Protoporphyrin IX sensitized photohemolysis: stoichiometry of the reaction and repair by reduced glutathione

Protoporphyrin IX acts as a sensitizer in the photohemolysis of bovine erythrocytes by binding to a limited number of membrane sites. The cholesterol-specific antibiotic lucensomycin competes with protoporphyrin in binding to the membranes. The possibility of cholesterol peroxidation as a primary event in photohemolysis is supported by the repairing effect of exogenous cholesterol and by the increased susceptibility of the photosensitized erythrocytes to lucensomycin. Glutathione, if present within the erythrocyte, postpones the onset of lysis; if added after irradiation, it may repair the membrane damage and prevent hemolysis. This effect appears to be related to a redox reaction (possibly involving glutathione peroxidase) between reduced glutathione and the cholesterol peroxide molecules.     

Role of the sterol superlattice in the partitioning of the antifungal drug nystatin into lipid membranes

Nystatin isolated from Streptomyces is a polyene antibiotic that is frequently used in the treatment and prophylaxis of fungal infections. Here, the fractional sterol concentration dependencies of the partition coefficient for partitioning of nystatin into ergosterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), cholesterol/DMPC, ergosterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC), and ergosterol/POPC/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylethano lam ine (POPE) multilamellar vesicles have been determined fluorometrically at 37 degrees C using approximately 0.3-1.0 mol % sterol concentration increments over a wide concentration range (e.g., 18-54 mol % sterol). This unconventional approach of varying membrane sterol content, in contrast to previous studies using large sterol concentration increments (e.g., 10 mol %), leads to a striking observation. The partition coefficient of nystatin changes dramatically with membrane sterol content in a well-defined alternating manner, displaying a local minimum at or very close to the critical sterol mole fractions (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % sterol) predicted for sterols regularly distributed in either hexagonal or centered rectangular superlattices. In ergosterol/DMPC bilayers, for example, there is a >3-fold increase in nystatin partitioning with a minute change (approximately 1 mol %) in sterol content on either side of the critical sterol mole fraction, 25.0 mol %. These results provide semifunctional evidence supporting the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073]. More importantly, these results reveal a new membrane phenomenon, that is, that nystatin partitioning is affected by the extent of sterol regular distribution in the plane of the membrane. This phenomenon occurs not only in saturated (e.g., DMPC) but also in unsaturated (e.g., POPC) lipid membranes, and persists in the presence of polar headgroup heterogeneity (e.g., POPC/POPE). This membrane property points to a new method for studying the interactions of polyene antibiotics with sterol-containing membranes, and the need to consider the membrane sterol content of the target cells when administering nystatin or other polyene antibiotics.     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40-50% of the existing cholesterol sites. As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9-10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

Steroidogenic acute regulatory protein (StAR) is a sterol transfer protein

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and beta-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP2), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heat-treated microsomes as well as to heat- and trypsin-treated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.     

Basis for rapid efflux of biosynthetic desmosterol from cells

Previous work shows that the efflux of biosynthetic desmosterol from cells is three times more efficient than that of cholesterol. To explain this difference, we labeled CHO-K1 cells with [3H]acetate precursor and measured sterols in the whole cells, plasma membranes and caveolae, and those released to high density lipoprotein (HDL3). The [3H]desmosterol-to-[3H]cholesterol ratio was similar in the plasma membrane and whole cells but was greater in HDL3, suggesting that the more efficient efflux of desmosterol is due to more rapid desorption from the plasma membrane. The ratio in caveolae was similar to that in whole cells, arguing against selective delivery of desmosterol to caveolae as an explanation for the more rapid efflux of this sterol. Additionally, to demonstrate that the enhanced release of desmosterol was not due to enhanced intracellular cycling, we made vesicles from CHO-cell plasma membranes labeled with [3H]desmosterol or [14C]cholesterol, and the rapid release of desmosterol was demonstrated in this system. To characterize sterol efflux from a simple lipid bilayer system, we measured the transfer of cholesterol and desmosterol between large unilamellar vesicles (LUV), and found that desmosterol transferred two to three times more rapidly than cholesterol. A similar differential was seen when HDL3 or low density lipoprotein (LDL) served as the acceptor. These results show that the greater efflux efficiency of biosynthetic desmosterol can be attributed to more efficient desorption from the plasma membrane, and that this difference is a property of the sterols' association with the lipid bilayer. In vivo, the rapid efflux of biosynthetic sterol intermediates, followed by efficient delivery to the liver, may constitute an important mechanism for preventing various types of pathology associated with these materials.     

