In vivo synthesis of stigmasterol in Nicotiana tabacum

Six-day-old tobacco seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]-sitosterol, but none of the radioactivity was recovered from stigmasterol. However, exogenously supplied [2-¹⁴C]-mevalonic acid was incorporated into both sitosterol and stigmasterol. Based on these results it is suggested that the biosynthetic pathway of stigmasterol is not via sitosterol but that both sterols have a common precursor.     

Sitosterol biosynthesis in Hordeum vulgare

Excised barley embryos cultured on a nutrient medium containing methionine-[CD₃] incorporated deuterium into the newly biosynthesized sterols. Two deuterium atoms were present in 24-methylenecycloartanol, 24-methylenelophenol and campesterol and a maximum of four deuterium atoms were incorporated into 24-ethylidenelophenol, stigmasterol and sitosterol. Mevalonic acid-[2-¹⁴C(4R)4-³H₁] was utilized by the barley embryos to give 28-isofucosterol with a ³H-¹⁴C atomic ratio of 3:5 and stigmasterol and sitosterol with a ³H-¹⁴C atomic ratio of 2:5. 24-Methylenelophenol and 24-ethylidenelophenol were isolated from barley seed and 24-ethylidenelophenol-[2,4-³H₃] was incorporated into sitosterol by barley seedlings. These results show that in the production of sitosterol a 24-ethylidenesterol intermediate is produced and it is suggested that this is isomerized to give a Δ^24,(25) sterol prior to reduction to the saturated C₂₉ sterol side chain.     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Effect of retinol and retinoic acid on permeability,electrical resistance and phase transition of lipid bilayers

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K⁺, I^? and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.     

Incorporation of [4-14C] cholesterol into steryl derivatives and saponins of oat (Avena sativa L.) plants

Sterols from both green and etiolated oat plants (Avena sativa) contain sitosterol, stigmasterol, cholesterol, ⁷-stigmastenol, ⁷-cholestenol and campesterol. In the saponin fraction avenacosides A and B and 26-desgluco-avenacosides A and B were detected. Etiolated plants incorporated [4-¹⁴C] cholesterol into steryl derivatives (esters, glycosides and acylated glycosides) and also into all of the 4 saponins. [4-¹⁴C] sitosterol, however, is incorporated only into steryl derivatives, but not in saponins. From this it is concluded that cholesterol, but not sitosterol is the in vivo precursor of oat saponins.Abbreviations GLC gas liquid chromatography - I.D. inner diameter - TMS trimethylsilyl - dpm decompositions per minute     

Role of cholesterol in the action of cereolysin on membranes.

The following evidence supports the concept that cholesterol in membranes is the receptor and target site for the cytolytic action of cereolysin. (i) Of the various phospholipids, gangliosides, and steroids tested, only cholesterol and closely related sterols (sitosterol and dihydrocholesterol) significantly inhibited the hemolytic activity of cereolysin. (ii) Acholeplasma laidlawii cells grown in the presence of cholesterol inhibited the hemolytic activity of cereolysin, but A. laidlawii grown in the absence of cholesterol did not. (iii) Incubation of A. laidlawii cells, grown in the absence of cholesterol, with a cholesterol-Tween 80 mixture reestablished the ability of the cells to bind cereolysin. (iv) Treatment of erythrocyte membranes and A. laidlawii cells containing cholesterol with cholesterol oxidase (EC 1.1.3.6, Brevibacterium species) abolished the ability of these membranes to bind cereolysin and inhibit the hemolytic activity of the toxin, (v) Cereolysin could bind to and alter the permeability of both right-side-out ghosts and inside-out vesicles prepared from human erythrocytes, in agreement with other data that cholesterol is present on both sides of the erythrocyte membrane, (vi) Cereolysin caused the release of [¹⁴C]glucose from liposomes containing cholesterol, and this release was dependent on the amount of cholesterol in the liposomes.     

