A comparison of a spin-label and a fluorescent cell membrane probe using pure and mixed monomolecular films

Monocular film studies of 12-nitroxide stearic acid and 12-(9-anthroyl) stearic acid reveal that deviations from the behavior of the parent molecule (stearic acid) are as much dictated by the polar, or nonpolar, nature of the probe group as by its size. In mixed films under membrane-like conditions, the spin label probe, 12-nitroxide stearic acid, exhibits positive deviations from ideality and should read too high a fluidity. The picture is, however, complicated by a tendency of this probe molecule to adopt a bent conformation, a tendency apparently enhanced by specific interactions with the lecithin zwitterion. 12-(9-anthroyl) stearic acid, in contrast, shows only negative deviations from ideality in mixed dipalmitoyl lecithin films and should read too low a fluidity.     

Structural insights for fatty acid binding in a Lys49-phospholipase A2: crystal structure of myotoxin II from Bothrops moojeni complexed with stearic acid

The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C(18)H(36)O(2)) has been determined at a resolution of 1.8 A. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA(2)s is analogous to phospholipids and transition state analogues to catalytically active PLA(2)s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA(2)s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA(2)s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in group I/II PLA(2)s, and to the possible interactions of Lys49-PLA(2)s with target membranes.     

Effects of stearic acid and beef tallow on iron utilization by the rat.

Two experiments were done in which anemic rats were fed diets containing safflower oil or stearic acid and low (10 ppm) or adequate (39-42 ppm) iron. Diets were 24% fat by weight. In the stearic acid diets, 2% (Experiment 1) or 4% (Experiment 2) of the fat was supplied by safflower oil to satisfy essential fatty acid requirements. Repletion of hemoglobin, hematocrit, and liver iron was assessed. Compared with safflower oil in both experiments, stearic acid had a significant positive effect (P less than 0.0001) on repletion of hemoglobin (Hb), hematocrit (Hct), and liver iron concentration; the effect on Hb and Hct was most pronounced when dietary iron was low. When expressed as g Hb/mg Fe intake, Hb repletion was affected by a significant interaction between fat and Fe (P less than 0.002) and was greatest in rats fed low iron stearic acid diets. In a third experiment, rats were fed low dietary iron and 24% safflower oil, 20% stearic acid + 4% safflower oil, 3.2% stearic acid + 20.8% safflower oil, or 20% beef tallow + 4% safflower oil. The 20% beef tallow provided 3.2% stearic acid in the total diet. The response of Hb and Hct were similar to those in the first two experiments for rats fed safflower oil or stearic acid. Rats fed beef tallow had significantly greater (P less than 0.05) Hb and Hct repletion than did rats fed safflower oil, although the degree of repletion was less than that observed in rats fed 20% stearic acid. There was no difference in iron repletion of rats fed 3.2% stearic acid and rats fed beef tallow. We conclude that stearic acid enhances iron utilization by rats.     

Temperature effect on a high stearic acid sunflower mutant.

Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.     

Enzymatic studies of high stearic acid sunflower seed mutants

Three high stearic acid sunflower (Helianthus annuus L.) mutants, CAS-3, CAS-4 and CAS-8, accumulating 28, 15 and 14 % of stearic acid in the seed lipids have been biochemically characterised. In vivo conversion rate of palmitic acid into stearic acid is not altered in the mutants but the conversion rate of stearic acid into oleic acid shows a reduction that correlated with the total stearic acid content of seed lipid mutants. Two enzymatic activities are found to be involved in the mutant phenotype, the acyl-ACP thioesterase (EC 3.1.2.14) and the stearoyl-ACP desaturase (EC 1.12.99.6). Our data suggest that the high stearic phenotype is due to the combined effect of a reduced stearoyl-ACP desaturase activity and an acyl-ACP thioesterase with higher activity on stearoyl-ACP. The same thioesterase activity increment, found on stearoyl-ACP, was also found on palmitoyl-ACP, suggesting that the affected thioesterase activity could be a FatB type.     

