In vitro and in vivo effects of unsaturated fatty acids on Schistosoma mansoni and S. haematobium lung-stage larvae

Incubation of Schistosoma mansoni lung-stage larvae in 90% corn oil for 6 hr was shown to elicit exposure of their, otherwise masked, apical membrane antigens to binding of anti-schistosome antibodies in the indirect membrane immunofluorescence test (IF). The possibility that unsaturated fatty acids (FA) are responsible for this effect was herein supported by IF data on ex vivo lung-stage larvae of S. mansoni and S. haematobium incubated for 1/2-2 hr with 80% corn oil, 50% olive oil, or 10-20 microM arachidonic acid. Treatment with unsaturated FA followed by filipin staining for cholesterol visualization indicated that unsaturated FA do not induce exposure of schistosomular surface membrane antigens via extraction of surface membrane cholesterol. Evidence using inhibitors and stimulators of neutral sphingomyelinase suggested that unsaturated FA perhaps activate worm tegument-bound neutral sphingomyelinase, leading to sphingomyelin hydrolysis and changes in surface membrane fluidity. Larval apical membrane antigens are, thus, allowed to diffuse freely in the plane of the membrane and bind specific antibodies in IE Excessive sphingomyelin hydrolysis might explain why high FA concentrations or long incubation periods eventually lead to larval death. The significant decrease (P < 0.01) in S. mansoni and increase (P < 0.02) in S. haematobium worm recovery in BALB/c mice given unsaturated FA-high and -poor diets, respectively, indicated these findings have in vivo relevance and led to the proposal that unsaturated FA likely plays a role in natural attrition of S. mansoni and S. haematobium lung-stage larvae.     

Methyl-beta-Cyclodextrin treatment and filipin staining reveal the role of cholesterol in surface membrane antigen sequestration of Schistosoma mansoni and S. haematobium lung-stage larvae

Ex vivo lung-stage larvae of Schistosoma mansoni and S. haematobium do not bind specific antibodies in the indirect membrane immunofluorescence test (IF), probably as a result of confinement of the surface membrane antigens in immobile, lipid-rich sites. Treatment with the membrane-impermeable, cholesterol-extracting drug methyl-beta-cyclodextrin (MBCD) and staining with filipin III (filipin), a fluorescent polyene antibiotic widely used for the detection and quantitation of cholesterol in biomembranes, allowed us to examine the role of cholesterol in surface membrane antigen sequestration of S. mansoni and S. haematobium ex vivo lung-stage larvae. Treatment of S. mansoni larvae with MBCD elicited appreciable cholesterol depletion as judged by filipin-cholesterol fluorescence diminution, which was accompanied by a considerable increase in specific antibody binding in IF, thus suggesting that cholesterol plays a predominant role in sequestration of the surface membrane antigens of S. mansoni lung-stage schistosomula. Despite that, MBCD induced an almost complete depletion of cholesterol from the outer membrane of S. haematobium larvae; no increase in specific antibody binding in IF was evident, implying that cholesterol is not responsible for masking surface membrane antigens of S. haematobium lung-stage larvae.     

Depletion of Schistosoma mansoni lung-stage schistosomula cholesterol by methyl-beta-cyclodextrin dramatically increases specific antibody binding to surface membrane antigens

Schistosoma mansoni lung-stage larvae appear to not bind antibodies from radiation vaccine or infection sera in the membrane immunofluorescence test. However, treatment of ex vivo lung-stage schistosomula with methyl-beta-cyclodextrin, a hydrophobic oligosaccharide that specifically extracts cholesterol from plasma membranes, induced readily detectable binding of specific antibodies in a concentration- and time-dependent manner. Surface membrane antigen binding of specific antibodies was also conclusively demonstrated by quantitative absorption of anti-schistosome sera with intact ex vivo larvae. These data together suggest that confinement of lung-stage schistosomula surface membrane antigens in cholesterol-rich sites allows only monovalent antibody binding, which can be detected by absorption and not by direct serology.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Effects of dietary fats on red blood cell membrane insulin receptor in normo- and hypercholesterolemic miniature swine

