Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)—that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R-OOHs). The concentration of tert-butyl hydroperoxide (t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R-OOH concentration in solutions of peroxylinolenic acid (13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, HpETE)—two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.     

Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid

Main conclusion

Arabidopsis was engineered to produce 21.2 % punicic acid in the seed oil. Possible molecular factors limiting further accumulation of the conjugated fatty acid were investigated. Punicic acid (18:3Δ^{9cis,11trans,13cis} ) is a conjugated linolenic acid isomer and is a main component of Punica granatum (pomegranate) seed oil. Medical studies have shown that punicic acid is a nutraceutical with anti-cancer and anti-obesity properties. It has been previously demonstrated that the conjugated double bonds in punicic acid are produced via the catalytic action of fatty acid conjugase (FADX), which is a homolog of the oleate desaturase. This enzyme catalyzes the conversion of the Δ¹²-double bond of linoleic acid (18:2Δ^9cis,12cis ) into conjugated Δ^11trans and Δ^13cis -double bonds. Previous attempts to produce punicic acid in transgenic Arabidopsis thaliana seeds overexpressing P. granatum FADX resulted in a limited accumulation of punicic acid of up to 4.4 %, accompanied by increased accumulation of oleic acid (18:1?^9cis ), suggesting that production of punicic acid in some way inhibits the activity of oleate desaturase (Iwabuchi et al. 2003). In the current study, we applied a new strategy to enhance the production of punicic acid in a high linoleic acid A. thaliana fad3/fae1 mutant background using the combined expression of P. granatum FADX and FAD2. This approach led to the accumulation of punicic acid at the level of 21 % of total fatty acids and restored the natural proportion of oleic acid observed in the A. thaliana fad3/fae1 mutant. In addition, we provide new insights into the high oleate phenotype and describe factors limiting the production of punicic acid in genetically engineered plants.     

The oxidative cleavage of folates. A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C⁹N¹⁰ bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N⁵-formyl-tetrahydrofolic acid are cleaved at the C⁹N¹⁰ bond to produce p-aminobenzoylglutamic acid. N⁵, N¹⁰-methenyl-tetrahydrofolic acid, N⁵,N¹⁰-methylene-tetrahydrofolic acid, and N¹⁰-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N¹⁰-formyl-folic acid which is completely stable to the oxidative treatment employed. N⁵-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N⁵-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-³H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-¹⁴C]folic acid to label the folates in vivo is presented.     

Activity of N-acylneuraminate-9-phosphatase (NANP) is not essential for de novo sialic acid biosynthesis

BackgroundSialylation of glycoproteins and glycolipids is important for biological processes such as cellular communication, cell migration and protein function. Biosynthesis of CMP-sialic acid, the essential substrate, comprises five enzymatic steps, involving ManNAc and sialic acid and their phosphorylated forms as intermediates. Genetic diseases in this pathway result in different and tissue-restricted phenotypes, which is poorly understood.Methods and resultsWe aimed to study the mechanisms of sialic acid metabolism in knockouts (KO) of the sialic acid pathway in two independent cell lines. Sialylation of cell surface glycans was reduced by KO of GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase), NANS (sialic acid synthase) and CMAS (N-acylneuraminate cytidylyltransferase) genes, but was largely unaffected in NANP (N-acylneuraminate-9-phosphatase) KO, as studied by MAA and PNA lectin binding. NANP is the third enzyme in sialic acid biosynthesis and dephosphorylates sialic acid 9-phosphate to free sialic acid. LC-MS analysis of sialic acid metabolites showed that CMP-sialic acid was dramatically reduced in GNE and NANS KO cells and undetectable in CMAS KO. In agreement with normal cell surface sialylation, CMP-sialic acid levels in NANP KO were comparable to WT cells, even though sialic acid 9-phosphate, the substrate of NANP accumulated. Metabolic flux analysis with ¹³C₆-labelled ManNAc showed a lower, but significant conversion of ManNAc into sialic acid.ConclusionsOur data provide evidence that NANP activity is not essential for de novo sialic acid production and point towards an alternative phosphatase activity, bypassing NANP.General significanceThis report contributes to a better understanding of sialic acid biosynthesis in humans.     

Candida albicans PHO81 is required for the inhibition of hyphal development by farnesoic acid

Farnesoic acid is a signaling molecule that inhibits the transition from budding yeast to filament formation in Candida albicans, but the molecular mechanism regulated by this substance is unknown. In this study, we analyzed the function of CaPHO81, which is induced by farnesoic acid. The pho81Δ mutant cells existed exclusively as filaments under favorable yeast growth conditions. Furthermore, the inhibition of hyphal growth and repression of CPH1, EFG1, HWP1, and GAP1 mRNA expression in response to farnesoic acid were defective in pho81Δ mutant cells. These data suggest a role for CaPHO81 in the inhibition of hyphal development by farnesoic acid.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.     

