Effect of norflurazon on resorcinolic lipid metabolism in rye seedlings

Norflurazon is a selective pyridazinone herbicide excessively employed in the control of many annual grasses and broad-leaved weeds. This chemical causes plant bleaching due to the inhibition of the carotenoid pigment biogenesis as well as induces irreparable changes to chloroplasts, which are considered the organelles where the biosynthesis of resorcinolic lipids takes place. Resorcinolic lipids, a group of phenolic compounds, constitute not only an essential part of the plant antifungal defense system, but also are an important component of the human cereal diet. The aim of this study was to investigate the effect of norflurazon on the biosynthesis of resorcinolic lipids in 5-day-old rye plants (Secale cereale L.) that were grown at three different temperatures under light or dark conditions. At all tested temperatures, norflurazon decreased the fresh biomass of light-grown rye seedlings and increased the weight of plants grown in darkness. Compared with respective controls, this herbicide caused an increase in total content of alkylresorcinols in both green and etiolated plants with the exception of dark-grown norflurazon-treated rye at 29 degrees C. The general level of saturated homologues was markedly decreased by norflurazon in all etiolated plants and in light-grown seedlings at 15 degrees C. Independent of thermal and light conditions, in all norflurazon-treated samples two alkylresorcinol derivatives predominated: 1,3-dihydroxy-5-n-heptadecylbenzene and 1,3-dihydroxy-5-n-nonadecylbenzene. Thus, our results suggest that norflurazon affected the metabolism of alkylresorcinols in rye seedlings and its action was dependent on external stimuli.     

Production and secretion of 5-n-alkylresorcinols by Fusarium culmorum

Fusarium culmorum F1 was found to produce and secrete into the culture medium several of 5-n-alkylresorcinols. The amount of resorcinolic lipids was 5.3 microg/g and 0.9 microg/l in mycelium and in post-culture liquid, respectively. First of all F. culmorum F1 produces saturated homologues with C15 to C25 side chains. The extract from the medium contained only homologues with shorter carbon chains (C13 to C17).     

Anti-inflammatory 5-(11'Z-heptadecenyl)- and 5-(8'Z,11'Z-heptadecadienyl)-resorcinols from mango (Mangifera indica L.) peels

Bioassay directed extraction and purification of mango peels revealed the 5-(11'Z-heptadecenyl)-resorcinol (1) and the known 5-(8'Z,11'Z-heptadecadienyl)-resorcinol (2) previously not described in Mangifera indica L. The structures of both compounds were determined by extensive 1D and 2D NMR studies and MS. Both compounds exhibited potent cyclooxygenase (COX)-1 and COX-2 inhibitory activity with IC(50) values ranging from 1.9 (2) to 3.5 microM (1) and from 3.5 (2) to 4.4 (1) microM, respectively, coming close to the IC(50) values of reference drugs. 5-Lipoxygenase (5-LOX) catalyzed leukotriene formation was only slightly inhibited. Structure-activity studies by referring to synthetic saturated homologues indicated that the degree of unsaturation in the alkyl chain plays a key role for COX inhibitory activity, whereas the influence of chain length was less significant.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

The protozoan toxin climacostol and its derivatives: Cytotoxicity studies on 10 species of free-living ciliates

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to resorcinolic lipids, a group of compounds that show antimicrobial, antiparasitic, and anticancer activities. We investigated the cytotoxic activity of the chemically synthesized toxin and its alkyl and alkynyl derivatives on C. virens and nine other common species of free-living freshwater ciliates. Our results show that the cytotoxic potency of climacostol can be modulated by the substitution of the double bond present in the aliphatic chain of the toxin with a single or a triple one that was previously obtained during the synthesis of the unsaturated and saturated derivatives of the parent molecule. We demonstrated that the cytotoxicity level of the molecules considered in this study appears to be inversely correlated to the unsaturation level of their aliphatic chains, and that the potency of their action is also related to the target organism.     

