Distribution of arachidonic and eicosapentaenoic acids in the lipids of mosses.

Lipid classes from four species of mosses, Mnium cuspidatum, and Mnium medium from Minnesota, and Hylocomium splendens and Pleurozium schreberi from Alaska, were analyzed. The total lipids of all species contained 30-40% arachidonic and eicosapentaenoic acids. However, the lipids from the Alaskan mosses contained about 75% neutral lipids (triacylglycerols, steryl esters and wax esters) whereas the lipids of the other species contained only 20% or less of these neutral lipids. Consistently, monogalactosyldiacylglycerols and phosphatidylethanol-amines were enriched in arachidonic acid and the galactolipids in eicosapentaenoic acid. The distribution of these acids in the phospholipids shows some preference for position 2. Together, the highly unsaturated C20 acids represented 80% of acyl groups in steryl esters. In triacylglycerols they were at average levels, while they were much less in sulfolipids and phosphatidylglycerols. Wax esters contained very little of the highly unsaturated acids but appreciable amounts of phytol and phytenic acid were found as wax constituents.     

Inhibition of phytoalexin synthesis in arachidonic Acid-stressed potato tissue by inhibitors of lipoxygenase and cyanide-resistant respiration

Arachidonic acid-stressed potato tuber discs synthesized the phytoalexin rishitin. This synthesis was inhibited by salicylhydroxamic acid (SHAM), and to a lesser extent by tetraethylthiuram disulfide (disulfiram). Disulfiram was less effective apparently because it was inactivated in the tuber discs. Disulfiram and SHAM both inhibited cyanide-resistant respiration of whole potato discs and lipoxygenase extracted from these discs. When low disulfiram concentrations were used, the lipoxygenase inhibition was quickly overcome, again because the disulfiram apparently was inactivated by oxidation.     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Enriching marine macroalgae with eicosatetraenoic (arachidonic) and eicosapentaenoic acids by chilling

Summary Twelve macroalgae belonging to the Chlorophyta, Phaeophyta and Rhodophyta were collected from the Arabian Gulf. Field samples and samples that were first incubated at 5° C and 24° C in the light for 1 week were analysed for lipids and fatty acids. The lipid contents varied according to the macroalga and, within the Chlorophyta and Phaeophyta, some representatives accumulated more lipids at 5° C and others at 24° C. All samples of algae had similar lipid composition with only quantitative differences. The temperature did not have a common effect on the lipid composition of representative algae, although changes in the relative concentration of specific classes were recorded. The Phaeophyta and Rhodophyta were as a rule richer than the Chlorophyta in eicosatetranoic (20:4) and eicosapentaenoic (20:5) but poorer in linolenic (18:3) acids. In most of the algae, incubation at 5° C was associated with lowering the proportion of palmitic acid (16:0) in the total lipids, and, but only in the Phaeophyta and Rhodophyta, increasing the concentration of 20:4 and 20:5. These polyunsaturated fatty acids occurred in high levels in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) of the Phaeophyta and Rhodophyta but not the Chlorophyta, the MGDG and DGDG of which were rich in 18:3 and hexadecatrienoic acid (16:3). Offprint requests to: R. H. Al-Hasan     

Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum

Several strains of species of the fungal genus Pythium, and of Phytophthora cinnamomi, were screened for content of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and eicosapentaenoic acid (EPA). The aim of the investigation was to establish alternative sources of these PUFAs, which are of importance in human nutrition. As a relatively prolific producer of EPA and AA, P. ultimum strain #144 was selected for a study of conditions that enhance their production over baseline levels that are present in the fungus when cultured for 6 d at 25 degrees C with rotary shaking (120 r.p.m.) in Vogel's medium containing sucrose as the carbon substrate. The levels of AA and EPA under these conditions were 133 +/- 27 and 138 +/- 25 mg l-1 (n = 5), respectively. Maximal production of these fatty acids was accomplished by the following sequence of steps. (1) Incubate the cultures for 6 d after inoculation under the conditions described above. Then (2) add glucose to the cultures (2%, w/v, final concentration) and incubate for a further 6 d at 13 degrees C. Under these conditions, the AA content of the mycelium was 205% higher than baseline levels and the EPA content was 198% higher. (3) Allow the cultures to remain stationary for 10 d which increases the AA content to 253% above baseline levels and the EPA content by 236%. Using such a procedure, 322 mg AA l-1 and 383 mg EPA 1-1 were produced.     

The metabolism of arachidonic and eicosapentaenoic acids in human neutrophils stimulated by A23187 and FMLP

A23187 stimulates the metabolism of endogenous as well as exogenous arachidonic acid (AA) and eicosapentaenolc acid (EPA) to their corresponding leukotrienes in human neutrophils. In contrast, conflicting results have been obtained concerning the effect of FMLP on the metabolism of these fatty acids. In the present study we compared the effect of A23187 and FMLP on the release and metabolism of these fatty acids in neutrophils. Stimulation of neutrophils with A23187, but not with FMLP, resulted in detectable levels of AA in the presence or absence of BW755C (a dual inhibitor of cyclooxygenase and lipoxygenase). The absolute amount of nonesterified AA in the extracts of neutrophils exposed to the agonist A23187 in the presence of BW755C was 20% higher than that obtained in the absence of BW755C, indicating that only a small fraction of the released AA was converted to lipoxygenase products. Furthermore, significant quantities of AA and EPA metabolites were detected only after treatment of neutrophils with A23187, but not with FMLP. Both A23187 and FMLP stimulated the conversion of exogenous EPA to 5-lipoxygenase products, with A23187 being somewhat more effective. In addition, significant differences were noted on the effect of EPA and DHA on the conversion of AA to its metabolites in A23187-stimulated neutrophils. Our results provide strong evidence that the amounts of eicosanoid precursors mobilized in response to FMLP are extremely small, if any, and this appears to be the likely explanation for the lack of eicosanoid detection by HPLC in FMLP-stimulated neutrophils.     

Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids

Lipoxygenase activity was characterized in the gill tissue of fresh-water trout. Incubation of arachidonic acid with gill preparations yielded 12-hydroxyeicosatetraenoic acid as the major product, suggesting a 12-lipoxygenase. Eicosapentaenoic acid was similarly converted to the 12-hydroxyeicosapentaenoic acid. Both arachidonic acid and docosahexaenoic acid were converted with equal apparent velocities and affinities into single monohydroxy derivatives. Analyses of the hydroxy product of docosahexaenoic acid were consistent with 14-hydroxydocosahexaenoic acid. This enzyme activity was localized to the cytosolic fraction and displayed a broad pH optimum around pH 7. The enzyme was insensitive to the cyclooxygenase inhibitors indomethacin and aspirin but activity was strongly inhibited in the presence of the lipoxygenase inhibitors, SnCl2 (5 mM), esculetin (10 microM) and eicosatetraynoic acid (100 microM).     

Novel study on the elicitation of hypersensitive response by polyunsaturated fatty acids in potato tuber.

A GC-MS procedure was carried out for the simultaneous and unequivocal quantitation of both potato phytoalexin (rishitin and lubimin) accumulation and the rate of disappearance of polyunsaturated fatty acids (PUFA) and some of their esters tested as possible elicitors. Potato 5-lipoxygenase and lipolytic acyl hydrolase play a key role in hypersensitive response (HR) induction. As expected, arachidonic acid, its hydrolysable esters, and eicosapentaenoic acid elicited much higher HR than the other PUFA tested, although the latter were equally affected by potato 5-lipoxygenase. Hydroxyl radicals appear to be actively involved in the browning process. The polyaminoacid poly-L-lysine did not show any eliciting activity.     

Factors affecting the detection of potato leafroll virus in potato foliage by enzyme-linked immunosorbent assay

Using antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme-linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10^-1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme. The detection end-point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10^-2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus-free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested. PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field-grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse-grown plants. In field-grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.     

A role for ca in the elicitation of rishitin and lubimin accumulation in potato tuber tissue

Calcium and strontium ions enhanced rishitin but not lubimin accumulation in tuber tissue of potato (Solanum tuberosum cv Kennebec) treated with arachidonic acid (AA). The same cations in the presence of poly-l-lysine (PL) enhanced the accumulation of lubimin more than rishitin. In contrast, Mg²⁺ did not affect AA-elicited rishitin and lubimin accumulation and inhibited the accumulation of these compounds following application of PL. AA-elicited potato tuber tissue remained sensitive to the stimulatory effects of Ca²⁺ and Sr²⁺ up to 24 h after application of AA, but PL-elicited tuber tissue was sensitive to Ca²⁺ and Sr²⁺ for only 6 hours after PL application. Ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid and La³⁺ both inhibited rishitin and lubimin accumulation elicited by AA. The inhibition by either agent was overcome by the addition of Ca²⁺. Calcium was more effective in overcoming lanthanum inhibition when applied simultaneously than when applied 12 hours later. Lanthanum was only effective in inhibiting rishitin and lubimin accumulation when applied within 3 hours of the application of AA. Inhibition of phytoalexin accumulation was greater when La³⁺ was applied simultaneously with AA compared to La³⁺ application after AA application to discs. These observations suggest that the mobilization of calcium may play a central regulatory role in the expression of phytoalexin accumulation following elicitation in potato tissue.     

Differential accumulation of potato tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor

Accumulation of messenger RNAs in potato tuber discs was analysed during the hypersensitive response induced by treatment with the biotic elicitor arachidonic acid. In vitro translation of polysomal poly(A)⁺ RNAs indicated that the accumulation of some sixteen mRNAs varied following treatment with arachidonic acid, and that the level of thirteen of these was increased. Two cDNA closes (pSTH-1 and-2) were isolated from a library of elicitor-treated tissue cDNAs. Northern blot analysis using these clones as molecular probes indicated that the levels of at least two mRNAs were markedly increased after elicitor treatment. In hybrid-released translation experiments, each of the cDNA clones selected more than one mRNA. Translation of these mRNAs yielded two polypeptides of M_r 45 000 (for the pSTH-1 clone), and three polypeptides of M_e 17 000 (for the pSTH-2 clone). The low molecular weight polypeptides may correspond to potato pathogenesis-related (PR) proteins.     

Content of arachidonic and eicosapentaenoic acids in polar lipids from Gracilaria (Gracilariales,Rhodophyta)

Fatty acid composition, especially the distribution of eicosapolyenoic acids in several species of Gracilaria, was analyzed in relation to their taxonomy. The species have been grouped into two types based on distribution of these polyenoic acids: Type 1, which contains palmitic, oleic and arachidonic acids as the major components, and Type II, which contains eicosapentaenoic acid in addition to Type I fatty acids. Octadecapolyenoic acids were detected only in trace amounts in each Type. A similar remarkable difference also was observed in the fatty acid composition of lipid classes. The major component of eicosapolyenoic acids in Type I was arachidonic acid in all lipid classes. In Type II, eicosapentaenoic acid was the major component in monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. Arachidonic and eicosapentaenoic acids were contained in large amounts in Type II phosphatidylcholine. Grouping of Gracilaria species into Type I and Type II is not entirely consistent with morphological and taxonomic features, but the difference in fatty acid composition is likely due to genetic rather than to environmental factors.     

Rhizosphere salinity and phytoalexin accumulation in soybean

Summary Rhizosphere salinity decreased the capacity of soybean to accumulate a pterocarpanoid phytoalexin (glyceollin) in the stem in response toPhytophthora megasperma var.sojae. Rapid (48h) accumulation was depressed by NaCl, Na₂SO₄, CaCl₂ and MgSO₄ applications. Time-course accumulations was slowed by applications. Time-course accumulation was slowed by application of 0.131M NaCl. Glyceollin accumulation was also reduced in plants subjected to a period of high salinity stress (0.177M NaCl, 72 h) after a period of nonsalinized growth. Calcium chloride completely suppressed glyceollin accumulation in normally-resistant plants but no susceptibility to the fungus was observed.     

Elongation and desaturation of arachidonic and eicosapentaenoic acids in rat liver. Effect of clofibrate feeding.

The fatty acid elongation-desaturation ability of 5,8,11,14-eicosatetraenoic (20:4(n-6)) and 5,8,11,14,17-eicosapentaenoic (20:5(n-3)) acids was determined in both liver microsomal and light mitochondrial (rich in peroxisomes) fractions of untreated and clofibrate treated rats. The elongation and the subsequent desaturation steps were performed in the corresponding favorable media. 20:5(n-3) elongation was about 2-times more extensive than that of 20:4(n-6). Clofibrate feeding for 10 days resulted in a marked decrease in the elongation rate with the two substrates, while the delta 4 desaturation rate was increased. There were small differences in the elongation rate between the microsomal and light mitochondrial fractions, however, the relative delta 4 desaturation rate was higher in the light mitochondrial fraction than microsomes.     

Effect of arachidonic, eicosapentaenoic and docosahexaenoic acids on the oxidative status of C6 glioma cells

n-3 polyunsaturated fatty acids (PUFAs) have been described to have beneficial effects on brain development and in the prevention and treatment of brain damage. C6 glioma cells were incubated with 100 microM of either C20:4n-6 (ARA), or C20:5n-3 (EPA), or C22:6n-3 (DHA) for different time periods to assess whether these acids altered the cellular oxidative state. The ARA and EPA were promptly metabolised to C22:4n-6 and C22:5n-3, respectively, whereas DHA treatment simply increased the amount of DHA in the cells. Cell viability was not affected by ARA, while a cytotoxic effect was observed 72 h after n-3 PUFAs supplementation. The levels of reactive oxygen species and thiobarbituric acid-reactive substances were significantly higher in DHA-treated cells than in EPA- and ARA-treated groups. This modification in the oxidative cellular status was also highlighted by a significant increase in catalase activity and a decrease in glutathione content in DHA-supplemented cells. Glucose-6-phosphate dehydrogenase activity, an enzyme involved in redox regulation, and O2*- release were significantly increased both in EPA and DHA groups. The effect of DHA was more severe than that of EPA. No significant changes were observed in the ARA group with respect to untreated cells. These data show that EPA and DHA induce alterations in the oxidative status that could affect the glial function.     

Factors affecting the development of spraing in potato tubers infected with potato mop-top virus

A larger proportion of tubers of Arran Pilot potato growing at the surface of soil infested with potato mop-top virus (PMTV) showed spraing symptoms (brown rings) at harvest than of tubers from below the surface. Infected tubers with or without spraing developed a spraing ring when stored in darkness, first for 1–2 wk at 18 d?C and then for 1–2 wk at any constant temperature between 5 and 13 d?C. Only a faint surface ring developed when either of these periods was decreased to 1 day; 4-day periods were needed to induce distinct symptoms. Internal tuber symptoms developed more slowly than surface symptoms, and their formation was favoured by cutting the tubers in half. Additional pigmented surface rings were produced outside the first ring by successive cycles of treatment at 18 and 9d?. Spraing did not develop when the first stage of treatment was at 22–25d?, when the tubers were kept first at 10d? and then at 5d?, when the treatment at 5–13d? preceded that at 18d?, or when the tubers were kept at constant temperatures ranging from 5 to 25d?. When tubers of six potato varieties were grown in PMTV-infested soil and then stored at temperatures designed to induce symptoms, the varieties known to be the most susceptible in the field were those which had the greatest tendency to develop spraing during storage. When infected tubers were exposed to light, typical spraing symptoms were not induced, but greening of the tuber surface was much delayed in localized ring-shaped areas, so that pale weals appeared. Spraing symptoms were produced, in favourable conditions, by the reaction of cells at the periphery of the PMTV-invaded zone. Internal spraing did not prevent PMTV invading tissue outside the brown arcs; its rate of spread was about 10 μm/h at 14–18d?.     

Age-dependent alterations of linoleic, arachidonic and eicosapentaenoic acids in renal cortex and medulla of spontaneously hypertensive rats

The lipid content as well as the fatty acid pattern of triglycerides, free fatty acids (FFA), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were estimated in renal cortex and medulla of spontaneously hypertensive rats (SHR) and normotensive Wistar rats (WR) at 4,8,26 and 52 weeks of age. In general, the level of triglycerides in renal medulla appeared higher when compared with the cortex. On the other hand, PC and PE, increasing with age, were usually higher in the cortex. A decreased percentage of linoleic acid (LA) in triglycerides, of arachidonic acid (AA) in PC and of eicosapentaenoic acid (EPA) in triglycerides, FFA, PC and PE could be found in the kidneys of SHR at 8 weeks of age, i.e. during the development of hypertension. This was accompanied with a rise of AA in FFA of SHR at 8 weeks of age, which occurred with delay in WR (at 26 weeks of age). From the data presented it can be concluded that systematic alterations in the availability of individual polyunsaturated fatty acids (PUFA) in various renal lipids might be related to the onset of hypertension in SHR which should be elucidated in more detail.