Filipin-induced lesions in planar phospholipid bilayers imaged by atomic force microscopy

Filipin is a macrolide polyene with antifungal activity belonging to the same family of antibiotics as amphotericin B and nystatin. Despite the spectroscopy and electron microscopy studies of its interaction with natural membranes and membrane model systems, several aspects of its biochemical action, such as the role of membrane sterols, remain to be completely understood. We have used atomic force microscopy (AFM) to study the effect of filipin on dipalmitoylphosphatidylethanolamine bilayers in the presence and absence of cholesterol. The bilayers were prepared by Langmuir-Blodgett deposition over mica and imaged under water. It was shown that filipin-induced lesions could only be found in membranes with cholesterol. In close agreement with electron microscopy results, we have reported the presence of densely packed circular protrusions in the membrane with a mean diameter of 19 nm (corrected for convolution with AFM tip) and 0.4 nm height. Larger circular protrusions (90 nm diameter and 2.5 nm height) and doughnut-shaped lesions were also detected. These results demonstrate that filipin-induced lesions in membranes previously observed by electron microscopy are not biased by artifacts resulting from sample preparation. Filipin aggregates in aqueous solution could also be imaged for the first time. These polydisperse spherical structures were observed in samples with and without cholesterol.     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Erythrocyte membrane lateral sterol domains: a dehydroergosterol fluorescence polarization study

Structural domains of cholesterol and their regulation in the erythrocyte membrane are poorly understood. Dehydroergosterol fluorescence polarization change was used to continuously monitor the kinetics of sterol exchange and sterol domain size in erythrocyte ghost membranes. Direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements was obtained without separation of donor and acceptor membranes. Three important observations were made. First, sterol exchange between small unilamellar vesicles (SUV) with the same cholesterol/phospholipid ratio as the erythrocyte membrane (1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol = 1:1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t1/2 = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t1/2 = 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, with a t1/2 of days. Second, the substitution of erythrocyte ghosts for SUV as an acceptor significantly altered the kinetic parameters of sterol exchange from donor SUV, graphically showing that both the properties of the acceptor and spontaneous desorption of cholesterol from the donor SUV influenced spontaneous cholesterol transfer. Third, studies of exchange between erythrocyte ghosts revealed multiple kinetic pools of sterol differing from those in the SUV: 4 +/- 2% of total sterol was rapidly exchangeable, with t1/2 = 32 +/- 9 min; 29 +/- 3% of total sterol was very slowly exchangeable, with t1/2 = 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, with a t1/2 of days.     

Physical connections between feeder cells and recipient normal mammary epithelial cells

Amphotericin B (AmB) is the most widely used polyene antibiotic to treat systemic fungal infections which affect an increasing number of immunocompromised patients. It is generally thought that AmB forms pores within the fungi membranes by interacting with ergosterol, the main sterol of fungi. However, it also interacts with the cholesterol contained in mammalian cells, hence its toxicity. In order to have a better understanding of the interactions prevailing between AmB and sterols, differential scanning calorimetry was used to study various mixtures incorporating from 6.5 to 25 mol% of AmB in pure dipalmitoylphosphatidylcholine (DPPC) vesicles and in ergosterol- or cholesterol-containing DPPC vesicles. The sterol concentration was kept constant at 12.5 mol% with respect to the phospholipid. Our results show that three phases co-exist when AmB is dispersed in the pure phospholipid. One corresponds to the phospholipid phase alone. The two others are characterised by a broad transition at temperatures higher than the main transition temperature of the pure phospholipid, corresponding to the drug in interaction with the aliphatic chains of the lipid. The fact that the transition temperatures of these additional components are higher than that of the pure phospholipid suggests that AmB interacts strongly with the aliphatic chains of the lipid, consistent with the idea prevailing in the literature that AmB by itself may form pores in a lipid matrix. When AmB interacts with cholesterol-containing bilayers the thermograms also present three components. Upon increasing the concentration of AmB, though, an important broadening of these components is observed which is explained in terms of destabilisation of the organisation of the aliphatic chains. The situation is strikingly different if ergosterol is present in the lipid matrix. The thermograms remain unmodified as the concentration of AmB is increased and a broad transition, now involving only two components when the thermograms are decomposed, is observed. An analysis of the results shows that various interacting units, e.g. AmB+DPPC and (AmB+ergosterol)+DPPC, are present within the membrane. These units involve the phospholipid and hence contribute to its structurisation. The important differences between the thermograms obtained with the ergosterol- as compared to the cholesterol-containing bilayers, in spite of the structural similarity of these two sterols, provides strong evidence for the selectivity of interaction of AmB with ergosterol as compared to cholesterol. It is thus clear that the action of AmB on cholesterol- as compared to ergosterol-containing membranes results from different mechanisms. Finally, UV-visible spectra of AmB in pure as well as sterol-containing DPPC vesicles show the presence of absorption bands that give support to the interpretation derived from the calorimetric data.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