The effect of merulinic acid on biomembranes

Merulinic acid (heptadecenylresorcinolic acid, resorcinolic acid) is one of the members of resorcinolic lipids, the natural amphiphilic long-chain homologues of orcinol (1,3-dihydroksy-5-methylbenzene). In the present study, membrane properties of merulinic acid were investigated. Merulinic acid exhibits strong haemolytic activity against sheep erythrocytes (EH₅₀ of 5±2 μM) regardless of the form of its application—direct injection into the erythrocyte suspension or injection as merulinic acid-enriched liposomes. The lysis of erythrocytes induced by merulinic acid was inhibited by the presence of divalent cations. The effectivity of the protection of erythrocytes was highest for Zn²⁺ and weakest for Mn²⁺.Merulinic acid at low concentrations also exhibits the ability for protection of cells against their lysis in hypoosmotic solutions. This protective effect is significant as, at 10 μM concentration of merulinic acid, the extent of osmotically induced cell lysis is reduced by approximately 40%. Merulinic acid induces increased permeability of liposomal vesicles. This effect was shown to be dependent on the composition of liposomal bilayer and it was stronger when lipid bilayer contained glycolipids (MGDG and DGDG) and sphingomyelin.Changes of TMA-DPH and NBD-PE fluorescence polarization show that the degree of merulinic acid incorporation into liposomal membrane is not very high. The polar “heads” of the molecules of investigated compounds are localized on the level of fatty acid's ester bonds in phospholipid molecules. Merulinic acid caused the increased fluorescence of the membrane potential fragile probe. This indicated an alteration of the surface charge and a decrease of the local pH at the membrane surface. This effect was visible in both low- and high-ionic strength environment. Merulinic acid causes also a decrease in activity of the membrane-bound enzyme acetylcholinesterase.     

Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1)

The binding properties of a wheat non-specific lipid-transfer protein (nsLTP1) for different mono- and diacylated lipids was investigated. Lipids varied by their chain length, unsaturation and/or polar head group. In the case of fatty acid or lysophospholipid with a C10 chain length, no interaction can be measured, while poor affinity is reported for a C12 chain length. The dissociation constant (Kd) is about 0.5 microM independent of chain length from C14 to C18. The same affinity is obtained for C18 fatty acids with one or two unsaturations, whatever the cis-trans double bond isomery. In all cases, the number of binding sites, n, by protein ranges between 1.6 and 1.9, suggesting that two lipids can fit within the protein. omega-Hydroxy-palmitic acid, a natural monomer of cutin polymer, is found to interact with nsLTP1 with a Kd of 1 microM and n = 2. In contrast with previous data that reported the binding of the anionic diacylated phospholipid, DMPG (Sodano et al., FEBS Lett. 416 (1997) 130-134), nsLTP1 is not able to bind dimyristoylphosphatidylcholine, dimyristoylphosphatidic acid, palmitoyl-oleoylphosphatidylcholine or palmitoyl-oleoylphosphatidylglycerol added as liposomes or solubilized in ethanol. However, when both nsLTP1 and lipids are first solubilized in methanol, and then in the buffer, it was evidenced that the protein can bind these lipids. These results suggest that lipid-lipid interactions play an essential role in the binding process of plant nsLTP1 as previously mentioned for other lipid-transfer proteins.     

2H nuclear magnetic resonance order parameter profiles suggest a change of molecular shape for phosphatidylcholines containing a polyunsaturated acyl chain.

Solid-state 2H nuclear magnetic resonance spectroscopy was used to determine the orientational order parameter profiles for a series of phosphatidylcholines with perdeuterated stearic acid, 18:0d35, in position sn-1 and 18:1 omega 9, 18:2 omega 6, 18:3 omega 3, 20:4 omega 6, 20:5 omega 3, or 22:6 omega 3 in position sn-2. The main phase transition temperatures were derived from a first moment analysis, and order parameter profiles of sn-1 chains were calculated from dePaked nuclear magnetic resonance powder patterns. Comparison of the profiles at 37 degrees C showed that unsaturation causes an inhomogenous disordering along the sn-1 chain. Increasing sn-2 chain unsaturation from one to six double bonds resulted in a 1.6-kHz decrease in quadrupolar splittings of the sn-1 chain in the upper half of the chain (or plateau region) and maximum splitting difference of 4.4 kHz at methylene carbon 14. The change in chain order corresponds to a decrease in the 18:0 chain length of 0.4 +/- 0.2 A with 18:2 omega 6 versus 18:1 omega 9 in position sn-2. Fatty acids containing three or more double bonds in sn-2 showed a decrease in sn-1 chain length of 0.7 +/- 0.2 A compared with 18:1 omega 9. The chain length of all lipids decreased with increasing temperature. Highly unsaturated phosphatidylcholines (three or more double bonds in sn-2) had shorter sn-1 chains, but the chain length was somewhat less sensitive to temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution of fatty acids from monolayers spread at the air-water interface: identification of phase transformations and the estimation of surface charge

