| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Selective increase in cholesterol at atherosclerosis-susceptible aortic sites after short-term cholesterol feeding Schwenke, D. C. (1995), Arterioscler Thromb Vasc Biol 15(11): 1928-37. Abstract: In rabbits, the aortic arch and branch sites of the descending thoracic and abdominal aortas are susceptible to atherosclerosis. This study investigated the hypothesis that the reported focal increase in LDL concentration and mean residence time at susceptible aortic sites after feeding cholesterol for 4 to 8 days precede atherosclerotic change as indicated by increased aortic cholesterol concentration. Cholesterol concentrations for all aortic sites of normal rabbits were similar (approximately equal to 2.8 mumol/g). No change in aortic cholesterol concentration could be detected after feeding cholesterol for 8 days. However, after feeding cholesterol for 12 and 16 days, cholesterol concentrations for abdominal branch sites were increased compared with abdominal branch sites of normal rabbits (4.47 +/- 0.50, n = 8, and 4.85 +/- 0.33, n = 11, mumol/g, respectively, versus 2.87 +/- 0.27, n = 12, mumol/g; P <.025 and P <.005, respectively). In contrast, the cholesterol concentration of atherosclerosis-resistant nonbranch abdominal aorta was unchanged after feeding cholesterol for 16 days and was much less than that of the branch sites (2.72 +/- 0.12 versus 4.85 +/- 0.33, mumol/g, n = 11; P <.001). Cholesterol concentrations for other susceptible sites were also increased after feeding cholesterol for 12 and 16 days. Cholesterol concentrations for susceptible sites were linearly related to a combined measure of duration and extent of hypercholesterolemia (P <.001 to P <.0001), whereas no such relationship could be detected for resistant sites. Most (59% to 93%) of the cholesterol accumulating in susceptible aortic sites after feeding cholesterol for 12 and 16 days was nonesterified, suggesting that the increased cholesterol concentration did not reflect development of foam cells or the insudation of plasma lipoproteins. This study suggests that the reported focal increases in LDL concentration and mean residence time at susceptible aortic sites during cholesterol feeding precede atherosclerosis. |
| Selective inhibition of cholesterol biosynthesis in brain cells by squalestatin 1 Crick, D. C., J. Suders, et al. (1995), J Neurochem 65(3): 1365-73. Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC50 = 37 nM), pig brain (IC50 = 21 nM), and C6 glioma cells (IC50 = 35 nM) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis-isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of 3HF-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab 1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with 3Hmevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC50 = 3-5 microM) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors Parker, R. A., R. W. Clark, et al. (1990), J Lipid Res 31(7): 1271-82. Abstract: Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or 14Cacetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it. |
| Selective inhibition of mammalian lanosterol 14 alpha-demethylase: a possible strategy for cholesterol lowering Walker, K. A., D. J. Kertesz, et al. (1993), J Med Chem 36(15): 2235-7. |
| Selective removal of cholesterol by plasmapheresis and the progression of atherosclerosis Nomura, H., P. S. Malchesky, et al. (1992), J Clin Apher 7(4): 194-200. Abstract: There is a strong correlation of plasma cholesterol levels with the risk of coronary heart diseases as shown by epidemiologic studies. This study was undertaken to evaluate the effect of plasma cholesterol lowering on the progression of atherosclerosis in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit. The effect of cholesterol lowering, which was accomplished by thermofiltration (on-line plasma separation with plasma filtration at 39 degrees C) was evaluated by comparison between treated and untreated control groups. Thermofiltration reduced significantly the mean plasma level of total cholesterol (284 vs. 655 mg/dl, P = 0.0005) and the percent aortic area occupied by atherosclerotic plaque (15.0 vs. 44.2%, P = 0.0003). The total lipid and cholesterol contents in the aortas in the treated group were also significantly lower than those in the control group. Microscopically, thickness measurements of the lesions showed that the mean thickness of the fibrous cap and the ratio of the thickness of the intima to that of the media were smaller for the treated group than the control group. This study demonstrated the slowing or stopping of the progression of atherosclerosis by lowering the plasma total cholesterol level in WHHL rabbits. |
