Glycerol increases the cytochemical detectability of cholesterol in the apical plasma membrane of uterine epithelial cells

Summary The apical plasma membrane of uterine epithelial cells in the rat has been treated with glycerol before fixation and then examined by freeze-fracture cytochemistry using digitonin and filipin. Many more lesions were produced by both cytochemicals following glycerol treatment than in untreated controls, and we suggest that this indicates an increased detectability of cholesterol. We consider the implications of the findings for the way in which glycerol acts on membranes and propose that glycerol promotes increased binding between cholesterol and the cytochemicals.     

Effects of piperine on the lipid composition and enzymes of the pyruvate-malate cycle in the testis of the rat in vivo

Effects of piperine at two oral doses (5 and 10 mg/kg body weight for 30 days) on the lipid composition and some lipogenic enzymes of the rat testis were studied. Piperine treatment depleted the total lipid content which was mainly due to the diminution of the total phospholipid concentration. All the classes of phospholipids were decreased markedly following high dose piperine treatment. In contrast, a marked increase in total cholesterol and cholesterol ester was evident with a concomitant fall in free cholesterol. A similar trend was found for the total glyceride glycerol and its fractions. Total glyceride glycerol and triacyl glycerol showed a significant increase at the expense of diacyl glycerol in rats treated with the high dose of piperine. Lipogenic enzymes, malate dehydrogenase (MDH), malic enzyme (ME) and isocitrate dehydrogenase (ICDH) were inhibited by the high dose and only MDH and ME activities were inhibited by the low dose treatment.     

Structural rearrangements induced by glycerol increase the permeability of bilayer lipid membranes for amphotericin

It has been shown that structural rearrangements induced by glycerol in bilayer lipid membranes (BLM) containing cholesterol facilitate the transmembrane transport of amphotericin B molecules in the direction of glycerol gradient. The addition of amphotericin B to the same side with glycerol results in a change in bilayer selectivity from the cation to the anion one. Besides, the final conductivity is blocked by tetraethylammonium from the solution with no amphotericin B added. It testifies to the transport of amphotericin molecules to the opposite side of the membrane. The transport effect depends on the cholesterol content in bilayer, ionic strength of the medium and slightly depends on temperature. It is concluded that transport of amphotericin B in such conditions differs from the diffusive one and is due to the formation of intermediate lipid phases in the course of structural rearrangements of bilayers.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.     

Effect of glycerol on the capacity and conductivity of lipid bilayer membranes

It is shown that glycerol addition to one side of BLM containing cholesterol leads to a significant decrease of its capacity, and the decrease rate is in indirect proportion to glycerol concentration. Washing out or addition of glycerol to another side results in a full or partial reconstitution of the capacity. The effect of glycerol on the BLM conductivity is manifested in its increase for membranes having specific conductivity above 2.10(-7) Om-1 X cm2. The peculiarity of glycerol is manifested in the fact that during definite period of time (20 +/- 30 min) the membrane is under "stress" condition with the characteristic current fluctuations, which may be compared with its medium meaning. Such condition is preserved up to the moment of BLM rupture. It is supposed that such effects are due to the big-scale reconstructions of membrane structure under the influence of glycerol.     

Prevention of the production of lipid peroxide in rooster spermatozoa

The effects on lipid peroxide in chicken sperm of seminal plasma, Ringer-Phosphate-Medium and some chemical agents such as membrane alterants (bovine serum albumin, cholesterol), chelating agents (calcium, magnesium), cryoprotectants (glycerol, dimethyl-sulfoxide) and citrate were examined. Seminal plasma, Ringer-Phosphate-Medium, calcium and citrate reduced peroxide production and provided minimal protection against it. On the contrary, glycerol, dimethylsulfoxide and magnesium partly inhibited the production of lipid peroxide, and bovine serum albumin and cholesterol had no inhibiting effect. These results suggest that a certain amount of seminal plasma, calcium and citrate can inhibit the endogenous lipid peroxidation in sperm and maintain fertilizing ability.     

The effect of glycerol on cytochrome P450scc (CYP11A1) spin state,activity, and hydration

We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity.     