Permeability and the mechanism of transport of boric acid across the plasma membrane of Xenopus laevis oocytes

Boron is an essential element for vascular plants and for diatoms, cyanobacteria, and a number of species of marine algal flagellates. Boron was recently established as an essential micronutrient for frogs (Xenopus laevis) and preliminary evidence suggests that it may be essential for all animals. The main form of B, which is available in the natural environment, is in the form of undissociated boric acid. The permeability coefficient and the mechanism of transport of boric acid, however, have not been experimentally determined across any animal membrane or cell. In the experiments described here, the permeability coefficient of boric acid in Xenopus oocytes was 1.5 × 10⁻⁶ cm/s, which is very close with the permeability across liposomes made with phosphatidylcholine and cholesterol (the major lipids in the oocyte membrane). Moreover, we investigated the mechanism of boric acid movement across the membrane of Xenopus oocytes and we compared it with the transport across artificial liposomes. The transport of boric acid across Xenopus oocytes was not affected by inhibitors such as HgCl₂, phloretin, or 4,4-diisothiocyanatostilbene-2,2′-d-sulfonic acid (DIDS). The kinetics of B uptake was linear with concentration changes, and the permeability remained the same at different external boric acid concentrations. These results suggest that B transport occurs via simple passive diffusion through the lipid bilayer in Xenopus oocytes.     

Permeability of Boric Acid Across Lipid Bilayers and Factors Affecting It

Boron enters plant roots as undissociated boric acid (H₃BO₃). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has been theorized that these differences reflect species differences in permeability coefficient of H₃BO₃ across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes made of phosphatidylcholine was 4.9 × 10⁻⁶ cm sec⁻¹, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10⁻⁶ to 9.5 × 10⁻⁹ cm sec⁻¹ with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition of the plasma membrane can affect total B uptake. Received: 15 October 1999/Revised: 11 February 2000     

Biosynthesis of makisterone A and 20-hydroxyecdysone from labeled sterols by the honey bee,Apis mellifera

In an effort to determine the sterol precursor(s) of the 28-carbon ecdysteroid, makisterone A, honey bee pupae (13 days post-oviposition) were injected with radiolabeled sterols and subsequently examined for labeled ecdysteroids. High performance liquid chromatography of the pupal extracts revealed that [³H]campesterol was converted to a compound that behaved chromatographically identical to authentic makisterone A, and [¹⁴C]cholesterol was incorporated into a compound chromatographically like 20-hydroxyecdysone. No incorporation of either 24-[³H]methylenecholesterol or [¹⁴C]sitosterol into an ecdysteroid was observed. The neutral sterols of uninjected honey bee pupae contained 49.8% 24-methylenecholesterol on a relative percent basis and, with three other C²⁸ and C²⁹ sterols, accounted for over 99% of the total sterols present.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Functional analysis of candidate ABC transporter proteins for sitosterol transport

Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters implicated in lipid movement and expressed in tissues with a role in sterol synthesis and absorption, might also be involved in sitosterol transport. Transport by the multidrug resistance P-glycoprotein (P-gp; Abcb1), the multidrug resistance-associated protein (Mrp1; Abcc1), the breast cancer resistance protein (Bcrp; Abcg2) and the bile salt export pump (Bsep; Abcb11) was assessed using several assays. Unexpectedly, none of the candidate proteins mediated significant sitosterol transport. This has implications for the pathology of sitosterolemia. In addition, the data suggest that otherwise broad-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols.     

Biosynthesis of sterols and triterpenes in Pelargonium hortorum

Although all parts of the geranium plant (Pelargonium hortorum) are capable of synthesizing sterols and triterpenes and their esters in vitro from mevalonic acid-[2-¹⁴C], the aerial portions are more active than other tissues. All plant parts were shown to incorporate mevalonic acid-[2-¹⁴C] into isoprenoids for at least 3 days. The leaves and petioles had the greatest incorporation on a wet weight basis. Chopped preparations showed comparable incorporations of mevalonate whereby rootlets incorporated about one half as much as most parts; the flower petals incorporated five times the average amount. In leaves the principal sterol synthesized was sitosterol. Metabolic studies with isolated leaves indicated a fairly rapid conversion of free tetracyclic triterpenes to 4-desmethyl-sterols, while β-amyrin was synthesized at a different rate than α-amyrin. Esterified tetracyclic triterpenes exhibited only a slight amount of conversion to 4-desmethylsterols.     