| Selective removal of cholesterol esters in an arteriosclerotic region of blood vessels with a free-electron laser Awazu, K., A. Nagai, et al. (1998), Lasers Surg Med 23(4): 233-7. Abstract: BACKGROUND AND OBJECTIVE: In advanced atheromatous atherosclerosis, a large amount of lipids, particularly cholesterol esters, accumulates on the arterial wall. The selective removal of cholesterol esters accumulated in the intracellular or extracellular spaces has clinical significance. In the present work, the authors investigated the removal of cholesterol esters by using a free-electron laser (FEL) in an arteriosclerotic region. STUDY DESIGN/MATERIALS AND METHODS: Thin films of cholesteryl oleate and albumin and the cross section of a rabbit artery were placed on an inverted microscope stage, and the changes caused by the FEL irradiation of 5.75 microm and 6.1 microm, with 1.5-3 mW on average, were monitored continuously by using a CCD camera in real time. RESULTS AND CONCLUSION: FEL irradiation at a wavelength of 5.75 microm, which is a stretching vibrational mode of the ester, was able to ablate cholesterol esters without affecting albumin. It can also remove cholesterol esters from rabbit arteriosclerotic arterial walls. |
| Selective screening for high cholesterol in Australian general practice: the Newcastle Cholesterol Prediction Study Kinlay, S. and R. F. Heller (1990), J Gen Intern Med 5(1): 1-8. Abstract: OBJECTIVE: To test whether information simply obtained from adults can identify those who are likely to have blood cholesterol levels higher than 6.5 mmol/L (250 mg/dl). DESIGN: Cross-sectional study. SETTING: Seven general practices in the lower Hunter Region of Australia. PARTICIPANTS: Of 693 men and women 25 to 65 years old attending the general practices, 616 (89%) participated. Twelve pregnant women and four without blood samples were excluded, leaving 600 subjects (208 men, 392 women). MEASUREMENTS AND MAIN RESULTS: Twenty-two percent of subjects had blood cholesterol higher than 6.5 mmol/L (250 mg/dl). In women, high cholesterol was mainly found among those over 40 years of age, but in men high cholesterol was more evenly spread across the different age groups. Stepwise logistic regression identified age, history of hypertension, and a past history of heart attack as significant independent predictors of high cholesterol. A simple model developed from these variables identified 81% of men and women (95% CI = 72-90%) with high cholesterol while testing only 49% of the population (95% CI = 44-54%). This model was developed in a random subset of 331 of the 600 subjects, and when applied to the remaining 269 subjects, it identified 77% of those with high cholesterol (95% CI = 67-87%) after testing 48% (95% CI = 42-54%). CONCLUSIONS: Selective screening using this simple model can identify adults who are likely to have high blood cholesterol and could complement case-finding or provide an alternative high-risk strategy for communities that cannot afford to screen all individuals. |
| Selective stimulation of caveolar endocytosis by glycosphingolipids and cholesterol Sharma, D. K., J. C. Brown, et al. (2004), Mol Biol Cell 15(7): 3114-22. Abstract: Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mbeta-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-alpha activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCalpha, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane. |