Effects of prolactin and androgens on seminal vesicular lipids of castrated mature bonnet monkeys Macaca radiata

Effects of prolactin (PRL), bromocriptine (Br), testosterone propionate (TP), dihydrotestosterone (DHT) and the combination of these androgens with PRL/Br on the total lipid, total cholesterol, total glyceride glycerols, total phospholipid and their fractions in seminal vesicles of castrated mature monkeys were studied. Glyceride glycerols formed the major portion (50%) of total lipids in normal monkeys. Cholesterol and phospholipids were of equal share (25%). Esterified cholesterol formed major share (75%) of total cholesterol. Diacyl glycerol was the major (60%) glyceride glycerol and phosphatidyl choline and ethanolamine were the major phospholipid classes. Except triacyl glycerol castration markedly decreased all the lipid classes. PRL restored normal free and esterified cholesterol and phosphatidyl inositol but Br invariably decreased all the lipid classes. TP/DHT treatment stimulated the free and esterified cholesterol more than the control; it restored the normal glyceride glycerols. Phosphatidyl inositol, choline and ethanolamine were stimulated by androgens and other phospholipid classes were brought to normal. Addition of PRL + TP/DHT markedly increased esterified cholesterol, phosphatidyl inositol, choline, ethanolamine and phosphatidic acid. In all these aspects, Br counteracted the effects of androgens and PRL.     

Lipid composition of the fishes Heterotis niloticus,Brycenus nurse,Gnathonemus cyprinoides and Sarotherodon galilaeus from a small lake in Nigeria

Lipid composition of Heterotis niloticus, Brycenus nurse, Gnathonemus cyprinoides and Sarotherodon galilaeus, which vary in scale thickness, were studied. Heterotis niloticus (very thick scales) had the lowest lipid content (13%), while G. cyprinoids (light scales) had the highest lipid content (26%). The common neutral lipids were cholesterol, free fatty acids and cholesterol esters, while diphosphatidyl glycerol, phosphatidyl glycerol and phosphatidyl ethanolamine were the most predominant phospholipids.     

Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes

The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P相似文献   

Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase

Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.     

Cholesterol-induced effects on the viscoelasticity of monoglyceride bilayers.

Changes in the viscoelastic properties of glycerol monooleate bilayers resulting from the incorporation of cholesterol into the membranes have been measured. The interface tension increases with the cholesterol concentration, reaching saturation for a 4.2:1 mole ratio of cholesterol:lipid in the film-forming solution. Incorporation of cholesterol in the membrane causes the appearance of a large intrinsic viscosity; this also increases with the sterol content of the membrane. Molecular models of lipid-sterol interactions and packing are considered to explain both the observed changes in membrane properties and similarities with comparable lipid systems.     

Relationship between testosterone and hepatic lipids in mature male bonnet monkeys, Macaca radiata (Geoffroy).

Administration of testosterone propionate (TP; 1 mg/kg body weight/day; im, for 30 days) to mature male bonnet monkeys, decreased total lipids, glyceride glycerol and cholesterol concentrations in hepatic tissue. The decrease in glyceride glycerol was due to decrease in monoacyl and triacyl glycerol. Both, free and esterified cholesterol were decreased after testosterone administration. Eventhough, total phospholipid was not significantly altered, phosphatidylserine and cardiolipin were increased, due to testosterone administration. Testosterone inhibited NADP-isocitrate dehydrogenase activity in liver. The findings suggest that testosterone acts as a lipolytic hormone and its action may be direct through its specific receptors in liver.     

The effects of cholesterol inclusion on the molecular organisation of bimolecular lipid membranes

Dielectric measurements on planar egg phosphatidylcholine bilayers formed from n-hexadecane solutions indicate that these bilayers contain very low equilibrium concentrations of alkane. In 100 mM KCl the capacitance of the hydrophobic region was found to be 7.0 ±0.2 mF/m². The addition of cholesterol (at 2:1 mole ratio) was found to affect only marginally the capacitance of the hydrophobic region of such bilayers. Precise measurements of the frequency dependence of the bilayer impedance at very low frequencies now allow the resolution of several electrically distinct substructural regions within the bilayer. Examination of the effects of cholesterol inclusion upon the electrical parameters of these substructural regions indicate that cholesterol spans the acetyl region (i.e. the region containing the glycerol bridge of the phosphatidylcholine molecules in the bilayer) with the hydroxyl group of the cholesterol molecules located inbetween the phosphate group and the glycerol oxygens of the phosphatidylcholine molecules. The capacitance of the hydrophobic region of both phosphatidylcholine and phosphatidylcholine/cholesterol bilayers formed from n-hexadecane solutions was found to decrease slightly as the external KCl concentration was decreased.     