The influence of dietary protein intake on milk production and blood composition of high-yielding dairy cows

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14c-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants greater than very low density lipoprotein greater than low density lipoprotein greater than high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.     

Bile salt-membrane interactions and the physico-chemical mechanisms of bile salt toxicity

We present evidence that ursodeoxycholate prevents toxicity of more hydrophobic bile salts by inhibiting micellar solubilization of membrane lipids. Using both centrifugal ultrafiltration and gel filtration methods we studied leakage of inulin from vesicles composed of egg phosphatidylcholine and cholesterol. We observed that the addition of tauroursodeoxycholate to taurodeoxycholate reduced leakage of inulin from large unilamelar vesicles compared to that seen with taurodeoxycholate alone. This protective effect was observed only at high membrane cholesterol:phospholipid ratios (> or = 0.5). By gel filtration we found that fractional leakage of inulin from vesicles was identical to fractional phospholipid solubilization, indicating that release of inulin from vesicles results from membrane dissolution rather than from increased permeability of otherwise intact membranes. Addition of tauroursodeoxycholate to taurodeoxycholate was found to suppress the dissolution of phospholipid from cholesterol-rich vesicles. Bile salts were found to absorb to vesicles with an affinity proportional to their relative hydrophobicity, as estimated by reverse phase HPLC. Adsorption affinity decreased progressively with increasing membrane cholesterol content. Different bile salts displaced each other from membranes in proportion to their respective binding, affinities. Tauroursodeoxycholate, which absorbed to membranes with low affinity, displaced taurodeoxycholate from vesicles only weakly. Based on these findings we postulate that bile salts may damage the liver through solubilization of canalicular membrane lipids. Ursodeoxycholate may protect the liver by inhibiting dissolution of the cholesterol-rich canalicular membrane by more hydrophobic endogenous bile salts. Biliary secretion of vesicles rich in phosphatidylcholine may buffer the intermicellar concentration of bile acids at levels below those required to disrupt the cholesterol-rich canalicular membrane; thus biliary vesicle secretion may have evolved as a mechanism to protect the biliary epithelium from injury by luminal bile salts.     

Identification of a receptor mediating absorption of dietary cholesterol in the intestine

Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.     

Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration

The major net flux of cholesterol in the intact animal or human is from the peripheral organs to the liver. This flux is made up of cholesterol that is either synthesized in these peripheral tissues or taken up as lipoprotein cholesterol. This study investigates whether it is the concentration of apolipoprotein (apo) A-I or high density lipoprotein in the plasma that determines the magnitude of this flux or, alternatively, whether events within the peripheral cells themselves regulate this important process. In mice that lack apoA-I and have very low concentrations of circulating high density lipoprotein, it was found that there was no accumulation of cholesterol in any peripheral organ so that the mean sterol concentration in these tissues was the same (2208 +/- 29 mg/kg body weight) as in control mice (2176 +/- 50 mg/kg). Furthermore, by measuring the rates of net cholesterol acquisition in the peripheral organs from de novo synthesis and uptake of low density lipoprotein, it was demonstrated that the magnitude of centripetal sterol movement from the peripheral organs to the liver was virtually identical in control animals (78 +/- 5 mg/day per kg) and in those lacking apoA-I (72 +/- 4 mg/day per kg). These studies indicate that the magnitude of net sterol flux through the body is not related to the concentration of high density lipoprotein or apolipoprotein A-I in the plasma, but is probably determined by intracellular processes in the peripheral organs that dictate the rate of movement of cholesterol from the endoplasmic reticulum to the plasma membrane.     

