The effects of topical application of various fatty acids on pheromone and glandular lipid biosynthesis in the moth Heliothis virescens

Binary mixtures of deuterium-labeled palmitic acid and an excess of different fatty acids were applied to the sex pheromone gland of female Heliothis virescens and the effects on the terminal steps of pheromone biosynthesis, including incorporation of fatty acids into the glandular lipids, observed. Relative to labeled palmitic acid applied alone, application of all the binary mixtures resulted in decreased levels of the labeled pheromone component, (Z)-11-hexadecenyl acetate (Z11-16:OAc), but there was generally no decrease in the amounts of labeled pheromone precursor, (Z)-11-hexadecenoate, nor labeled palmitate in the glandular lipids. These data suggest that the excess of fatty acid in the gland inhibits Delta11-desaturation. However, in the case of excess myristoleic acid, the amount of labeled (Z)-11-hexadecenoate increased significantly, suggesting that this acid inhibited fatty acid reduction. Dose-response tests with certain of the fatty acids were consistent with the above interpretations and further indicated that the gland had a high capacity for rapidly activating and incorporating excess fatty acids into the glandular lipids. Finally, application of the various fatty acids resulted in increased levels of these acids in the gland and, in the cases of myristoleic, palmitoleic and myristic acids, it also resulted in increased levels of the corresponding aldehydes, which had previously been detected in the gland of female H. virescens. This suggests that the fatty acid reductase in H. virescens is not highly specific for the major component, and that the final ratio of pheromone components is determined in part by the availability of their corresponding fatty acids in the gland.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

Metabolism of fatty acids by bovine spermatozoa

The incorporation of (14)C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.     

Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Alteration of the fatty acid composition of Escherichia coli by growth in the presence of normal alcohols.

The addition of normal alcohols in the series n-butanol to n-octanol to cultures of Escherichia coli ML308 grown on defined or lipid-free medium (at 17, 27, and 37 degrees C) caused an alteration in the fatty acid composition of this organism: the ratio of saturated to unsaturated fatty acids increased. Changes in the relative quantities of individual fatty acid species elicited by increasing concentrations of these alcohols were as follows: (i) myristic acid remained constant: (ii) palmitic acid increased; and (iii) the combined amount of palmitoleic plus cis-methylene hexadecanoic acids changed in a way which was reflected inversely by changes in the amount of cis-vaccenic acid. Comparable changes were not observed when cells were grown in the presence of n-nonanol and n-decanol in the concentration range tested. The changes observed upon addition of normal alcohols (n-butanol to n-octanol) paralleled, in part, the alterations in fatty acid composition observed when growth temperature was increased.     

Control of female pheromones in Drosophila melanogaster by homeotic genes

We have investigated the role of the Antennapedia and Bithorax complexes (ANT-C and BX-C) on the production of cuticular hydrocarbons in Drosophila melanogaster. In males, there is little, if any, influence of these complexes on the hydrocarbon pattern. In females, there are large and opposite effects of these complexes on diene production: two ANT-C mutations cause an increase in diene production and a reduction of monoenes, whereas most BX-C mutations result in a decrease in dienes and an increase in monoenes, although their sum remains constant. The effect is the highest in Mcp and iab6 females. It is suggested that a factor originating from the prothorax might activate the conversion of monoenes to dienes in females. The abdomen seems to have a crucial role in the production or control of pheromones: abdominal segments four to seven have the main effects, with a most dramatic effect for segments four and five.     

Biochemistry of development in insects. Stereospecific incorporation of fatty acids into triacylglycerols.

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Exogenous myristic acid acylates proteins in cultured rat hepatocytes

Fatty acid acylation is a functionally important modification of proteins. In the liver, however, acylated proteins remain largely unknown. This work was aimed at investigating fatty acid acylation of proteins in cultured rat hepatocytes. Incubation of these cells with [9,10-3H] myristic acid followed by two-dimensional electrophoresis separation of the delipidated cellular proteins and autoradiography evidenced the reproducible and selective incorporation of radioactivity from the precursor into 18 well-resolved proteins in the 10--120 kDa range and the 4--7 pH range. Radiolabeling of these proteins resulted from covalent linkage to the precursor [9,10-3H] myristic acid or to its elongation product, palmitic acid. The majority of the covalent linkages between the proteins and the fatty acids were broken by base hydrolysis, which indicated that the linkage was of thioester or ester-type. Only one of the studied proteins was attached to myristic acid via an amide linkage which resisted the basic treatment but was broken by acid hydrolysis. After incubation with [9,10-3H] palmitic acid, only two proteins previously detected with myristic acid were radiolabeled. Finally, the identified acylated proteins may be grouped into two classes: proteins involved in signal transduction (the alpha subunit of a heterotrimeric G protein and several small G proteins) and cytoskeletal proteins (cytokeratins, actin).     