Dietary Stearic Acid Leads to a Reduction of Visceral Adipose Tissue in Athymic Nude Mice

Stearic acid (C18:0) is a long chain dietary saturated fatty acid that has been shown to reduce metastatic tumor burden. Based on preliminary observations and the growing evidence that visceral fat is related to metastasis and decreased survival, we hypothesized that dietary stearic acid may reduce visceral fat. Athymic nude mice, which are used in models of human breast cancer metastasis, were fed a stearic acid, linoleic acid (safflower oil), or oleic acid (corn oil) enriched diet or a low fat diet ad libitum. Total body weight did not differ significantly between dietary groups over the course of the experiment. However visceral fat was reduced by ∼70% in the stearic acid fed group compared to other diets. In contrast total body fat was only slightly reduced in the stearic acid diet fed mice when measured by dual-energy x-ray absorptiometry and quantitative magnetic resonance. Lean body mass was increased in the stearic acid fed group compared to all other groups by dual-energy x-ray absorptiometry. Dietary stearic acid significantly reduced serum glucose compared to all other diets and increased monocyte chemotactic protein-1 (MCP-1) compared to the low fat control. The low fat control diet had increased serum leptin compared to all other diets. To investigate possible mechanisms whereby stearic acid reduced visceral fat we used 3T3L1 fibroblasts/preadipocytes. Stearic acid had no direct effects on the process of differentiation or on the viability of mature adipocytes. However, unlike oleic acid and linoleic acid, stearic acid caused increased apoptosis (programmed cell death) and cytotoxicity in preadipocytes. The apoptosis was, at least in part, due to increased caspase-3 activity and was associated with decreased cellular inhibitor of apoptosis protein-2 (cIAP2) and increased Bax gene expression. In conclusion, dietary stearic acid leads to dramatically reduced visceral fat likely by causing the apoptosis of preadipocytes.     

Oral cavity discrimination of vapor-phase long-chain 18-carbon fatty acids

Linoleic, oleic, and stearic fatty acids, presented vapor-phase retronasally, were discriminable from blanks and each other, but the same concentrations, oral-cavity-only (OCO), were not discriminable from blanks. It remained possible that higher concentrations might be discriminable OCO. To evaluate this, participants attempted to discriminate undiluted linoleic, oleic, or stearic acids, vapor-phase OCO, from blanks. For each fatty acid, participants received 5 stimulus delivery containers (SDCs) in 2 trials; 4 SDC held blanks, the fifth, a fatty acid. As a "positive control" in 2 trials, participants received vapor-phase OCO peppermint extract and blanks. For all trials, the task was to select the 1 different SDC. It was found that the 1 different SDC was selected in 24% of stearic, 32% of linoleic, 47% of oleic acid, and in 92% of peppermint trials; discriminations (the 1 different SDC selected in both trials) occurred in 0%, 16%, 26%, and 84% of pairs, respectively. Correct selections for oleic acid differed from chance, P = 0.0004, but not for linoleic acid, P = 0.125, or stearic acid, P = 0.345, Bonferroni corrected. Vapor-phase oleic acid can be an oral cavity trigeminal stimulus, linoleic acid might be (uncorrected P = 0.0384), but vapor-phase stearic acid cannot be.     

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

The inhibition of stearoyl-coenzyme A desaturase by phenyllactate and phenylpyruvate.

The inhibition of oleic acid from stearic acid was studied using a rat liver microsomal preparation. Phenyllactic acid and phenylpyruvic acid are partially inhibitory. The may be related to the low oleic acid to stearic acid ratios found in brains of phenylketonuric persons and experimentally phenylketonuric rats.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

A spin-label study of sciatic nerves from quaking, jimpy and trembler mice.

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant (T) between 0 degrees C and 50 degrees C. Within this range of temperatures, we obtained identical values of 2 T for nerves from normal and jimpy mice, whereas 2 T was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice. The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.     

Mapping the <Emphasis Type="Italic">Fas</Emphasis> locus controlling stearic acid content in soybean

Increasing the stearic acid content to improve soybean [ Glycine max (L) Merr] oil quality is a desirable breeding objective for food-processing applications. Although a saturated fatty acid, stearic acid has been shown to reduce total levels of blood cholesterol and offers the potential for the production of solid fat products (such as margarine) without hydrogenation. This would result in the reduction of the level of trans fat in food products and alleviate some current health concerns. A segregating F(2) population was developed from the cross between Dare, a normal stearic acid content cultivar, and FAM94-41, a high stearic acid content line. This population was used to assess linkage between the Fas locus and simple sequence repeat (SSR) markers. Three SSR markers, Satt070, Satt474 and Satt556, were identified to be associated with stearic acid (P < 0.0001, r(2) > 0.61). A linkage map consisting of the three SSR markers and the Fas locus was then constructed in map order, Fas, Satt070, Satt474 and Satt556, with a LOD score of 3.0. Identification of these markers may be useful in molecular marker-assisted breeding programs targeting modifications in soybean fatty acids.     

