Differential Effect of Plant Lipids on Membrane Organization: SPECIFICITIES OF PHYTOSPHINGOLIPIDS AND PHYTOSTEROLS*

The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains.     

Plant sterols: Friend or foe in CNS disorders?

In mammals, the central nervous system (CNS) is the most cholesterol rich organ by weight. Cholesterol metabolism is tightly regulated in the CNS and all cholesterol available is synthesized in situ. Deficits in cholesterol homeostasis at the level of synthesis, transport, or catabolism result in severe disorders featured by neurological disability. Recent studies indicate that a disturbed cholesterol metabolism is involved in CNS disorders, such as Alzheimer’s disease (AD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). In contrast to circulating cholesterol, dietary plant sterols, can cross the blood–brain barrier and accumulate in the membranes of CNS cells. Plant sterols are well-known for their ability to lower circulating cholesterol levels. The finding that they gain access to the CNS has fueled research focusing on the physiological roles of plant sterols in the healthy and diseased CNS. To date, both beneficial and detrimental effects of plant sterols on CNS disorders are defined. In this review, we discuss recent findings regarding the impact of plant sterols on homeostatic and pathogenic processes in the CNS, and elaborate on the therapeutic potential of plant sterols in CNS disorders.     

Metabolic profiling and enhanced production of phytosterols by elicitation with methyl jasmonate and silver nitrate in whole plant cultures of Lemna paucicostata

In this study, the effects of methyl jasmonate (MJ) and silver nitrate (SN) treatment on metabolic profiles and yields of phytosterols such as campesterol, stigmasterol, and β-sitosterol in whole plant cultures of Lemna paucicostata were investigated using gas chromatography–mass spectrometry coupled with multivariate statistical analysis. The MJ and SN treatments retarded the growth of L. paucicostata plants, while they enhanced the yields of three phytosterols, compared to control. Higher yields of phytosterols were attained at day 28 compared to day 42. Moreover, stigmasterol yield was the highest at 0.85 mg/g from day 28 plants grown under MJ + SN co-treated culture. Among the various metabolites, the levels of palmitic and stearic acids, which might participate in a defense mechanism, were higher in the MJ + SN condition than in control. To determine the optimal timing of MJ + SN addition, MJ + SN was added on days 21, 28, and 35 after inoculation. The total yield and productivity of phytosterol reached maximum levels when the MJ + SN was added at day 35. The highest productivity of stigmasterol (6.08 mg/L) was also achieved when MJ + SN was added on day 35.     

Preparation and mass spectrometry of 14 pure and 18O(2)-labeled oxidation products from the phytosterols beta-sitosterol and stigmasterol

To lower cholesterol, phytosterols are currently introduced as food additives, where they may become oxidized. In addition, specific biological effects of oxyphytosterols are suggested by the recent molecular clarification of the phytosterol storage disease as a dysfunctional mutation of an active sterol reexporter potentially regulated by oxidized phytosterols. We therefore studied the hydroxybenzotriazole-mediated PbO(2)-driven oxidation of phytosterols and compared it to the oxidation of cholesterol. We prepared, identified, and purified standards of 14 oxidation products of two major phytosterols. The gas chromatographic mass spectrometric characteristics of the 7alpha- and 7beta-hydroxy-, 5alpha,6alpha-epoxy, 5beta,6beta-epoxy, 7keto-, 3beta,5alpha,6beta-trihydroxy-, 3keto-, and 7-dehydro-derivatives of beta-sitosterol and stigmasterol are presented. The method also provided a convenient access to prepare 18O-labeled oxyphytosterols of high chemical and isotopic purity and can easily be extended to further phytosterols and -stanols. This enables the gas chromatography-mass spectrometry analysis of oxyphytosterols and the study of their biological effects.     

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5 kbar at 30 °C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-β-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.     

A concise synthesis of β-sitosterol and other phytosterols

A convenient synthesis of sidechain-modified phytosterols is achieved via a temporary masking of the stigmasterol 5,6-alkene as an epoxide. Following performance of the desired modification, the alkene is regenerated through a mild deoxygenation. The approach is applied to the syntheses of β-sitosterol and campesterol acetate, and suggests a facile route to the (Z)-isomers of Δ^22-23 phytosterols.     

Effects of ABA on primary terpenoids and Δ<Superscript>9</Superscript>-tetrahydrocannabinol in <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. at flowering stage

This work examined the effects of exogenously applied abscisic acid (ABA) on the content of chlorophyll, carotenoids, α-tocopherol, squalene, phytosterols, Δ⁹-tetrahydrocannabinol (THC) concentration, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate synthase (DXS) activity in Cannabis sativa L. at flowering stage. Treatment with 1 and 10 mg l⁻¹ ABA significantly decreased the contents of chlorophyll, carotenoids, squalene, stigmasterol, sitosterol, and HMGR activity in female cannabis plants. ABA caused an increase in α-tocopherol content and DXS activity in leaves and THC concentration in leaves and flowers of female plants. Chlorophyll content decreased with 10 mg l⁻¹ ABA in male plants. Treatment with 1 and 10 mg l⁻¹ ABA showed a decrease in HMGR activity, squalene, stigmasterol, and sitosterol contents in leaves but an increase in THC content of leaves and flowers in male plants. The results suggest that ABA can induce biosynthesis of 2-methyl-d-erythritol-4-phosphate (MEP) pathway secondary metabolites accumulation (α-tocopherol and THC) and down regulated biosynthesis of terpenoid primary metabolites from MEP and mevalonate (MVA) pathways (chlorophyll, carotenoids, and phytosterols) in Cannabis sativa.     

