Ultrastructural examination of cytoskeletal linkage of L-selectin and comparison of L-selectin cytoskeletal association to that of other human and bovine lymphocyte surface antigens

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells

The effects of linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA) were compared to that of palmitic acid (PA), on inflammatory responses in human monocytic THP-1 cells. When cells were pre-incubated with fatty acids for 2-h and then stimulated with lipopolysaccharide for 24-h in the presence of fatty acids, secretion of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNFalpha) was significantly decreased after treatment with LA, ALA, and DHA versus PA (P < 0.01 for all); ALA and DHA elicited more favorable effects. These effects were comparable to those for 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and were dose-dependent. In addition, LA, ALA, and DHA decreased IL-6, IL-1beta, and TNFalpha gene expression (P < 0.05 for all) and nuclear factor (NF)-kappaB DNA-binding activity, whereas peroxisome proliferator-activated receptor-gamma (PPARgamma) DNA-binding activity was increased. The results indicate that the anti-inflammatory effects of polyunsaturated fatty acids may be, in part, due to the inhibition of NF-kappaB activation via activation of PPARgamma.     

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Delta6, Delta5 and Delta4 front-end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271-280], and two predicted elongases specific for C18 Delta6 and C20 Delta5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Delta6 and Delta5 desaturases but contain Delta4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Delta4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57-52% identity, and group together with C20 Delta5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Delta8 and Delta11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaDelta6 elongase, showed a high specificity for C18 Delta6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Delta6 and Delta5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Delta5 elongases, which, together with Delta4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.     

Anti-HCV activities of selective polyunsaturated fatty acids

HCV infection can lead to chronic infectious hepatitis disease with serious sequelae. Interferon-alpha, or its PEGylated form, plus ribavirin is the only treatment option to combat HCV. Alternative and more effective therapy is needed due to the severe side effects and unsatisfactory curing rate of the current therapy. In this study, we found that several polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are able to exert anti-HCV activities using an HCV subgenomic RNA replicon system. The EC(50) (50% effective concentration to inhibit HCV replication) of AA was 4microM that falls in the range of physiologically relevant concentration. At 100microM, alpha-linolenic acid, gamma-linolenic, and linoleic acid only reduced HCV RNA levels slightly and saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid had no inhibitory activities toward HCV replication. When AA was combined with IFN-alpha, strong synergistic anti-HCV effect was observed as revealed by an isobologram analysis. It will be important to determine whether PUFAs can provide synergistic antiviral effects when given as food supplements during IFN-based anti-HCV therapy. Further elucidation of the exact anti-HCV mechanism caused by AA, DHA, and EPA may lead to the development of agents with potent activity against HCV or related viruses.     

Oxygen radical chemistry of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) are readily susceptible to autoxidation. A chain oxidation of PUFA is initiated by hydrogen abstraction from allylic or bis-allylic positions leading to oxygenation and subsequent formation of peroxyl radicals. In media of low hydrogen-donating capacity the peroxyl radical is free to react further by competitive pathways resulting in cyclic peroxides, double bond isomerization and formation of dimers and oligomers. In the presence of good hydrogen donators, such as alpha-tocopherol or PUFA themselves, the peroxyl radical abstracts hydrogen to furnish PUFA hydroperoxides. Given the proper conditions or catalysts, the hydroperoxides are prone to further transformations by free radical routes. Homolytic cleavage of the hydroperoxy group can afford either a peroxyl radical or an alkoxyl radical. The products of peroxyl radicals are identical to those obtained during autoxidation of PUFA; that is, it makes no difference whether the peroxyl radical is generated in the process of autoxidation or from a performed hydroperoxide. Of particular interest is the intramolecular rearrangement of peroxyl radicals to furnish cyclic peroxides and prostaglandin-like bicyclo endoperoxides. Other principal peroxyl radical reactions are the beta-scission of O2, intermolecular addition and self-combination. Alkoxyl radicals of PUFA, contrary to popular belief, do not significantly abstract hydrogens, but rather are channeled into epoxide formation through intramolecular rearrangement. Other significant reactions of PUFA alkoxyl radicals are beta-scission of the fatty chain and possibly the formation of ether-linked dimers and oligomers. Although homolytic reactions of PUFA hydroperoxides have received the most attention, hydroperoxides are also susceptible to heterolytic transformations, such as nucleophilic displacement and acid-catalyzed rearrangement.     

Downstream processing of algal polyunsaturated fatty acids

Little information exists on recovering polyunsaturated fatty acids from microalgae; however, methods for concentration and purification of PUFAs from fish oil have been extensively reported. This review examines recovery and purification of microalgae derived PUFAs, but techniques developed for use with fish oil are also reviewed as being potentially useful for concentration and purification from microalgae. The two main techniques for concentrating and purifying-urea fractionation and high performance liquid chromatography-are discussed in depth and attention is focused on the process developed by the authors for obtaining highly pure PUFA. Other potentially useful techniques, such as supercritical fluid extraction and lipase-catalyzed processing are detailed.     

Inhibitory action of polyunsaturated fatty acids on IMP dehydrogenase

We screened the inhibitor of mouse inosine 5'-monophosphate dehydrogenase (IMPDH) type II from natural compounds, and found that a fatty acid, linoleic acid (C18:2), inhibited IMPDH activity. In the C18:2 fatty acid derivatives, all trans-configuration (i.e., linoelaidic acid), ester form, alcohol form, and addition of the hydroxyl group of linoleic acid had no effect on inhibitory activity. Therefore, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Among the various carbon atom lengths and double bonds of fatty acids examined, the strongest inhibitor was C20:2-fatty acid, eicosadienoic acid, and 50% inhibition was observed at a concentration of 16.1 microM. Eicosadienoic acid induced the inhibition of IMPDH activity and was competitive with respect to IMP (K(i)=3.1 microM). For inhibitory effect, the C20-fatty acids ranked as follows: C20:2>C20:3>C20:1> C20:4>C20:5, and C20:0 showed no inhibition. The energy-minimized three-dimensional structures of linear-chain C20-fatty acids were calculated, and it was found that a length of 20.7-22.5A and width of 4.7-7.2A in the fatty acid molecular structure was suggested to be important for IMPDH inhibition. Docking simulation of C20-fatty acids and mouse IMPDH type II, which was homology modeled from human IMPDH type II (PDB code: 1NF7), was performed, and the fatty acid could bind to Cys331, which is a amino acid residue of the active site, competitively with IMP. Based on these results, the IMPDH-inhibitory mechanism of fatty acids is discussed.     

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.     

Tritioboration and synthesis of tritium-labeled polyunsaturated fatty acids

Methyl esters of polyunsaturated fatty acids labeled with tritium were prepared by partial stereoselective reduction of the corresponding acetylenic esters with tritiated disiamylborane, followed by protonolysis with tritiated acetic acid. The labeling was strictly specific, and the tritium atoms were located only at the carbon atoms forming the unsaturated bond(s). Synthesis of some tritiated fatty acid methyl esters with methylene-interrupted trans-cis double bonds is also reported.     

Economical downstream processing of microbial polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are important nutrients for humans and animals. Microorganisms, such as yeast, filamentous fungi, and microalgae, have successfully been modified to produce PUFAs. Apart from strain improvement and fermentation optimization, efficient and cost-effective downstream processing will determine whether production can advance from the laboratory to the factory.     

The role of polyunsaturated fatty acids in fish

The physical properties of polyunsaturated and saturated fatty acids are compared in relation to melting points and fluidity. The role of polyunsaturated fatty acids on membrane fluidity and membrane bound enzyme activity is discussed. The influence of the environment, particularly temperature, on poikilothermic animals is considered in relation to membrane fatty acid composition and metabolism. The metabolic role of polyunsaturated fatty acids of the (n-3) series and their interaction with arachidonate metabolism is discussed.