Biological origins of normal-chain hydrocarbons: a pathway model based on cuticular wax analyses of maize silks

Long-chain normal hydrocarbons (e.g. alkanes, alkenes and dienes) are rare biological molecules and their biosynthetic origins are obscure. Detailed analyses of the surface lipids that accumulate on maize silks have revealed that these hydrocarbons constitute a large portion (>90%) of the cuticular waxes that coat this organ, which contrasts with the situation on maize seedling leaves, where the cuticular waxes are primary alcohols and aldehydes. The normal hydrocarbons that occur on silks are part of a homologous series of alkanes, alkenes and dienes of odd-number carbon atoms, ranging between 19 and 33 in number. The alkenes and dienes consist of a homologous series, each of which has double bonds situated at defined positions of the alkyl chains: alkenes have double bonds situated at the sixth, ninth or 12th positions, and dienes have double bonds situated at the sixth and ninth, or ninth and twelfth positions. Finding a homologous series of unsaturated aldehydes and fatty acids suggests that these alkenes and dienes are biosynthesized by a series of parallel pathways of fatty-acid elongation and desaturation reactions, which are followed by sequential reduction and decarbonylation. In addition, the silk cuticular waxes contain metabolically related unsaturated long-chain methylketones, which probably arise via a decarboxylation mechanism. Finally, metabolite profiling analyses of the cuticular waxes of two maize inbred lines (B73 and Mo17), and their genetic hybrids, have provided insights into the genetic control network of these biosynthetic pathways, and that the genetic regulation of these pathways display best-parent heterotic effects.     

Incorporation of acetate into fatty acids and lecithin by lung slices from fetal and newborn lambs

Incorporation of acetate-1-(14)C into phospholipids and fatty acids by lung slices from fetal and newborn lambs and from ewes was studied in vitro. The distribution of radioactivity in the fatty acids of neutral lipids, phospholipids, and lecithin was determined. Acetate-1-(14)C was incorporated into myristic, palmitic, and C(18) fatty acids. Of the lecithin fatty acids, myristic and palmitic were the major radioactive fatty acids. The results indicate that the lung of fetal lambs is able to synthesize lecithin containing saturated fatty acids, a major constituent of pulmonary surfactant. A marked increase in the rate of acetate incorporation into lecithin was observed during maturation, and these rates were higher than those obtained in the ewes. A possible relationship between developmental changes in lecithin biosynthesis and pulmonary surfactant is discussed.     

Influence of ethanol on the hopanoid content and the fatty acid pattern in batch and continuous cultures of Zymomonas mobilis

By thin layer chromatographic, gas-liquid chromatographic, and mass spectrometric methods 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) was shown to occur in Zymomonas mobilis. This compound contributed up to 20% to the total lipids.The fatty acid pattern and the content of hopanoids (hopene, hopanol, and THBH) were determined in batch and continuous cultures. In late exponential cells from batch cultures the relative amount of palmitic acid was increased partially at the expense of cis-vaccenic acid, when the initial glucose concentrations were increased. In a batch culture, THBH reached a maximum value in the early exponential growth phase.In an anaerobic continuous culture with a low glucose feed concentration, the THBH content and the relative amount of cis-vaccenic acid were low. The contribution of both compounds increased strongly with increasing glucose feed concentrations (i.e. at higher steady-state ethanol concentrations). The same result was found with aerobic continuous cultures which produced significant amounts of acetaldehyde and acetic acid, in addition to ethanol and carbon dioxide.It was concluded that stability and permeability of the cytoplasmic membrane of the ethanol producing bacterium Z. mobilis was regulated by variations in the distribution of hopanoids and fatty acids.Abbreviations 14:0 myristic acid - 16:0 palmitic acid - 18:1 cisvaccenic acid - THBH 1,2,3,4-tetrahydroxypentane-29-hopane     

The biosynthesis of the hydrocarbons in males and females of the millipede Graphidostreptus tumuliporus

The in vivo hydrocarbon biosynthesis in the millipede Graphidostreptus tumuliporus was studied after the injection of 1-¹⁴C-acetate, 16-¹⁴C-, and 1-¹⁴C-palmitic acid.From all precursors used an active incorporation into the unsaturated hyrocarbons (alk-1-enes, alkadienes, and alkatrienes) was observed, whereas no radioactivity was incorporated into the saturated alkanes at all, in accordance with their supposed exogenous origin (food). From the distribution of the radiolabel over both the various hydrocarbon classes and the individual hydrocarbon components it was concluded that in this millipede hydrocarbons are synthesized from fatty acids (irrespective of their chain structure) by an elongation-decarboxylation mechanism in which an α-oxidation step is involved, whilst during the decarboxylation process a terminal double bond is introduced. Thus, saturated fatty acids give rise to alk-1-enes (as is evidenced by an overwhelming incorporation of palmitic acid into the alk-1-enes), monoenoic fatty acids to alkadienes, and dienoic fatty acids to alkatrienes.The proposed mechanism for hydrocarbon biosynthesis in G. tumuliporus has not yet been described in other organisms.     

Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Delta(9)Z-C(16:1)) and oleic acid (Delta(9)Z-C(18:1)), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C(16:0)) and stearic acid (C(18:0)), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Delta(9) and Delta(11)) and substrate chain-length preferences (i.e., C(16:0) and C(18:0)); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Delta(11)Z-C(18:1)), whereas neither Delta(11)Z-C(16:1) nor palmitoleic acid (Delta(9)Z-C(16:1)) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Mechanism of biosynthesis of unsaturated fatty acids in Pseudomonas sp. strain E-3, a psychrotrophic bacterium.

Biosynthesis of palmitic, palmitoleic, and cis-vaccenic acids in Pseudomonas sp. strain E-3 was investigated with in vitro and in vivo systems. [1-14C]palmitic acid was aerobically converted to palmitoleate and cis-vaccenate, and the radioactivities on their carboxyl carbons were 100 and 43%, respectively, of the total radioactivity in the fatty acids. Palmitoyl coenzyme A desaturase activity was found in the membrane fraction. [1-14C]stearic acid was converted to octadecenoate and C16 fatty acids. The octadecenoate contained oleate and cis-vaccenate, but only oleate was produced in the presence of cerulenin. [1-14C]lauric acid was aerobically converted to palmitate, palmitoleate, and cis-vaccenate. Under anaerobic conditions, palmitate (62%), palmitoleate (4%), and cis-vaccenate (34%) were produced from [1-14C]acetic acid, while they amounted to 48, 39, and 14%, respectively, under aerobic conditions. In these incorporation experiments, 3 to 19% of the added radioactivity was detected in released 14CO2, indicating that part of the added fatty acids were oxidatively decomposed. Partially purified fatty acid synthetase produced saturated and unsaturated fatty acids with chain lengths of C10 to C18. These results indicated that both aerobic and anaerobic mechanisms for the synthesis of unsaturated fatty acid are operating in this bacterium.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

Cuticular lipids of adults and puparia of the Australian sheep blowfly Lucilia cuprina (Wied.)

The presence of a strong contact component in the sex and ovipositing behavior of the sheep blowfly Lucilia cuprina Wied. prompted an investigation into the chemical composition of the cuticular wax of the adult male and female flies as well as that of the blowfly puparia. Thin-layer chromatography indicated that the lipids in all the waxes examined comprise hydrocarbons, nonglyceryl esters, triglycerides, free fatty acids, and hydroxy compounds, probably diglycerides and monoglycerides. Phospholipids were not detected. Straight-and branched-chain saturated compounds, the latter often pre-dominating, are present in the hydrocarbon, free fatty acid, and ester fractions. Unsaturated molecules were absent. The hydrocarbons resemble those of the cricket to some extent, but the absence of unsaturated compounds is in striking contrast to both the cricket and the cockroach. Pheromones may be present in the low molecular weight fatty acids obtained on brief extraction of the insects.     

Changes in the fatty acid composition of Drosophila melanogaster during development and ageing.

In Drosophila melanogaster the saturated fatty acids increase in amount early in pupal development relative to the concentrations in late third instar larvae, then decline to the levels characteristic of one-day-old adults. Conversely, the monounsaturated fatty acids decline in content early in pupal development, then increase late in the pupal period. Lauric acid (12:0), myristic acid (14:0) and palmitoleic acid (16:1) become more prominent and oleic acid (18:1) and palmitic acid (16:0) less prominent as the adult ages. At about 40 days of adult age myristic acid (14:0) begins to decrease and oleic acid (18:1) to increase. Within 20 days of eclosion males and females contain different amounts of myristic acid (14:0), palmitic acid (16:0) and oleic acid (18:1).     

Insulin effect on some biochemical and biophysical characteristics of lung surfactant

The incorporation of [14C]palmitic acid into rat alveolar wash total phospholipids and phospholipid fractions has been followed for 6, 8, 10 and 12 hr after insulin administration, indicating a considerable enhancement. The fatty acid profiles of phosphatidylcholines, phosphatidylethanolamines and phosphatidylglycerols were found changed after the hormone administration. Eight hours post insulin treatment the precursor incorporation was highest in all phospholipid fractions studied, as well as the contribution of long chain fatty acids. Dynamic monolayer studies of the lung wash lipid extracts indicated a maximally expanded lipid film corresponding to the highly unsaturated phospholipids present.