelin, treated with hepatic lipase in the presence of albumin to remove the products of lipolysis, reisolated, and then incubated with cultured rat hepatoma cells. Relative to control HDL, modification of HDL with hepatic lipase stimulated both the amount of HDL-free cholesterol taken up by the cell and the esterification of HDL-free cholesterol but did not affect the delivery of sphingomyelin. Experiments utilizing HDL reconstituted with 14C-free cholesterol and [3H]cholesteryl oleoyl ether suggest that hepatic lipase enhances the incorporation of HDL-esterified cholesterol. However, the amount of free cholesterol delivered as a result of treatment with hepatic lipase was 4-fold that of esterified cholesterol. On the basis of HDL composition, the cellular incorporation of free cholesterol was about 10 times that which would occur by the uptake and degradation of intact particles. The preferential incorporation of HDL-free cholesterol did not require the presence of lysophosphatidylcholine. To correlate the events observed at the cellular level with alterations in lipoprotein structure, high-resolution, proton-decoupled 13C nuclear magnetic resonance spectroscopy (90.55 MHz) was performed on HDL3 in which the cholesterol molecules were replaced with [4-13C]cholesterol by particle reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS)