Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus.

Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis. Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases. In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS. The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate. The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid. Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods. The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains. In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively. The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A. fumigatus. The availability of detailed structures of both glycosylinositol phosphorylceramides [Levery, S. B., Toledo, M. S., Straus, A. H., and Takahashi, H. K. (1998) Biochemistry 37, 8764-8775] and cerebrosides from P. brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs. These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P. brasiliensis.     

Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity

Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.     

A bifunctional delta-fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus. A new member of the cytochrome b5 superfamily.

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis-unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Delta12-acetylenase. The gene encoding this protein, together with a Delta6-desaturase, was cloned by a PCR-based approach with primers derived from conserved regions in Delta5-, Delta6-fatty-acid desaturases and Delta8-sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N-terminal extension of unknown function, a cytochrome b5-domain, and a C-terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Delta6-desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Delta9-unsaturated C16- and C18-fatty acids by inserting an additional Delta6cis-double bond. One of these transgenic yeast clones was also able to introduce a Delta6-triple bond into gamma-linolenic and stearidonic acid. This resulted in the formation of 9,12,15-(Z,Z,Z)-octadecatrien-6-ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Delta6-acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Delta6cis-double bond into 9,12,(15)-C18-polyenoic acids as well as converting a Delta6cis-double bond to a Delta6-triple bond.     

Menstrual cycle-associated expression of 2-hydroxy fatty acyl phytosphingosine-containing GlcCer, LacCer and Gb3Cer in human uterine endometrium.

In the previous study, we found that sulfatide was characteristically expressed in the secretory phase of human uterine endometrium and that the metabolism of glycosphingolipids was strictly controlled by sex steroid hormones. Therefore, the neutral glycosphingolipid composition of human uterine endometrium in the proliferative and secretory phases was analyzed and was found to be characteristic in both phases. The major neutral glycolipids were GlcCer, LacCer, Gb3Cer and Gb4Cer. The concentrations of GlcCer, LacCer and Gb3Cer in the secretory phase were higher than those in the proliferative phase. Furthermore, on TLC, GlcCer, LacCer and Gb3Cer in the proliferative phase gave three bands, the 3rd band, which migrated to the lowest position, being much more predominant in the secretory phase. The individual band materials in both phases were purified by silica gel column chromatography, and their structures were analyzed by FABMS and GLC. The lower-migrating bands of GlcCer, LacCer and Gb3Cer were found to contain molecules with 2-hydroxy fatty acyl phytosphingosine, indicating that hydroxylation of the fatty acid and sphingosine moieties to give 2-hydroxy fatty acid- and phytosphingosine-containing glycosphingolipids, respectively, is induced selectively in the secretory phase on a change in the hormonal environment.     

花生△12脂肪酸脱氢酶基因高效表达载体构建与原核表达

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△^12脂肪酸脱氢酶（FAD2）是亚油酸合成的关键酶,催化油酸（18：1）在△^12位上脱氢生成亚油酸（18：2）,但由于△^12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△^12脂肪酸脱氢酶基因（GenBank接受号为AY1006）构建高效表达载体,把花生△^12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET／HO-A融合表达载体,并转化到大肠杆菌表达菌BL21（DE3）pLysS中,在IPTG诱导下,pRSET／HO-A融合表达载体在BL21（DE3）pLysS菌株中高效表达了△^12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,所编码的酶具有△^12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11．8％。花生△^12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.     

A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega13 desaturation in methylotrophic yeast and tobacco

Pinolenic acid (PA; 18:3Delta(5,9,12)) and coniferonic acid (CA; 18:4Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Delta(9,12)) and alpha-linolenic acid (ALA; 18:3Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1Delta(9) but not 18:1Delta(11). Unexpectedly, CrDES also showed Delta(7)-desaturase activity on 20:2Delta(11,14) and 20:3Delta(11,14,17) to produce 20:3Delta(7,11,14) and 20:4Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega13' double bonds, these results indicate that CrDES has omega13 desaturase activity for omega9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.     

Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-Delta(3)-unsaturation correlates with the yeast-mycelium phase transition

Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as beta-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-delta(3)-octadecenoate. These results suggest that the yeast-mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-delta(3)-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (< 5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-delta(8)-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.     

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.     

Epidermal sphingomyelins are precursors for selected stratum corneum ceramides

Epidermal ceramides (Cer) comprise a heterogeneous family of seven species, including two unique omega-hydroxylated Cer, that are key components of the stratum corneum (SC) intercellular lamellar membranes responsible for the epidermal permeability barrier. Although both glucosylceramide (GlcCer) and the phospho-sphingolipid sphingomyelin (SM) are potential precursors of SC Cer, based on reported chemical structures of epidermal GlcCer and SC Cer, it is assumed that all major subfractions of SC Cer are generated from lamellar body-derived GlcCer. Yet, we and others have shown that SM-derived Cer are required for normal barrier homeostasis. Moreover, two pools of SM, one from plasma membrane, the other from lamellar body-derived contents, are potentially available for Cer production. To clarify the role of SM as a potential precursor of bulk or specific SC Cer, we compared Cer moieties in epidermal SM, Cer generated from epidermal SM by sphingomyelinase treatment, Cer within SC, and Cer that persist in Gaucher SC, where GlcCer cannot generate Cer due to an absence of beta-glucocerebrosidase. Using gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance for Cer characterization, epidermal SM comprise three major subfractions with distinctive amide-linked (N-acyl) fatty acid (FA) compositions: that is, either long-chain FA (SM-1; C(22;-26)), short-chain FA (SM-2; primarily C(16)), and short-chain alpha-hydroxy FA (SM-3; C(16;-18)). In contrast, only trace quantities of omega-hydroxy FA were present. For each SM subfraction, the sphingoid base was either sphingosine or sphinganine, but phytosphingosine was not detected. Comparison of these SM with corresponding sphingomyelinase-generated epidermal Cer and SC Cer revealed that the Cer moieties of SM-1 and SM-3 are equivalent to Cer 2 (NS) and Cer 5 (AS), respectively. Moreover, both Cer 2 and Cer 5 occurred in Gaucher SC, whereas other Cer subfractions did not occur.These results indicate that two epidermal SM, that is, SM-1 and SM-3, are important precursors of two corresponding Cer in mammalian SC, that is, Cer 2 and Cer 5, but other Cer species, including the omega-hydroxy Cer species, do not derive from SM.     

The desaturase from Bacillus subtilis, a promising tool for the selective olefination of phospholipids

The Delta5-desaturase from Bacillus subtilis has been cloned in Escherichia coli BL21 cells and its enzyme activity has been investigated as a function of temperature and oxygenation by analyzing methyl ester adducts from the total lipid extract in GC-MS measurements. The present data bring out that the activity of recombinant Delta5-desaturase, at 20-22 degrees C and 20% oxygen, is surprisingly high yielding 22% of C16:1,Delta5 (5-cis-palmitoleic acid) and 13% C18:2, Delta5 Delta11 (efedrenic acid). Lower amounts of other mono- and doubly-Delta5-unsaturated fatty acids were also detected. These findings demonstrate that Delta5-desaturase can accept a multiplicity of substrates and is endowed with an unprecedented activity among other acyl-lipid desaturases thus representing a unique tool for the production of rare Delta5 unsaturated fatty acid derivatives.     

Distinct electrophoretic isozyme profiles of Fusarium graminearum and closely related species