Effect of estrogen and progesterone on the expression of hepatic and extrahepatic sterol 27-hydroxylase in baboons (Papio sp)

Sterol 27-hydroxylase plays an important role in cholesterol metabolism in hepatic and extrahepatic tissues. To determine whether female sex steroid hormones influence its expression, we measured plasma and hepatic 27-hydroxycholesterol, hepatic mRNA levels, activity of sterol 27-hydroxylase, and adrenal mRNA levels of this enzyme in baboons (n = 6 per group) treated with placebo, estrogen, estrogen + progesterone, and progesterone. We also measured hepatic cholesterol concentration and hepatic acyl coenzyme A:cholesterol acyltransferase (ACAT) activity to determine their relationship with hepatic sterol 27-hydroxylase activity. Plasma 27-hydroxycholesterol concentration was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P = .090) and LDL (P = .026) cholesterol concentrations. Similarly, hepatic sterol 27-hydroxylase activity was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (P = .056) and LDL (P = .052) cholesterol concentrations but was positively correlated with hepatic and plasma 27-hydroxycholesterol concentrations (P < .001). Hepatic ACAT activity was increased by progesterone (P < .004) and was positively correlated with plasma (P = .002) and LDL (P = .009) cholesterol concentrations but was negatively correlated with hepatic sterol 27-hydroxylase activity (P = .035). Hepatic and adrenal gland mRNA levels for sterol 27-hydroxylase were increased by estrogen alone or in combination with progesterone (P < .05). Hepatic sterol 27-hydroxylase activity was positively correlated with hepatic mRNA levels (P < .001), an observation suggesting that estrogen increases the activity of sterol 27-hydroxylase by increasing its synthesis. Hepatic cholesterol concentration was not influenced by the hormone treatment. These observations suggest that estrogen alone or in combination with progesterone increases the synthesis of sterol 27-hydroxylase in hepatic and extrahepatic tissues, and the increased activity of hepatic sterol 27-hydroxylase resulting from the increased synthesis is associated with a hypolipidemic effect on plasma LDL levels. Furthermore, progesterone alone increases the hepatic ACAT activity, but given in combination with estrogen progesterone does not have the same effect on hepatic ACAT activity. The effect of estrogen on hepatic ACAT activity may be mediated by sterol 27-hydroxylase and its effect on cholesterol metabolism (decreased cholesterol synthesis and increased output of cholesterol in the bile) in liver.     

Effect of cholesterol and cholestyramine feeding and of fasting on sterol synthesis in the liver, lleum, and lung of the guinea pig

The effects of feeding diest containing either cholesterol (0.24% w/w) or cholestyramine (2.5% w/w) and of fasting on sterol synthesis in the liver, ileum, and lung of both male and female guinea pigs have been studied by measuring the incorporation by tissue slices of 14C-labeled acetate into total digitoninpredipitable sterols. Cholesterol feeding significantly decreased (P less than 0.05) sterol synthesis in the liver, ileum, and lung of the males and in the ileum of females. Cholestyramine feeding stimulated the rate of hepatic sterol synthesis 13-fold but did not significantly affect sterologenesis in the ileum. Sterol synthesis in the lung was significantly increased (P less than 0.05) but to a much lesser extent than in the liver. Fatty acid synthesis in the liver, ileum, and lung was not significantly affected by either cholesterol or cholestyramine feeding. In guinea pigs fasted for 24 hr, sterol synthesis was inhibited in all three tissues, the most pronounced effect occurring in the liver. Only in the lung was fatty acid synthesis significantly decreased (P less than 0.001) by fasting. Cholesterol feeding resulted in increased concentrations of cholesterol in the plasma and liver. Cholestyramine feeding reduced plasma cholesterol concentration by 81% in females and by 64% in males. However, it did not significantly change the tissue cholesterol concentrations. Fasting resulted in a significant increase (P less than 0.05) in plasma cholesterol concentration but did not effect the concentration of cholesterol in the tissues. It was concluded that in the normal guinea pig, the feedback inhibition produced by both cholesterol and also possibly by bile acids suppresses sterol synthesis in the liver to very low rates compared to those in the small intestine, where sterologenesis is not only less sensitive to the cholesterol negative feedback system than that in the liver, but also is not subject to regulation by the bile acid negative feedback system.     

The pleckstrin homology domain of oxysterol-binding protein recognises a determinant specific to Golgi membranes

BACKGROUND: Peripheral membrane proteins are targeted to the cytoplasmic face of specific intracellular membranes. The organelle-specific ligands recognised by peripheral proteins include other proteins and lipids. Oxysterol-binding protein (OSBP) translocates from the cytoplasm to the Golgi apparatus on binding oxygenated derivatives of cholesterol. The mechanism by which OSBP recognises the Golgi is unknown. It does, however, contain a pleckstrin homology (PH) domain, which in other proteins has been found to mediate regulated membrane binding, although in all previously studied examples the binding is to the plasma membrane. RESULTS: The PH domain of OSBP and of a yeast homologue, Osh1p, were sufficient to target proteins specifically to mammalian Golgi membranes. In addition, high level expression disrupted Golgi architecture and prevented forward traffic of cargo protein. In vitro, the OSBP PH domain bound to Golgi membranes in a manner apparently dependent on phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or a related phosphatidylinositide. The OSBP PH domain bound to PI(4,5)P2 in liposomes with a submicromolar dissociation constant. CONCLUSIONS: The PH domains of OSBP and its yeast homologue recognise a determinant which is specific to Golgi membranes and important for Golgi function. The determinant appears to be a combination or a phosphatidylinositol polyphosphate and a second, Golgi-specific feature.     

Influence of the physical structure of phospholipid membranes on peroxidation induced by Fe2+ ions

Kinetics of free radical lipid peroxidation, induced by Fe2+ in presence of ascorbic acid, was studied in phospholipid membranes. The maximal rate of peroxidative oxidation was observed at 2.5 muM concentration of Fe2+, in this case a half of the maximal amount of peroxidative oxidation products was formed within 20-30 min at 20 degrees and at 200 muM concentration of ascorbic acid. The rate of peroxidative oxidation depended on addition of substances modifying the membrane structure (linoleic acid, cetyl trimethylammonium, Tween-60, derivatives of phenothiazol). Charge of the membrane surface was shown to have a distinct effect on the peroxidative oxidation. Loosening of membranes by non-ion detergent (Tween-60) increased the rate of the process, whereas the increase of the membranes rigidity by cholesterol did not cause any effect. Uneffectiveness of cholesterol is discussed with relation to diffusion of radicals, participating in peroxidative oxidation, from depth of the membrane to its surface and in the opposite direction.     

Active transport mechanisms in the intestinal mucosa of the small intestine and their functional disorders

The efficacy of an anion-exchange gel, Secholex, as a hypocholesterolemic agent was assessed in 46 patients in 4 different studies and the effects were compared with those of cholestyramine. All patients had severe Type II-a or II-b hyperlipoproteinemia. In short-term metabolic studies Secholex (15 g/day) and cholestyramine (16 g/day) decreased serum cholesterol levels and increased total fecal sterol output and serum methyl sterol concentration to a similar extent, but cholestyramine was more effective than Secholex in increasing fecal bile acid excretion. In crossover studies, the two drugs appeared to be equally effective in lowing serum cholesterol levels but the patients mostly preferred Secholex. Twenty patients were treated with Secholex over a two-year period. The average decrease in serum cholesterol levels from the mean pretreatment value of 406 mg/100 ml was 15% during the first year, and 13% during the second year. In 5 patients the serum cholesterol was permanently lowered by more than 20% (good responders), while in 7 patients the average reduction of serum cholesterol level during Secholex administration was less than 10% (non-responders). The serum triglyceride level was slightly decreased by Secholex in Type II-b patients but was unaltered in Type II-a patients. At the end of the treatment period, serum iron and vitamin B12 levels were normal but the serum folic acid concentration was reduced in eight of 20 patients. A dose--response study indicated that a similar cholesterol-lowering effect was obtained with daily doses of 9 and 15 g of Secholex. It is concluded that Secholex is a relatively safe drug which effectively reduces serum cholesterol levels in two-thirds of patients with severe hypercholesterolemia.