The contraction or decrease in area of fatty acid monolayers maintained at a constant surface pressure of 16 dynes/cm was studied as a function of fatty acid chain length, unsaturation, temperature, and the hydrogen ion concentration in the subphase. The data were consistent with the hypothesis that fatty acid solution from the monolayer into the subphase was the mechanism for film loss. Autoxidative reactions did not contribute significantly to film loss since contraction occurred with saturated fatty acid monolayers and with unsaturated fatty acid monolayers in an anaerobic environment. The decrease in area per unit time or the solution rate was inversely proportional to chain length and directly proportional to the degree of unsaturation. Arrhenius plots showed activation energies of 1.5-2.5 kcal mole(-1) for tetradecanoic, octadecenoic, and octadecadienoic acids, and 25 kcal mole(-1) for hexadecanoic acid. The solution rate from the monolayer increased in a sigmoidal fashion with an increase in subphase pH, and the apparent surface pK(a) was estimated as the point where the solution rate was half-maximum. Apparent surface pK(a) values were: hexadecanoic acid, 9.7; octadecenoic acid, 8.3; tetradecanoic acid, 7.9; and octadecadienoic acid, 8.0.     

Influence of chain length and unsaturation on sphingomyelin bilayers

Sphingomyelins (SMs) are among the most common phospholipid components of plasma membranes, usually constituting a mixture of several molecular species with various fatty acyl chain moieties. In this work, we utilize atomistic molecular dynamics simulations to study the differences in structural and dynamical properties of bilayers comprised of the most common natural SM species. Keeping the sphingosine moiety unchanged, we vary the amide bonded acyl chain from 16 to 24 carbons in length and examine the effect of unsaturation by comparing lipids with saturated and monounsaturated chains. As for structural properties, we find a slight decrease in average area per lipid and a clear linear increase in bilayer thickness with increasing acyl chain length both in saturated and unsaturated systems. Increasing the acyl chain length is found to further the interdigitation across the bilayer center. This is related to the dynamics of SM molecules, as the lateral diffusion rates decrease slightly for an increasing acyl chain length. Interdigitation also plays a role in interleaflet friction, which is stronger for unsaturated chains. The effect of the cis double bond is most significant on the local order parameters and rotation rates of the chains, though unsaturation shows global effects on overall lipid packing and dynamics as well. Regarding hydrogen bonding or properties related to the lipid/water interface region, no significant effects were observed due to varying chain length or unsaturation. The significance of the findings presented is discussed.     

Application of highly purified anti-galactocerebroside antibody to the analysis of lipid-lipid or lipid-protein interactions. Significance of lipid matrix

The significance of the lipid matrix in the reaction of liposomal antigen, antibody and complement (Ag-Ab-C) was analyzed using purified anti-glactocerebroside antibody and synthetic lipids and the following results were obtained. 1. For the optimal Ag-Ab-C reaction it was necesary that galactocerebroside (galxCMH) and lecithin molecules were well dispersed by virtue of cholesterol (Chol) and that the molar ratio of cholesterol to the sum of galactocerebroside and lecithin was more than one. 2. The Ag-Ab-C reactivity changed depending upon the chain length of lecithin, and the maximal reaction was observed in the case of dilauroylphosphatidylcholine. When the fatty acyl chain of lecithin was either shorter or longer than that of dilaurosylphosphatidylcholine, the reactivity was reduced. 3. The Ag-Ab-C reactivity was increased by elongation of the fatty acyl chain of galactocerebroside and an abrupt change was found to be around the carbon number 8 to 10 of the fatty acyl chain. 4. The Ag-Ab-C reactivity was elevated by the increase in unsaturation of fatty acyl moiety. 5. There is a tendency that an increase in the charge of the lipid matrix leads to the reduction of the Ag-Ab-C reactivity. 6. The results suggest that the physicochemical properties of lipids and especially the lipid-lipid interaction in the hydrophobic region of the lipid matrix play an important role in the Ag-Ab-C reaction.     

The effect of the phase transition on the hydration and electrical conductivity of phospholipids.