It has been demonstrated that the type of dietary fat affects insulin receptors in various tissues in normal humans and animals by altering membrane fluidity. This study compares the effects of n-3 fatty acids from fish oil and n-6 fatty acids from corn oil on red blood cell membrane insulin receptors in normal and hypercholesterolemic minipigs. A group of minipigs were made hypercholesterolemic by feeding cholesterol and lard for 2 months; the other group served as controls and was fed stock diet. Both groups were then fed experimental diets containing either corn oil or menhaden oil or a mixture of the two for 23 additional weeks. Blood was collected at 0, 2, 12 and 23 weeks after the start of the experimental diets and membranes were prepared from the red blood cells. Insulin binding to red blood cell membranes was measured by radioreceptor assay. Plasma insulin was measured by radioimmunoassay. Insulin binding to red blood cell membrane was compared with the fluidity of the membrane measured and reported earlier. There was no significant effect of cholesterol feeding on plasma insulin concentrations. After 23 weeks on experimental diet plasma insulin was significantly higher in minipigs fed menhaden oil compared to those fed corn oil. No such effect was observed in hypercholesterolemic minipigs. No significant effect of either hypercholesterolemia or fish oil was observed on red blood cell insulin binding. A significant negative relationship was observed between insulin binding and anisotropy at 4°C for all probes but at 37°C significant negative relationship was observed only with polar probes. The data suggest that n-3 fatty acids from fish oil significantly increases plasma insulin in minipigs compared to n-6 fatty acids from corn oil. However, the unsaturation has no significant effect on insulin receptors on erythrocytes. Similarly, prior hypercholesterolemic state also has no effect on plasma insulin levels or the insulin binding to red blood cell membranes.     

The surface coat of chylomicrons: electron microscopy

Electron microscope studies were performed on thoracic duct lymph and on washed chylomicrons from dogs fed corn oil. High-resolution electron micrographs showed the presence of a surface coat that differed from the core material and did not resemble a plasma membrane. This was true for both chylomicrons in whole lymph and those that had been subjected to repeated washing. Apparently, the chylomicrons, while passing from the intracellular to the extracellular space, do not acquire their surface coat from pinched off cellular membrane.     

Lung-stage protein profile and antigenic relationship between Ascaris lumbricoides and Ascaris suum

The protein profile and antigenic properties of lung-stage larvae of Ascaris lumbricoides and A. suum were studied using 2-dimensional electrophoresis and immunoblot analysis, respectively. The protein profiles of the 2 parasites were identical except for the presence of only 1 major protein spot specific for each. There was a complete cross-reactivity between the 2 parasites at the immunological level, and no specific antigen was recognized using specific antibody raised against the 2 parasites in rabbits.     

Lipid composition of liver microsomes in rats fed a high monounsaturated fatty acid diet

The fatty acid and cholesterol contents of tissue membranes are the determinants of membrane stability and functionality. This study was designed to evaluate the influence of a high monounsaturated fatty acid diet on the fatty acid composition of rat liver microsomes and on their cholesterol and lipid phosphorus content. Weanling animals were fed for 5 weeks with high fat diets containing olive oil or corn oil. Saturated fatty acids were increased and oleic acid decreased in microsomal total phospholipids and in the three major phosphoglycerides, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), of rats fed corn oil as compared to the olive oil group. The percentage of linoleic acid was higher in the corn oil group, but only for total phospholipids and PC. Linoleic and alpha-linolenic metabolites were significantly increased in total phospholipids of olive oil-fed animals with respect to those fed corn oil. These changes were responsible for the low unsaturation index found in microsomal phospholipids of the corn oil group. The diet did not affect the microsome cholesterol or the lipid phosphorus content. These results show that, in olive oil-fed rats, the cholesterol content and the degree of unsaturation of liver microsomes was similar to that observed in weanling animals; this probably suggests an adequate maintenance of functionality of membranes in olive oil-fed animals.     

Lung-stage expression of a major schistosome surface antigen

The topographical expression of a glycoprotein of 180,000 molecular weight on the surface of lung-stage Schistosoma mansoni schistosomula was determined by immunofluorescence microscopy using a monoclonal antibody. Postfixation treatment with graded ethanols enhanced specific immunofluorescent staining of adult worms, and was required for detection of the antigen on the surfaces of lung-stage schistosomula. The epitope recognized by the monoclonal antibody was also present on the surfaces of adult Schistosoma haematobium, but not on those of Schistosoma japonicum.     

Dietary docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft recruitment and IL-2 production

To date, the proximal molecular targets through which dietary n-3 polyunsaturated fatty acids (PUFA) suppress the inflammatory process have not been elucidated. Because cholesterol and sphingolipid-enriched rafts have been proposed as platforms for compartmentalizing dynamically regulated signaling assemblies at the plasma membrane, we determined the in vivo effects of fish oil and highly purified docosahexaenoic acid (DHA; 22:6n-3) on T cell microdomain lipid composition and the membrane subdomain distribution of signal-transducing molecules (protein kinase C (PKC)theta;, linker for activation of T cells, and Fas/CD95), before and after stimulation. Mice were fed diets containing 5 g/100 g corn oil (control), 4 g/100 g fish oil (contains a mixture of n-3 PUFA) plus 1 g/100 g corn oil, or 4 g/100 g corn oil plus 1 g/100 g DHA ethyl ester for 14 days. Dietary n-3 PUFA were incorporated into splenic T cell lipid raft and soluble membrane phospholipids, resulting in a 30% reduction in raft sphingomyelin content. In addition, polyclonal activation-induced colocalization of PKCtheta; with lipid rafts was reduced by n-3 PUFA feeding. With respect to PKCtheta; effector pathway signaling, both AP-1 and NF-kappaB activation, IL-2 secretion, and lymphoproliferation were inhibited by fish oil feeding. Similar results were obtained when purified DHA was fed. These data demonstrate for the first time that dietary DHA alters T cell membrane microdomain composition and suppresses the PKCtheta; signaling axis.     

Influence of mitochondrial membrane fatty acid composition on proton leak and H2O2 production in liver

Mitochondrial membrane fatty acid composition has been proposed to play a role in determining mitochondrial proton leak rate. The purpose of this study was to determine if feeding rats diets with different fatty acid sources produces changes in liver proton leak and H(2)O(2) production. Six-month-old male FBNF(1) rats were fed diets with a primary fat source of either corn or fish oil for a 6-month period. As expected, diet manipulations produced substantial differences in mitochondrial fatty acid composition. These changes were most striking for 20:4n6 and 22:6n3. However, proton leak and phosphorylation kinetics as well as lipid and protein oxidative damage were not different (P > 0.10) between fish and corn oil groups. Metabolic control analysis, however, did show that control of both substrate oxidation and phosphorylation was shifted away from substrate oxidation reactions to increased control by phosphorylation reactions in fish versus corn oil groups. Increased mitochondrial H(2)O(2) production was observed in corn versus fish oil-fed rats when mitochondria were respiring on succinate alone or on either succinate or pyruvate/malate in the presence of antimycin A. These results show that mitochondrial H(2)O(2) production and the regulation of oxidative phosphorylation are altered in liver mitochondria from rats consuming diets with either fish or corn oil as the primary lipid source.     

Immunochemical studies of the surface and cytoplasmic membranes of Entamoeba invadens (Rodhain 1934).

The antigenic properties of cell fractions prepared from axenically grown Entamoeba invadens were investigated using various immunoelectrophoretic methods. All membrane fractions displayed varying degrees of antigenicity, the most predominant being a cytoplasmic light-vesicle fraction. The surface membrane showed the least diversified range of antigenic components and was also the least immunogenic fraction as judged by the reactivity of the antisera produced. Using tandem-crossed immunoelectrophoresis the antigenic profiles of the membrane fractions were compared. No evidence was obtained for the presence of either lipid or carbohydrate in the antigenic moieties of any of the membrane fractions. Using a series of sequential solvent extractions it was concluded that both divalent metallochelate linkages and interiorly located hydrophobic associations were principally involved in binding the major antigenic components within the light-vesicle membrane. An enzymic function was assigned to certain of the membrane antigens, the immunoprecipitates obtained showing both acid phosphohydrolase and non-specific esterase acitivity toward a variety of substrates.     

Rat enterocytes secrete SLPs containing alkaline phosphatase and cubilin in response to corn oil feeding

Surfactant-like particles (SLP) are unilamellar secreted membranes associated with the process of lipid absorption and isolated previously only from the apical surface of enterocytes. In this paper, the intracellular membrane has been isolated from corn oil-fed animals, identified by its content of the marker protein intestinal alkaline phosphatase (IAP). Another brush-border protein, cubilin, and its anchoring protein megalin have been identified as components of extracellular SLP, but only cubilin is present to any extent in intracellular SLP. During fat absorption, IAP is modestly enriched in intracellular SLP, but full-length cubilin (migrating at 210 kDa in fat-fed mucosal fractions) falls by one-half, although fragments of cubilin are abundant in the intracellular SLP. Both IAP and cubilin colocalize to the same cells during corn oil absorption and colocalize around lipid droplets. This localization is more intense during feeding of corn oil with Pluronic L-81, a detergent that allows uptake of fatty acids and monoglycerides from the lumen, but blocks chylomicron secretion. Confocal microscopy confirms the colocalization of IAP and the ligand for cubilin, intrinsic factor. Possible roles for cubilin in intracellular SLP include facilitating movement of the lipid droplet through the cell and binding to the basolateral membrane before reverse endocytosis.     

Dietary n-3 polyunsaturated fatty acids modify fatty acid composition in hepatic and abdominal adipose tissue of sucrose-induced obese rats

The fatty acid profile of hepatocytes and adipocytes is determined by the composition of the dietary lipids. It remains unclear which fatty acid components contribute to the development or reduction of insulin resistance. The present work examined the fatty acid composition of both tissues in sucrose-induced obese rats receiving fish oil to determine whether the effect of dietary (n-3) polyunsaturated fatty acids (PUFAs) on the reversion of metabolic syndrome in these rats is associated to changes in the fatty acid composition of hepatocyte and adipocyte membrane lipids. Animals with metabolic syndrome were divided into a corn–canola oil diet group and a fish oil diet group, and tissues fatty acids composition were analyzed after 6 weeks of dietary treatment. Fatty acid profiles of the total membrane lipids were modified by the fatty acid composition of the diets fed to rats. N-3 PUFAs levels in animals receiving the fish oil diet plus sucrose in drinking water were significantly higher than in animals under corn–canola oil diets. It is concluded that in sucrose-induced obese rats, consumption of dietary fish oil had beneficial effects on the metabolic syndrome and that such effects would be conditioned by the changes in the n-3 PUFAs composition in hepatic and adipose tissues because they alter membrane properties and modify the type of substrates available for the production of active lipid metabolites acting on insulin resistance and obesity.     

Influence of menhaden oil on mitochondrial respiration in BHE rats

The effects of corn or menhaden oil and thyroxine treatment on hepatic mitochondrial respiration was studied. BHE rats were fed a 64% sucrose, 6% corn, or menhaden oil diet until they were 60-70 days of age. Succinate-supported mitochondrial respiration was studied at 3 degrees C intervals from 4 to 40 degrees C. Upper and lower activation energies and transition temperatures were determined through the calculation of Arrhenius plot. Menhaden oil plus daily thyroxine injection resulted in higher and lower activation energies than the other treatments. This combined treatment also resulted in lower state 3 and higher state 4 respiration rates and tighter coupling of respiration to ATP synthesis. These effects were thought to be due to the effect this treatment combination had on membrane fluidity.     

Effects of Temperature,Time and Composition on Food Oil Surface Tension

Equilibrium and time-dependent surface tension properties at the lipid-vapor interface were investigated, due to their importance in many food applications. Common cooking oils and triglycerides, with or without added oil-soluble amphiphiles, were studied as a function of time and temperature. Surface tension was found to decrease linearly as temperature was increased, and this linear dependence was analyzed to yield thermodynamic information on the surface excess energy and entropy. The different types of cooking oils were nearly indistinguishable with regard to their surface entropy and energy, but an effect of acyl chain length was observed from data for different purified triglyceride oils. These results were consistent with separate results on pure fatty acids of different chain lengths and degree of unsaturation. Lipid amphiphiles, natively present or deliberately added at low concentration to oil, did not cause a change in either dynamic or equilibrium surface tension of corn or olive oil. We conclude that such amphiphilic molecules, despite their presence within the food oil, lack significant surface activity at their native concentration when presented with the surface between oil and air. A decrease in tension in corn oil was seen when mixed in solution with the short-chain caprylic acid (octanoate), but the decrease was notable (>4%) only when this short-chain fatty acid was added at high concentration (≥ 1 M). Added sorbitan monooleate (Span 80) or dioctyl sulfosuccinate sodium salt (AOT) surfactants, on the other hand, decreased equilibrium surface tension by up to 12% and 18%, respectively, at low concentrations (<0.125 M).     

Stimulation of acyl-CoA oxidase by alpha-linolenic acid-rich perilla oil lowers plasma triacylglycerol level in rats

The effect of dietary polyunsaturated fatty acids (PUFA) on hepatic peroxisomal oxidation was investigated with respect to the postprandial triacylglycerol levels. Male Sprague--Dawley rats were fed semipurified diets containing either 1% (w/w) corn oil, or 10% each of beef tallow, corn oil, perilla oil, and fish oil for 4 weeks and 4 days. Hepatic and plasma triacylglycerol levels were reduced in rats fed fish and perilla oil diets compared with corn oil and beef tallow diets. The peroxisomal beta-oxidation, catalase activity, and acyl-CoA oxidase (AOX) activity were markedly increased by fish oil feeding. To a lesser extent, perilla oil elevated AOX activity in a 4-day feeding although the effect gradually decreased in a 4-week feeding. Similarly, the mRNA levels were increased in rats fed fish and perilla oils. AOX activity was negatively correlated with postprandial triacylglycerol levels. In addition, the stimulation of AOX was highly associated with the content of long chain n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in hepatic microsome. These effects were evident within 4 days of initiating feeding. Therefore, alpha-linolenic perilla oil exerts a similar effect to fish oil in stimulating hepatic activity and gene expression of AOX by enriching long chain n-3 PUFA in hepatic membrane fraction, which can partly account for the reduction of postprandial triglyceridemia.     

Entamoeba histolytica: specific antigen recognized by a monoclonal antibody

Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.     

Reinforcing effect for corn oil stimulus was concentration dependent in an operant task in mice

Corn oil is reported to elicit a conditioned place preference (CPP) in a CPP test in mice. To further investigate a reinforcing effect of corn oil, we studied whether the corn oil acts as a reinforcer under a progressive ratio (PR) schedule in the operant task. BALB/c mice were trained to lever press for sucrose and corn oil. After reaching a stable break-point for 100% corn oil, the PR test was conducted for various concentrations of corn oil (0%-100%). The reinforcing effect of corn oil was increased in a concentration-dependent manner under the PR schedule. A mineral oil and 0.3% xanthan gum as vehicles did not show any reinforcing effect in the PR test, suggesting that oily and viscous texture was not related to the reinforcing property of corn oil. The break-point for corn oil was attenuated by pretreatment with (-)-sulpiride, a D(2) antagonist, in the PR test. On the other hand, SCH23390, a D(1) antagonist, did not influence the break-point. Furthermore, the pretreatment with (-)-sulpiride or SCH23390 did not influence the intake of corn oil in a one-bottle test for 30 min, suggesting that the dopaminergic system is involved in the reinforcing effect but not the consumption of corn oil in mice. In conclusion, operant response to corn oil is concentration-dependently enhanced under the PR schedule. This reinforcing effect of corn oil is at least partly mediated through the dopaminergic systems via D(2) receptors.     

Genetic mapping of antigenic determinants on a membrane protein

The antigenic determinants recognized by two monoclonal antibodies were mapped on LamB, an outer membrane protein of Escherichia coli. The procedure consisted of performing immunoprecipitation experiments with extracts of strains which produced truncated fragments of LamB, either in a free form (deletion and nonsense mutants) or fused to another polypeptide (malK-lamB and lamB-lacZ fusion strains). The conclusion is that the two antigenic determinants are located within 70 residues from the COOH-terminal end of LamB, which contains a total of 421 amino acids. Since these two antigenic sites were previously demonstrated to be exposed at the cell surface, it follows that a COOH-terminal portion of LamB must be located on the outer surface of the outer membrane.