A new viscosupplement based on partially hydrophobic hyaluronic acid: a comparative study

A novel partially hydrophobized derivative of hyaluronic acid (HYADD? 4), containing a low number of C16 side-chains per polysaccharide backbone, provides injectable hydrogels stabilized by side-chain hydrophobic interactions. The rheological properties of Hymovis?, a physical hydrogel based on the hyaluronic acid derivative HYADD? 4, were evaluated using as reference a solution of the parent natural polysaccharide, hyaluronic acid. The rheological measurements were performed both in flow and oscillation regimes at the physiological frequency values of the knee, typically spanning the range from 0.5 Hz (walking frequency) to 3 Hz (running frequency). Moreover, the viscoelastic features of Hymovis? were compared with the market-available viscosupplementation products in view of its use in joint diseases.The different behavior of the investigated materials in crossover frequency measurements and in structure recovery experiments can be explained on the basis of the structural and dynamic properties of the polymeric systems.     

Stearic acid attenuates cholestasis-induced liver injury

Inflammation is involved in cholestasis-induced hepatic damage. Stearic acid has been shown to possess anti-inflammatory potential. We assessed whether stearic acid has protective effects against cholestasis-related liver damage. Cholestasis was produced by bile duct ligation (BDL) in male Sprague-Dawley rats for 3 weeks. Daily administration of stearic acid was started 2 weeks before injury and lasted for 5 weeks. In comparison with the control group, the BDL group showed hepatic damage as evidenced by elevation in serum biochemicals, ductular reaction, fibrosis, and inflammation. These pathophysiological changes were attenuated by chronic stearic acid supplementation. The anti-fibrotic effect of stearic acid was accompanied by reductions in α-smooth muscle actin-positive matrix-producing cells and critical fibrogenic cytokine transforming growth factor beta-1 production. Stearic acid also attenuated BDL-induced leukocyte accumulation and NF-κB activation. The data indicate that stearic acid attenuates BDL-induced cholestatic liver injury. The hepatoprotective effect of stearic acid is associated with anti-inflammatory potential.     

Mutations in SACPD-C Result in a Range of Elevated Stearic Acid Concentration in Soybean Seed

Soybean oil has a wide variety of uses, and stearic acid, which is a relatively minor component of soybean oil is increasingly desired for both industrial and food applications. New soybean mutants containing high levels of the saturated fatty acid stearate in seeds were recently identified from a chemically mutagenized population. Six mutants ranged in stearate content from 6–14% stearic acid, which is 1.5 to 3 times the levels contained in wild-type seed of the Williams 82 cultivar. Candidate gene sequencing revealed that all of these lines carried amino acid substitutions in the gene encoding the delta-9-stearoyl-acyl-carrier protein desaturase enzyme (SACPD-C) required for the conversion of stearic acid to oleic acid. Five of these missense mutations were in highly conserved residues clustered around the predicted di-iron center of the SACPD-C enzyme. Co-segregation analysis demonstrated a positive association of the elevated stearate trait with the SACPD-C mutation for three populations. These missense mutations may provide additional alleles that may be used in the development of new soybean cultivars with increased levels of stearic acid.     

Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart

The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.     

Adsorption of a PEO–PPO–PEO triblock copolymer on metal oxide surfaces with a view to reducing protein adsorption and further biofouling

Abstract

Biomolecule adsorption is the first stage of biofouling. The aim of this work was to reduce the adsorption of proteins on stainless steel (SS) and titanium surfaces by modifying them with a poly(ethylene oxide) (PEO)–poly(propylene oxide) (PPO)–PEO triblock copolymer. Anchoring of the central PPO block of the copolymer is known to be favoured by hydrophobic interaction with the substratum. Therefore, the surfaces of metal oxides were first modified by self-assembly of octadecylphosphonic acid. PEO–PPO–PEO preadsorbed on the hydrophobized surfaces of titanium or SS was shown to prevent the adsorption of bovine serum albumin (BSA), fibrinogen and cytochrome C, as monitored by quartz crystal microbalance (QCM). Moreover, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry were used to characterize the surfaces of the SS and titanium after competitive adsorption of PEO–PPO–PEO and BSA. The results show that the adsorption of BSA is well prevented on hydrophobized surfaces, in contrast to the surfaces of native metal oxides.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.