Cellulose-acetate electrophoresis (CAE) was used to investigate isozyme polymorphisms among different isolates of Fusarium cerealis, F. culmorum, F. graminearum and F. pseudograminearum from around the world. After initial testing of 22 enzymes in three buffer systems for activity and resolution of bands, 12 proved to be appropriate for analysis of the full sample set. Remarkably uniform isozyme patterns were obtained intraspecifically, irrespective of the geographical origin of the isolates or the host/substratum from which they were isolated. This result indicated that isolates within a given species are descendant from a same ancestral population. Comparing the different electrophoretic types (ETs), adenylate kinase (AK), NADP dependent glutamate dehydrogenase (NADP GDH), peptidase B (PEP B), peptidase D (PEP ID) and phosphoglucomutase (PGM) proved to be diagnostic for at least one species examined. However, only PEP D was useful alone as a marker to distinguish the four taxa studied providing a rapid and simple CAE based diagnostic protocol. Cluster analysis of band sharing coefficients grouped the isolates into four distinct groups corresponding to the 4 species studied. Isolates of F. cerealis were clustered between those of F. culmorum and F. graminearum corroborating their known close relationship to both species. For common ETs, the similarity values between F. cerealis and F. culmorum and between F. cerealis and F. graminearum were the same. Furthermore, the similarity values and the resulting phenogram indicated that F. graminearum is more closely related to F. cerealis and F. culmorum than to F. pseudograminearum, thus the morphological similarity of F. graminearum and F. pseudograminearum does not reflect their generic relationship. This fact supports the species status of F. pseudograminearum.     

Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference

Species identity and trichothecene toxin potential of 560 members of the Fusarium graminearum species complex (FGSC) collected from diseased wheat, barley and maize in South Africa was determined using a microsphere-based multilocus genotyping assay. Although three trichothecene types (3-ADON, 15-ADON and NIV) were represented among these isolates, strains with the 15-ADON type predominated on all three hosts. A significant difference, however, was identified in the composition of FGSC pathogens associated with Gibberella ear rot (GER) of maize as compared to Fusarium head blight (FHB) of wheat or barley (P<0.001). F. graminearum accounted for more than 85% of the FGSC isolates associated with FHB of wheat and barley (N=425), and was also the dominant species among isolates from maize roots (N=35). However, with the exception of a single isolate identified as an interspecific hybrid between Fusariumboothii and F. graminearum, GER of maize (N=100) was exclusively associated with F. boothii. The predominance of F. graminearum among FHB isolates, and the near exclusivity of F. boothii among GER isolates, was observed across all cultivars, collection dates, and provinces sampled. Because these results suggest a difference in host preference among species of the FGSC, we hypothesize that F. graminearum may be less well adapted to infect maize ears than other members of the FGSC.     

Cryptoregiochemistry of the delta11-myristoyl-CoA desaturase involved in the biosynthesis of Spodoptera littoralis sex pheromone

Many moth species biosynthesize their sex pheromones by the action of unique desaturases. These membrane-bound family of enzymes are especially interesting, since some of them produce (E)-unsaturated fatty acids either exclusively or along with the (Z)-isomer. In this article we present the first mechanistic study on one of these enzymes, namely, the Delta11-myristoyl-CoA desaturase of the moth Spodoptera littoralis. Intermolecular primary isotope effect determinations were performed in competition experiments. The unusual use of odd-number fatty acids, tridecanoic acid and deuterium-labeled tridecanoic acid, in these experiments showed the existence of a large isotope effect for the carbon-hydrogen bond cleavage at C11, but no isotope discrimination occurred in the removal of C12-H. The results of the competitive experiments are consistent with the hypothesis that this Delta11-desaturase involves a first slow, isotope-sensitive C11-H bond cleavage, with probable formation of an unstable intermediate, followed by a second fast C12-H bond removal. We suggest that a single enzyme may be responsible for the formation of both (Z)- and (E)-11-tetradecenoic acids by accommodating both gauche and anti conformers of the substrate, respectively. It is also possible that two mechanistically identical discrete enzymes are involved in each desaturation. In this case, the geometry of the resulting double bond would result from the different conformation adopted by the acyl substrate at each enzyme active site.     

Functional characterization of desaturases involved in the formation of the terminal double bond of an unusual 16:3Delta(9,12,150) fatty acid isolated from Sorghum bicolor root hairs

Sorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Delta(9)) to hexadecadienoic acid (16:2Delta(9,12)), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Delta(9,12,15)). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone.     

A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.     

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.