| Selective thyroid hormone receptor-beta activation: a strategy for reduction of weight, cholesterol, and lipoprotein (a) with reduced cardiovascular liability Grover, G. J., K. Mellstrom, et al. (2003), Proc Natl Acad Sci U S A 100(17): 10067-72. Abstract: Few treatments for obesity exist and, whereas efficacious therapeutics for hyperlipidemia are available, further improvements are desirable. Thyroid hormone receptors (TRs) regulate both body weight and cholesterol levels. However, thyroid hormones also have deleterious effects, particularly on the heart. The TR beta subtype is involved in cholesterol lowering and possibly elevating metabolic rate, whereas TR alpha appears to be more important for control of heart rate (HR). In the current studies, we examined the effect of TR beta activation on metabolic rate and HR with either TR alpha 1-/- mice or the selective TR beta agonist KB-141 in mice, rats, and monkeys. 3,5,3'-triiodi-l-thyronine (T3) had a greater effect on increasing HR in WT than in TR alpha-/- mice (ED15 values of 34 and 469 nmol/kg/day, respectively). T3 increased metabolic rate whole body oxygen consumption (MVO2) in both WT and TR alpha-/- mice, but the effect in the TR alpha 1-/- mice at the highest dose was half that of the WT mice. Thus, stimulation of MVO2 is likely due to both TR alpha and -beta. T3 had equivalent potency for cholesterol reduction in WT and TR alpha-/- mice. KB-141 increased MVO2 with selectivities of 16.5- and 11.2-fold vs. HR in WT and TR alpha 1-/- mice, respectively. KB-141 also increased MVO2 with a 10-fold selectivity and lowered cholesterol with a 27-fold selectivity vs. HR in rats. In primates, KB-141 caused significant cholesterol, lipoprotein (a), and body-weight reduction (up to 7% after 1 wk) with no effect on HR. TR beta-selective agonists may constitute a previously uncharacterized class of drugs to treat obesity, hypercholesterolemia, and elevated lipoprotein (a). |
| Selective uptake from LDL is stimulated by unsaturated fatty acids and modulated by cholesterol content in the plasma membrane: role of plasma membrane composition in regulating non-SR-BI-mediated selective lipid transfer Seo, T., W. Velez-Carrasco, et al. (2002), Biochemistry 41(25): 7885-94. Abstract: We previously reported that unsaturated fatty acids stimulated low-density lipoprotein (LDL) particle uptake in J774 macrophages by increasing LDL receptor activity. Since free fatty acids (FFA) also change plasma membrane properties, a putative cholesteryl ester (CE) acceptor for selective uptake (SU), we questioned the ability of FFA to modulate SU from LDL. Using (3)Hcholesteryl ether/(125)I-LDL to trace CE core and whole particle uptake, we found that oleic acid and eicosapentaenoic acid, but not saturated stearic acid, increased SU by 30% over control levels. An ACAT inhibitor, Dup128, abolished FFA effects on SU, indicating that increased SU by FFA was secondary to changes in cell-free cholesterol (FC). Consistent with these observations, ACAT inhibition increased cell FC and reduced LDL SU by half. The important role of plasma membrane composition was further demonstrated in that beta-cyclodextrin- (beta-CD-) mediated FC removal from the plasma membrane increased SU from LDL and was further stimulated by U18666A, a compound that inhibits FC transport between lysosomes and the plasma membrane. In contrast, cholesterol-saturated beta-CD markedly reduced LDL SU. In contrast to LDL SU, oleic acid, ACAT inhibition, U18666A, or beta-CD had no effects on HDL SU. Moreover, HDL SU was inhibited by antimouse SR-BI antibody by more than 50% but had little effect on LDL SU. In C57BL/6 mice fed a high fat diet, plasma FFA levels increased, and SU accounted for an almost 4-fold increased proportion of total cholesterol delivery to the arterial wall. Taken together, these data suggest that LDL SU is mediated by pathways independent of SR-BI and is influenced by plasma membrane FC content. Moreover, in conditions where elevated plasma FFA occur, SU from LDL can be an important mechanism for cholesterol delivery in vivo. |
| Selective uptake of cholesteryl ester from low density lipoprotein is involved in HepG2 cell cholesterol homeostasis Charest, M. C., D. Rhainds, et al. (1999), Eur J Biochem 263(2): 402-9. Abstract: Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein-CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL-CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a specific antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL-CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr-deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL-CE selective uptake is coupled to cell cholesterol homeostasis. |
| Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: complexation and stabilization of insulin Akiyoshi, K., S. Kobayashi, et al. (1998), J Control Release 54(3): 313-20. Abstract: Insulin (Ins) spontaneously and easily complexed with the hydrogel nanoparticle of hydrophobized cholesterol-bearing pullulan (CHP) in water. The complexed nanoparticles (diameter 20-30 nm) thus obtained formed a very stable colloid. The thermal denaturation and subsequent aggregation of Ins were effectively suppressed upon complexation. The complexed Ins was significantly protected from enzymatic degradation. Spontaneous dissociation of Ins from the complex was barely observed, except in the presence of bovine serum albumin. The original physiological activity of complexed Ins was preserved in vivo after i.v. injection. |
| Self-diffusion nuclear magnetic resonance, microstructure transitions, and solubilization capacity of phytosterols and cholesterol in Winsor IV food-grade microemulsions Spernath, A., A. Yaghmur, et al. (2003), J Agric Food Chem 51(8): 2359-64. Abstract: Microemulsions are of growing interest to the food industry as vehicles for delivering and enhancing solubilization of natural food supplements with nutritional and health benefits. The incorporation of molecular phytosterols, cholesterol-lowering agents, in food products is of great interest to the food industry. In this work is demonstrated the use of water dilutable food-grade microemulsions consisting of ethoxylated sorbitan ester (Tween 60), water, R-(+)-limonene, ethanol, and propylene glycol as vehicles for enhancing the phytosterols solubilization. Phytosterols were solubilized up to 12 times more than the dissolution capacity of the oil R-(+)-limonene for the same compounds. The solubilization capacity of phytosterols and cholesterol along a dilution line in a pseudo-ternary phase diagram on this dilution line the weight ratio of R-(+)-limonene/ethanol/Tween 60 is constant at 1:1:3 was correlated to the microstructure transitions along the dilution line. Structural aspects were studied by self-diffusion NMR spectroscopy. The ability of phytosterols to compete with cholesterol for penetration into bile salt micelles in the gut may be limited to rich aqueous systems (O/W microemulsion). |
| Self-reported adherence to cholesterol-lowering medication in patients with familial hypercholesterolaemia: the role of illness perceptions Senior, V., T. M. Marteau, et al. (2004), Cardiovasc Drugs Ther 18(6): 475-81. Abstract: BACKGROUND: The objectives of this study are to describe levels of adherence to cholesterol-lowering medication and to identify predictors of adherence in patients with familial hypercholesterolaemia (FH). DESIGN: Descriptive questionnaire study. METHODS: 336 adults patients with FH attending one of five outpatient lipid clinics in South East England underwent a clinical assessment by a nurse and completed a questionnaire. The questionnaire assessed self-reported adherence to cholesterol-lowering medication, anxiety, depression, and patient perceptions of heart disease. RESULTS: Overall, participants reported high levels of medication adherence, although 63% reported some level of non-adherence. Total medication adherence (never deviating from the regimen) was more likely to be reported by older participants, those with no formal educational qualifications, those with a personal history of cardiovascular disease, those with a lower total cholesterol level, and those with a greater difference between untreated cholesterol levels and current cholesterol levels. The illness perceptions associated with reported total adherence were lower perceived risk of raised cholesterol, perceiving greater control over FH, and perceiving genes and cholesterol to be important determinants of a heart attack. Emotional state was not associated with medication adherence. In logistic regression analysis, predictors of total medication adherence were having personal history of cardiovascular disease, having no formal qualifications, and perceiving genes to be important determinants of a heart attack. CONCLUSIONS: Both clinical factors and patients' illness perceptions were associated with self-reported cholesterol-lowering medication adherence. The association with illness perceptions was small and many of these associations may be a consequence, rather than a cause, of greater adherence. Given this, intervention strategies aimed at helping patients' to establish routines for medication taking may be more effective in increasing adherence than interventions designed to alter perceptions related to taking statins. |