Salinity increases the triterpenoid content of a salt secretor and a non-salt secretor mangrove

The present study describes the effect of salinity on the triterpenoid content of the salt secretor mangrove Avicennia marina and the non-secretor Rhizophora stylosa. Mangrove seedlings were grown for eight months in 0%, 0.5%, 1.5%, 2.0% and 3.0% salt concentration. The growth of both species was increased by salt with maximal stimulation at 1.5%, and this elevation appeared to be attenuated by increasing the salt concentration above 1.5%. The triterpenoid compositions of three types of chemical structures, lupane (lupeol, lupenone), oleanane (β-amyrin, taraxerol, germanicol), and ursane (α-amyrin), were studied. In addition, the phytosterol components campesterol, stigmasterol and β-sitosterol were analyzed. The total triterpenoid contents in the roots and leaves of A. marina for the 0% group were 87.0 and 66.2 μg g⁻¹, respectively, and increased significantly to 173.1 and 142.6 μg g⁻¹ with 3% salinity. The higher salinity also significantly increased the total concentration of phytosterols in the leaves and roots of this species. A similar increase in the concentration of both triterpenoids and phytosterols was observed in the roots and leaves of R. stylosa with increasing salt concentration. Thus, the triterpenoid concentration was increased by salinity in the roots and leaves of both A. marina and R. stylosa irrespective of their differences in salt management by salt excretion or by a non-excretion mechanism. Comparison of the triterpenoid concentration in four species of growing mangrove seedlings revealed a correlation between the total triterpenoid content and the salt tolerance based on the habitat zonation on Iriomote Island. A. marina thrives closest to sea and had the highest content of triterpenoids (173.1 μg g⁻¹ in 3% salt group). Therefore, it is likely that the triterpenoid content play an important role in mangrove plants for protection from salinity in both salt-secretors and non-secretors.     

Studies on β-sitosterol and ceramide-induced alterations in the properties of cholesterol/sphingomyelin/ganglioside monolayers

Phytosterol—β-sitosterol promotes apoptosis in various cancer cells and inhibits their growth. Supplementation of cancer cells with this compound causes modifications in membrane composition, namely, substitution of cholesterol (Chol), decrease of sphingomyelin (SM) content and increase of ceramide (Cer) level. The aim of this work was to investigate the influence of partial replacement of cholesterol by plant sterol, substitution of sphingomyelin by ceramide and both these factors simultaneously on the properties of the monolayers composed of major lipids identified in breast cancer membranes, namely Chol/SM/GM3 mixtures. Brewster Angle Microcopy experiments and the analysis of the isotherms recorded during films compression and resulting parameters evidenced that β-sitosterol weakens the interactions between molecules, decreases films stability and condensation. The influence of ceramide on sterol/SM/GM3 films was reflected in strong modifications of their texture, however, the morphology of monolayer was determined by the structure of sterol present in the system. It was also found, that simultaneous replacement of 50 mol% of Chol and SM by phytosterol and Cer, respectively, induces lipids segregation, which is manifested in large diversity of phases observed in BAM images. To facilitate the analysis of the data collected for multicomponent monolayers, the properties of selected sterol/GM3, sterol/Cer, SM/GM3, Cer/GM3 binary films were also investigated. The obtained results evidenced that the studied herein modifications in the composition of Chol/SM/GM3 monolayer, reflecting compositional alterations induced by phytosterol in cancer membranes, strongly affect the organization of model system, therefore they should be considered in the studies on anticancer mechanism of β-sitosterol.     

Inhibition of Escherichia coli O157:H7 motility and biofilm by β-Sitosterol glucoside

Background

Escherichia coli O157:H7 (EHEC) is a food borne pathogen, which causes diarrhea and hemolytic uremic syndrome (HUS). There is an urgent need of novel antimicrobials for treatment of EHEC as conventional antibiotics enhance shiga toxin production and potentiate morbidity and mortality.

Methods

Six bioactive compounds were isolated, identified from citrus and evaluated for the effect on EHEC biofilm and motility. To determine the possible mode of action, a series of genes known to affect biofilm and motility were overexpressed and the effect on biofilm/motility was assessed. Furthermore, the relative expression of genes involved in motility and biofilm formation was measured by qRT-PCR in presence and absence of phytochemicals, to examine the repression caused by test compounds.

Results

The β-sitosterol glucoside (SG) was identified as the most potent inhibitor of EHEC biofilm formation and motility without affecting the cell viability. Furthermore, SG appears to inhibit the biofilm and motility through rssAB and hns mediated repression of flagellar master operon flhDC.

Conclusion

SG may serve as novel lead compound for further development of anti-virulence drugs.

General significance

Plant sterols constitute significant part of diet and impart various health benefits. Here we present the first evidence that SG, a plant sterol has significant effect on EHEC motility, a critical virulence factor, and may have potential application as antivirulence strategy.