Preparation of human cord sera for enzymatic triglyceride assays: removal of free glycerol by ultrafiltration

Enzymatic triglyceride assays that generate glycerol from triglycerides as a part of the enzymatic process in quantitating serum triglyceride levels give elevated values when external free glycerol is present. Our objective was to develop an ultrafiltration technique that would remove exogenous and/or endogenous free glycerol from small aliquots of human cord sera so that accurate serum triglyceride values could be obtained with the commercially available triglyceride assay kits. Exogenous glycerol was completely removed from cord sera when the samples were washed twice with saline in Amicon Centricon-30 microconcentrators. This ultrafiltration technique lowered cord serum triglyceride levels significantly (P less than 0.001), but had no effect on cord total serum cholesterol levels. A comparison of washed and unwashed cord sera by either polyacrylamide or agarose gel electrophoresis indicated that the serum protein and lipoprotein profiles were not altered by the ultrafiltration process.     

The behavior of lipoproteins, cholesterol, triglycerides, free fatty acids and free glycerol in serum of rats with experimental hyperthyroxinemia and hypothyreosis]

In hyperthyroxinemic and hypothyreotic rats the lipoprotein level in serum was investigated using agarosegel-electrophoresis and changes in serum level of cholesterol, triglycerides, free fatty acids and glycerol were determined. In hyperthyroxinemic animals the beta-lipoproteins were found in the same level as the control animals, while the prae-beta-fraction was significantly elevated and the alpha-lipoproteins lowered. The cholesterol was significantly reduced, the triglycerides and the glycerol significantly increased. The free fatty acids were slightly elevated. In hypothyreotic animals the beta-and prae-beta-fraction of lipoproteins was significantly elevated. The alpha-lipoproteins were found diminished. Cholesterol and triglyceride values were also significantly increased. The levels of free fatty acids and glycerol did not differ in both groups of animals.     

New spectrophotometric assays of acid lipase and their use in the diagnosis of Wolman and cholesteryl ester storage diseases

Trinitrophenylaminolauric acid (TNPAL) was linked to glycerol or cholesterol and the resulting yellow compounds were used as substrates for several lipases and cholesteryl esterase in cells from normal individuals and patients with Wolman's or cholesteryl ester storage diseases. Normal cells (lymphoid cell lines or skin fibroblasts) showed two peaks of lipase or cholesteryl esterase activity at about pH 4.0 and 6.0 each. The activity of the most acidic enzyme, which hydrolyzed natural or synthetic triacylglycerols as well as cholesteryl esters, was considerably reduced in cells derived from patients with Wolman's or cholesteryl ester storage diseases. Simple spectrophotometric procedures were developed for using tri-TNPAL glycerol or TNPAL cholesterol to identify homozygotes of these two respective diseases.     

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.     

Interaction of cholesterol with conformationally restricted phospholipids in vesicles.

The interaction of cholesterol with conformationally restricted analogs of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in the liquid-crystalline phase has been studied in vesicles. These analogs contain one of three cyclopentane triols in place of the glycerol moiety found in natural phospholipids and make possible an analysis of whether a limitation of the conformational mobility in the glycerol backbone region affects the interaction with cholesterol. When cholesterol was incorporated into vesicles from cyclopentanoid phospholipids in which the acyl group vicinal to the head group is trans, the first-order rate constant for Cl- efflux is decreased similarly to that in vesicles from 'natural' DPPC or DPPG (about 50%). However, when the head group is in the unnatural 2 position, cholesterol has a much smaller effect on the rate of Cl- efflux (a decrease of about 20%). Cholesterol decreased the rate constants for valinomycin-mediated 86Rb+ efflux from vesicles of the cyclopentanoid PC analogs and of DPPC to a similar extent. The half-time values for spontaneous intervesicle cholesterol exchange were not markedly different using vesicles prepared with the natural glycerophospholipids and with the cyclopentano-phospholipids, suggesting that the geometrical orientation of the acyl chains or the head group has little influence on cholesterol desorption from the lipid/water interface.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids. Permeability and 13C-nuclear magnetic resonance measurements

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.