Early stages of ecdysteroid biosynthesis: The role of 7-dehydrocholesterol

The synthesis of [4-¹⁴C]cholesta-4,6-dien-3-one and [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one is described. Both [4-¹⁴C]cholest-4-en-3-one and [4-¹⁴C]cholesta-4,6-dien-3-one were not incorporated significantly into ecdysteroids compared to [1α,2α-³H]cholesterol in fifth instar and maturing adult female Schistocerca gregaria. Similarly, [4-¹⁴C]3β-hydroxy-5α-cholestan-6-one was not incorporated significantly in the latter system. The results suggest that none of the three ¹⁴C-substrates are intermediates in ecdysteroid biosynthesis from cholesterol, although possible complications from permeability barriers cannot be discounted. [4-¹⁴C, 7-³H]7-dehydrocholesterol has been synthesized and incorporated into ecdysteroids in adult female Schistocerca gregaria and in Spodoptera littoralis pupae. Although approximately half the tritium was eliminated during ecdysteroid synthesis in S. gregaria, there was essentially complete retention of the tritium in Spodoptera. The results support the direct incorporation of 7-dehydrocholesterol into ecdysteroids and not via cholesterol. A possible explanation for the loss of appreciable tritium in S. gregaria is discussed.     

Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP > CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.     

Production of phytosterols by mature Digitalis purpurea L. plants

Summary The relative rates of production by mature Digitalis purpurea plants of cholesterol, campesterol, stigmasterol and sitosterol isolated from the glycoside and lipid fractions of the plant extract, were estimated. Plants were exposed to an atmosphere of ¹⁴CO₂ in a growth chamber and the radioactivity of the individual sterols assessed at intervals over 25 days on a gas-liquid radio chemical chromatography (GLRC). Incorporation of ¹⁴CO₂ occurred within 12 hours into both fractions of the extract. The 5-ene sterols were produced at a similar rate over a period of 25 days but the lipid fraction was about 100% more radioactive than the glycoside fraction.     

Investigation of the role of micellar phospholipid in the preferential uptake of cholesterol over sitosterol by dispersed rat jejunal villus cells

The uptake of radioactive cholesterol and sitosterol by rat jejunal villus cells was examined using mixed micellar solutions containing sodium taurocholate, equimolar mixtures of the two sterols, and a variety of phospholipid types. The addition of phospholipid to the incubation solutions reduced the cellular absorption of both sterols and gave rise to uptake kinetics that were linear with time. In the presence of egg yolk phospholipid, uptake of the sterols by villus cells occurred with a modest preference for cholesterol over sitosterol. The ratio of accumulated cholesterol/sitosterol increased from 1.0 initially to 1.23 +/- 0.04 (n = 18) after a 30-min incubation at 37 degrees C. The selectivity displayed in the villus cells increased significantly as egg phosphatidylethanolamine was added to the egg phosphatidylcholine (PC) preparation in micellar solution. It was markedly decreased when dipalmitoyl PC or the primarily saturated egg yolk sphingomyelin were incorporated into the micelles. In every case examined, phospholipid was taken up by the cells concurrently with the sterols. The selectivity between cholesterol and sitosterol was maintained when the donor species were multilamellar vesicles composed of egg PC and the sterols, but not when the donor particles were albumin-stabilized sterol dispersions or taurocholate solutions in the absence of PC. The results show that the selective absorption of cholesterol over the plant sterol occurs only in the presence of unsaturated phospholipid. The phospholipid may act by influencing the permeability of the cellular membranes to the two sterols or the rate of sterol desorption from the phospholipid-containing micellar or liposomal carriers.(ABSTRACT TRUNCATED AT 250 WORDS)