| Self-reported frequency of serum cholesterol testing, awareness of test results, and laboratory cholesterol values in two South Carolina communities Heath, G. W., E. Vartiainen, et al. (1993), Public Health Rep 108(4): 465-70. Abstract: Self-reported frequency of cholesterol testing and awareness of test results were collected from 5,246 adults 18 years and older in two semirural communities in South Carolina. Serum cholesterol was also measured for about 60 percent of this group. More than half of these persons had serum cholesterol values greater than 200 milligrams per deciliter (mg per dL) and 21 percent had values greater than 240 mg per dL. One-third of the population had had their cholesterol level measured within the past year; 40 percent reported that their cholesterol level had never been measured. Among persons whose cholesterol was 240 mg per dL or more, 39 percent reported that their cholesterol had never been measured or that they did not know if it had been measured, 37 percent reported that their cholesterol had been measured but that they were not told that it was high, and 18 percent reported that their cholesterol had been measured and that they were advised to reduce it. Among persons whose cholesterol was 200 mg per dL or more, and who reported that they had cardiovascular disease, 25 percent reported that they were advised to reduce their cholesterol. These results emphasize the need to increase the proportion of the population who have had their cholesterol level measured, who know their test results, and who have been properly counseled about the results. |
| Seminal plasma choline phospholipid-binding proteins stimulate cellular cholesterol and phospholipid efflux Moreau, R., P. G. Frank, et al. (1999), Biochim Biophys Acta 1438(1): 38-46. Abstract: Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins (BSP-A1/-A2, BSP-A3 and BSP-30-kDa, collectively called BSP proteins) that potentiate sperm capacitation induced by high-density lipoproteins. We showed recently that BSP proteins stimulate cholesterol efflux from epididymal spermatozoa and play a role in capacitation. Here, we investigated whether or not BSP proteins could stimulate cholesterol and phospholipid efflux from fibroblasts. Cells were radiolabeled (3Hcholesterol or 3Hcholine) and the appearance of radioactivity in the medium was determined in the presence of BSP proteins. Alcohol precipitates of bovine seminal plasma (designated crude BSP, cBSP), purified BSP-A1/-A2, BSP-A3 and BSP-30-kDa proteins stimulated cellular cholesterol and choline phospholipid efflux from fibroblasts. Efflux mechanistic differences were observed between BSP proteins and other cholesterol acceptors. Preincubation of BSP-A1/-A2 proteins with choline prevented cholesterol efflux, an effect not observed with apolipoprotein A-I. Also, the rate of BSP-induced efflux was rapid during the first 20 min, but leveled off thereafter in contrast to a relatively slow, but constant, rate of cholesterol efflux mediated by apolipoprotein A-I, apolipoprotein A-I-containing reconstituted lipoproteins (LpA-I) and high-density lipoproteins. These results indicate that fibroblasts are a good cell model to study the mechanism of lipid efflux mediated by BSP proteins. |
| Semiologic value of LDL cholesterol and apolipoprotein B in risk of atherosclerosis Frey, J. and R. Couderc (1998), Ann Biol Clin (Paris) 56(5): 517-20. |
| Semliki forest virus budding: assay, mechanisms, and cholesterol requirement Lu, Y. E. and M. Kielian (2000), J Virol 74(17): 7708-19. Abstract: All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells. |
| Sensitive assay for cholesterol in biological membranes reveals membrane-specific differences in kinetics of cholesterol oxidase Crockett, E. L. and J. R. Hazel (1995), J Exp Zool 271(3): 190-5. Abstract: Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure cholesterol, originally described for whole cells (Heider and Boyett 1978 J. Lipid Res., 19:514-518; Gamble et al. 1978 J. Lipid Res., 19:1068-1070) and serum (Huang et al. 1975 Clin Chem., 21:1605-1608). This method has a detection limit of 0.3 microgram cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order. |
| Sensitivity of volume-regulated anion current to cholesterol structural analogues Romanenko, V. G., G. H. Rothblat, et al. (2004), J Gen Physiol 123(1): 77-87. Abstract: 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. |