Adsorption isotherms for various saturated phosphatidylcholines have been obtained. Lipids above and below their phase transition temperature differ only in the amount of water adsorbed and not in the nature of their adsorption isotherms. Cholesterol has an effect similar to that of increasing unsaturation in the hydrocarbon chains. Decreasing the length of the hydrocarbon chains for lipids below their phase transition temperature has no effect on the isotherms. If the chain length is short enough so that the lipids are above their transition temperature, however, a large increase in water adsorption occurs. All of the phospholipids exhibit a rapid increase of electrical conductivity for a few water molecules adsorbed per lipid molecule. All of the phospholipids show a saturation in conductivity at greater amounts of adsorbed water; the shape of the saturation region depends on whether the lipids are above or below their phase transition temperature. The activation energy for the electrical conductivity process depends on whether the hydrated lipids are in the "liquid-like" of the crystalline state, being lower for phospholipids in the liquid-like state. If the lipids are hydrated above their phase transition temperatures, their activation energies are lower than if they are hydrated below the transition temperature. Cholesterol lowers the activation energy. The phosphatidylcholines can be characterized by different activation energies, depending both upon their physical state and the presence of unsaturation in their hydrocarbon chains.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

Alkylresorcinols in fruit pulp and leaves of Ginkgo biloba L

These studies were undertaken to characterise resorcinolic lipids (5-n-alk(en)ylresorcinols) composition and to determine their seasonal fluctuations in fruit pulp and leaves of Ginkgo biloba L. Resorcinolic lipid concentrations were consistently higher in fruit pulp than in leaves. In pulp, several mono- and di-unsaturated homologs of alkylresorcinols were the predominant group of analysed lipids. Contrary to the fruit pulp, only 5-n-pentadecylresorcinol was demonstrated in leaves. Initially, the alkylresorcinol's content both in pulp and leaves increased until June-July and decreased following seeds ripening. This trend continued until senescence of leaves in late September and October.     

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.     

The estimation of sphingolipids by gas chromatography-chemical ionization mass spectrometry of their derived aldehydes with particular reference to the ceramides of children's plasma

A method for the estimation of sphingolipids based on the quantitation, by gas-liquid chromatography/chemical ionization, of the long-chain aldehydes released from the native lipids, is described. The method is highly sensitive (6–7 pmol per injection), specific, and can, in addition, provide information on the chain length and degree of unsaturation of the long-chain bases.     

Plant Growth Inhibition and Membrane Penetration by a Homologous Series of Dichlorobenzoate Esters

The homologous series of n-alkyl esters (C₁–C₁₀) of 3,4-dichlorobenzoic acid was synthesized and their effects in inhibiting the growth of Nicotiana meristems were studied. The inhibition of growth was considered in terms of penetration of chemicals into the plant tissue and subsequent cell membrane disruption. Penetration was investigated by applying the emulsified ester to the meristem and then measuring the compound recovered with the isolated surface lipids. Decreasing amounts of 3,4-dichlorobenzoate esters penetrated into the plant as the alkyl chain length of the ester moeity was increased. Essentially no penetration occurred with the n-C₇ through C₁₀ esters tested. The effect of the ester homologues on cell membranes was studied by measuring the efflux of betacyanin from beet root cells. Decreasing amounts of pigments were released as the alkyl chain length of the ester was increased. Minimal cell membrane disruption was found for the C₇–C₁₀ esters. Inhibition of the plant meristem may result from the more rapid penetration of the short chain ester homologues into the plant.     

Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.     

Structural and thermodynamic determinants of chain-melting transition temperatures for phospholipid and glycolipids membranes

For optimum function, biological membranes need a fluid environment, which is afforded by the liquid-disordered phase of lipids with low chain-melting temperatures or the liquid-ordered phase that is formed by combining high chain-melting lipids with cholesterol. The dependence of chain-melting transition temperature on lipid chain structure is therefore of central importance. The currently available database, including sphingolipids and glycolipids, is summarised here by parameterising systematic dependences on molecular structure in terms of suitable thermodynamic models. Chain-length dependence, chain asymmetry of lipids forming partially interdigitated and mixed interdigitated gel phases, chain unsaturation, positional dependence of methyl branching, headgroup-attached and α-branched chains, and length of zwitterionic headgroups are all covered. This type of information is essential for biophysical approaches to functional lipidomics.