(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Characterization of a branched-chain amino-acid transporter SBAT1 (SLC6A15) that is expressed in human brain

The SLC6 gene family comprises membrane proteins that transport neurotransmitters, amino acids, or osmolytes. We report the first functional characterization of the human SLC6A15 gene, which codes for a sodium-coupled branched-chain amino-acid transporter 1 (SBAT1). SBAT1 expression is specific to the brain. When expressed in Xenopus oocytes, SBAT1 mediated Na+-coupled transport of hydrophobic, zwitterionic alpha-amino and imino acids. SBAT1 exhibited a strong preference for branched-chain amino acids (BCAA) and methionine (K0.5 80-160 microM). SBAT1 excluded aromatic or charged amino acids, beta-amino acids, glycine, and GABA. SBAT1-mediated transport of amino or imino acids was extremely temperature-dependent (Q10=9) and was inhibited at acidic pH. PKC activation reduced the plasma-membrane population of SBAT1 protein. SBAT1-mediated transport of BCAA, particularly leucine, may be an important source of amino nitrogen for neurotransmitter synthesis in glutamatergic and GABAergic neurons.     

Characterization of a novel lipid A structure isolated from Azospirillum lipoferum lipopolysaccharide

The structure of lipid A from Azospirillum lipoferum, a plant-growth-promoting rhizobacterium, was investigated. It was determined by chemical analysis, mass spectrometric methods, as well as 1D and 2D NMR spectroscopy. Because of the presence of substituents, the investigated lipid A differs from typical enterobacterial lipid A molecules. Its backbone is composed of a beta-(1,6)-linked D-glucosamine disaccharide but lacks phosphate residues. Moreover, the reducing end of the backbone (position C-1) is substituted with alpha-linked d-galacturonic acid. 3-hydroxypalmitoyl residues are exclusively connected to amino groups of the glucosamine disaccharide. Hydroxyls at positions C-3 and C-3' are esterified with 3-hydroxymyristic acids. Primary polar fatty acids are partially substituted by nonpolar fatty acids (namely, 18:0, 18:1 or 16:0), forming acyloxyacyl moieties.     

“Phloem sap analysis of Schleichera oleosa (Lour) Oken, Butea monosperma (Lam) Taub. and Ziziphus mauritiana (Lam) and hemolymph of Kerria lacca (Kerr) using HPLC and tandem mass spectrometry”

Females of lac insects especially of Kerria lacca (Kerr) secret a resin known as lac for their own protection, which has tremendous applications. Lac insect completes its lifecycle on several host taxa where it exclusively feeds on phloem sap but Schleichera oleosa (Lour.) Oken, Butea monosperma (Lam.) and Ziziphus mauritiana (Lam.) are its major hosts. Analysis of phloem sap constituents as well as hemolymph of lac insect is important because it ultimately gets converted into lac by insect intervention. Main phloem sap constituent’s viz. sugars and free amino acids and hemolymph of lac insect were analyzed using HPLC and tandem mass spectrometry, respectively. The results were transformed to relative percentage of the total sugars and free amino acids analyzed in each sample for comparison among lac insect hemolymph and the phloem sap of the three different host taxa. Sucrose (58.9 ± 3.6–85.6 ± 0.9) and trehalose (62.3 ± 0.4) were the predominant sugars in phloem sap of three taxa and hemolymph of lac insect, respectively. Glutamic acid (33.1 ± 1.4–39.8 ± 1.4) was found to be main amino acid among the phloem sap of three taxa while tyrosine (61 ± 2.6) was the major amino acid in hemolymph of lac insect. The relative percentage of non-essential amino acids (60.8 %–69.9 %) was found to be more in all the three host taxa while essential amino acids (30.1 %–35.4 %) were present at a lower relative percentage. In contrast to this, the relative percentage of essential amino acids (81.9 %) was observed to be higher as compared to non-essential amino acids (17.7 %) in lac insect hemolymph. These results led to the detection of lac insect’s endosymbionts. Moreover, this study revealed a clue regarding the importance of development of a synthetic diet for this insect so that a precise pathway of lac biosynthesis could be investigated for thorough understanding.     

Nickel speciation in the xylem sap of the hyperaccumulator Alyssum serpyllifolium ssp. lusitanicum growing on serpentine soils of northeast Portugal

Nickel speciation was studied in the xylem sap of Alyssum serpyllifolium ssp. lusitanicum, a Ni-hyperaccumulator endemic to the serpentine soils of northeast Portugal. The xylem sap was collected from plants growing in its native habitat and characterized in terms of carboxylic and amino acids content. The speciation of nickel was studied in model and real solutions of xylem sap by voltammetric titrations using Square Wave Voltammetry (SWV). The results showed that Ni transport in the xylem sap occurs mainly as a free hydrated cation (about 70%) and complexed with carboxylic acids, mainly citric acid (18%). Altogether, oxalic acid, malic acid, malonic acid and aspartic acid complexed less than 13% of total Ni. A negligible amount bounded to the amino acids, like glutamic acid and glutamine (<1%). Histidine did not play a role in Ni translocation in the xylem sap of A. serpyllifolium under field conditions. Amino acids are one of the main forms of N transport in the xylem sap, and under field conditions, N is usually a limited nutrient. We hypothesize that the translocation of Ni in the xylem sap as a free ion or chelated with carboxylic acids is ‘cheaper’ in terms of N resources.     

Structure of a major glycolipid from Thermus oshimai NTU-063

The structure of a major glycolipid isolated from the thermophilic bacteria Thermus oshimai NTU-063 was elucidated. The sugar and fatty acid compositions were determined by GC-MS and HPLC analysis on their methanolysis and methylation derivatives, respectively. After removal of both O- and N-acyl groups by alkaline treatment, the glycolipid was converted to a fully acetylated tetraglycosyl glycerol derivative, the structure of which was then determined by NMR spectroscopy (TOCSY, HSQC, HMBC). Thus, the complete structure of the major glycolipid from T. oshimai NTU-063 was established as beta-Glcp-(1-->6)-beta-Glcp-(1-->6)-beta-GlcpNAcyl-(1-->2)-alpha-Glcp-(1-->1)-glycerol diester. The N-acyl groups on the 2-amino-2-deoxy-glucopyranose residue are C15:0 and C17:0 fatty acids, whereas the fatty acids of glycerol diester are more heterogeneous including both straight and branched fatty acids from C15:0 to C18:0.     

Non-protein amino acids of Bocoa (Leguminosae; Papilionoideae).

The non-protein amino acids of the legume genus Bocoa (Papilionoideae; Swartzieae) were surveyed by LC-MS and GC-MS using extracts of herbarium leaf fragments. Bocoa alterna (Benth.) R.S. Cowan, B. decipiens R.S. Cowan, B. limae R.S. Cowan, B. mollis (Benth.) R.S. Cowan and B. racemulosa (Huber) R.S. Cowan were found to contain 2,4-methanoproline, 2,4-methanoglutamic acid, cis-1-amino-3-hydroxymethyl-cyclobutane-1-carboxylic acid and delta-N-acetylornithine. The former three compounds have otherwise only been reported from Ateleia and Cyathostegia and, therefore, the results support the relationship with these genera found in recent phylogenetic analysis of DNA sequence data. In contrast, Bocoa viridiflora (Ducke) R.S. Cowan was found to contain trans-5-hydroxypipecolic acid and trans-4-cis-5-dihydroxypipecolic acid, while trans-4-hydroxypipecolic acid and an unidentified compound were the major non-protein amino acids in B. prouacensis Aublet. The non-protein amino acid chemistry of these two species was therefore more similar to a representative of Swartzia examined, S. macrosema Harms, which also contained mono- and dihydroxypipecolic acids. The monotypic Candolleodendron brachystachyum (DC.) R.S. Cowan, considered related to Bocoa, accumulated trans-5-hydroxypipecolic acid. LC-MS data on flavonoids obtained from four of the extracts revealed the presence of flavone C-glycosides in B. viridiflora and B. prouacensis but only flavonoid O-glycosides in B. alterna and B. mollis. The chemical division of Bocoa concurs with studies of other character types and recent molecular phylogenies.     

Analysis of monosaccharides, fatty constituents and rare O-acetylated sialic acids from gonads of the starfish Asterias rubens

A previous study (Bergwerff et al., Biochimie 74 (1992) 25-37) reported that sialic acids present in Asterias rubens gonads were essentially composed of 8-methyl-N-glycolylneuraminic acid (Neu5Gc8Me), a large part of it being acetylated in position 9. Using GC/MS of heptafluorobutyrate derivatives (Zanetta et al., Glycobiology 11 (2001) 663-676) on the chloroform/methanol soluble and insoluble fractions, we showed that most sialic acids were found in the latter and demonstrated that all sialic acids were derived from N-glycolylneuraminic acid, most of them being 8-methylated, but that the majority were also acetylated in position 4 or 7 (or both positions). GC/MS analyses of the constituents liberated using acid-catalysed methanolysis verified that major glycoprotein-bound glycans were N-linked and of the gluco-oligomannosidic type. Major fatty acids were poly-unsaturated (especially C20:4) and long-chain bases were C22:1 phytosphingosine and C22:2 6-hydroxysphingenine. Major monosaccharides found in the chloroform/methanol extract (quinovose and fucose) were derived from steroidal saponins.     

Identification and preliminary functional analysis of alternative splicing of Siah1 in Xenopus laevis

Siah proteins are vertebrate homologs of the Drosophila ‘seven in absentia’ gene. In this study, we characterized two splicing forms, Siah1a and Siah1b, of the Xenopus seven in absentia homolog 1 gene (Siah1). Overexpression of xSiah1a led to severe suppression of embryo cleavage, while that of xSiah1b was not effective even at a high dose. Competition analysis demonstrated that co-expression of xSiah1a and 1b generated the same phenotype as overexpression of xSiah1a alone, suggesting that xSiah1b does not interfere with the function of xSiah1a. Since xSiah1b has an additional 31 amino acids in the N-terminus compared to xSiah1a, progressive truncation of xSiah1b from the N-terminus showed that inability of xSiah1b to affect embryo cleavage was associated with the length of the N-terminal extension of extra amino acids. The possible implication of this finding is discussed.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.     

Structure-activity relationship between carboxylic acids and T cell cycle blockade

This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.     

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

Structural analysis of a unique hybrid-type ganglioside with isoglobo-, neolacto-, and ganglio-core from the gills of the Pacific salmon (Oncorhynchus keta)

Monosialosyl gangliosides from the gills of the Pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. The unknown acidic glycolipids (M14 and M15) with slower mobility than GM1a on thin-layer chromatography were separated by Iatrobeads column chromatography and were characterized by compositional analysis, methylation analysis, chemical, and enzymatic degradation, negative-ion LSIMS, and (1)H nuclear magnetic resonance spectroscopy. Both M14 and M15 contained a same oligosaccharide core with isoglobo-, neolacto-, and ganglio-series as follows: [carbohydrate structure: see text]. The only difference between M14 and M15 was in fatty acid acylation. Analysis of the fatty acids indicated a predominance of C24:1 fatty acid in M14 and shorter chain saturated fatty acids, C14:0 and C16:0, in M15.     

N-Glycosylation of secretion enhancer peptide as influencing factor for the secretion of target proteins from Saccharomyces cerevisiae

hIL-1beta-derived polypeptide, when fused to the N-terminal end of target proteins, exerts a potent secretion enhancer function in Saccharomyces cerevisiae. We investigated the effect of N-glycosylation of the secretion enhancer peptide on the secretion of target proteins. The N-terminal 24 amino acids (Ser5-Ala28) of human interleukin 1beta (hIL-1beta) and interleukin 1 receptor antagonist (IL-1ra) were used as secretion enhancer for synthesizing recombinant human granulocyte-colony stimulating factor (rhG-CSF) from S. cerevisiae. The mutation of potential N-glycosylation site, by substituting Gln for either Asn7 of N-terminal 24 amino acids of hIL-1beta (Asn7Gln) or Asn84 of IL-1ra (Asn84Gln), resulted in a dramatic reduction of rhG-CSF secretion efficiency. In contrast, the mutant containing an additional N-glycosylation site on the N-terminal 24 amino acids of hIL-1beta (Gln15Asn) secreted twice as much rhG-CSF into culture media as wild type hIL-1beta. These results show that N-glycosylation of the secretion enhancer peptide plays an important role in increasing the secretion efficiency of the downstream target proteins. The results also suggest that judicious choice of enhancer peptide and the control of its glycosylation could be of general utility for secretory production of heterologous proteins from S. cerevisiae.     

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl-l-leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for "leucine export") was induced by leucine, l-alpha-amino-n-butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.     

Cuticular wax composition of Salix varieties in relation to biomass productivity

The leaf cuticular waxes of six Salix clones (one Salix miyabeana, one Salix dasyclados, one Salix eriocephala, two Salix purpurea, and one interspecific hybrid of Salix eriocephala x interior) with different biomass productivities were characterized by gas chromatography-mass spectrometry. Total wax content ranged from 6.3 to 16.8 microg cm(-2), and two distinct patterns of wax were measured. The wax from leaves of S. dasyclados 'SV1' differed from all other clones and was dominated by fatty acids (42%), high concentrations of n-alkanes (25%) and n-alcohols (28%), with low n-aldehyde content (4%). All other clones produced cuticular wax dominated by n-alcohols (32-51%), particularly 1-hexacosanol, with fatty acids (14-37%) and n-aldehydes (19-26%) present in lower abundances. Clones of Salix grown under identical environmental conditions produce noticeably different amounts of cuticular wax. In contrast to previous studies of Salix, total wax content was independent of biomass productivity, measured as basal area, suggesting that wax production is not directly linked with woody biomass production by shrub willows under these site conditions.