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Gene Sequence and Properties of an s-Triazine Ring-Cleavage Enzyme from Pseudomonas sp. Strain NRRLB-12227

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO₂ and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K_m of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Intracellular Phospholipase A1 and Acyltransferase, Which Are Involved in Caenorhabditis elegans Stem Cell Divisions, Determine the sn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI), an important constituent of membranes, contains stearic acid as the major fatty acid at the sn-1 position. This fatty acid is thought to be incorporated into PI through fatty acid remodeling by sequential deacylation and reacylation. However, the genes responsible for the reaction are unknown, and consequently, the physiological significance of the sn-1 fatty acid remains to be elucidated. Here, we identified acl-8, -9, and -10, which are closely related to each other, and ipla-1 as strong candidates for genes involved in fatty acid remodeling at the sn-1 position of PI. In both ipla-1 mutants and acl-8 acl-9 acl-10 triple mutants of Caenorhabditis elegans, the stearic acid content of PI is reduced, and asymmetric division of stem cell-like epithelial cells is defective. The defects in asymmetric division of these mutants are suppressed by a mutation of the same genes involved in intracellular retrograde transport, suggesting that ipla-1 and acl genes act in the same pathway. IPLA-1 and ACL-10 have phospholipase A₁ and acyltransferase activity, respectively, both of which recognize the sn-1 position of PI as their substrate. We propose that the sn-1 fatty acid of PI is determined by ipla-1 and acl-8, -9, -10 and crucial for asymmetric divisions.     

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ₂ and PGA₂, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.     

Diterpene acids from Helianthus species and their microbiological conversion by Gibberella fujikuroi, mutant B1-41a

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A₉, and A₁₂ have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.     

Chemotaxonomical investigations on the occurrence of sialic acids in protostomia and deuterostomia

Several tissues of different protostome species were tested in respect to the occurence of sialic acid. There is no sialic acid in the turbellarian Euplanaria gonocephala and the mollusc Helix pomatia. In contrast to other tissues the digestive gland of the decapod crustaceans Astacus leptodactylus and Uca tangeri contains 60–80 μg of sialic acid per gram wet weight. Radioactive tracer experiments showed that Astacus leptodactylus cannot synthesize sialic acid by using the specific precursor N-acetyl-mannosamine but obviously resorbs exogenous sialic acid taken up with the food and stores it in the digestive gland. It is suggested that protostomes in general are devoid of endogenous sialic acid whereas it is abundant in deuterostomes. Contradictory to existing results low amounts of a resorcinol positive substance, probably a sialic acid, were detected in the deuterostome tunicate Phallusia mammilata.     

Brown Pigments Produced by Yarrowia lipolytica Result from Extracellular Accumulation of Homogentisic Acid

Yarrowia lipolytica produces brown extracellular pigments that correlate with tyrosine catabolism. During tyrosine depletion, the yeast accumulated homogentisic acid, p-hydroxyphenylethanol, and p-hydroxyphenylacetic acid in the medium. Homogentisic acid accumulated under all aeration conditions tested, but its concentration decreased as aeration decreased. With moderate aeration, equimolar concentrations of alcohol and p-hydroxyphenylacetic acid (1:1) were detected, but with lower aeration the alcohol concentration was twice that of the acid (2:1). p-Hydroxyphenylethanol and p-hydroxyphenylacetic acid may result from the spontaneous disproportionation of the corresponding aldehyde, p-hydroxyphenylacetaldehyde. The catabolic pathway of tyrosine in Y. lipolytica involves the formation of p-hydroxyphenylacetaldehyde, which is oxidized to p-hydroxyphenylacetic acid and then further oxidized to homogentisic acid. Brown pigments are produced when homogentisic acid accumulates in the medium. This acid can spontaneously oxidize and polymerize, leading to the formation of pyomelanins. Mn²⁺ accelerated and intensified the oxidative polymerization of homogentisic acid, and lactic acid enhanced the stimulating role of Mn²⁺. Alkaline conditions also accelerated pigment formation. The proposed tyrosine catabolism pathway appears to be unique for yeast, and this is the first report of a yeast producing pigments involving homogentisic acid.     

Biosynthesis of iridoid glucosides in Galium mollugo,G. spurium var. Echinospermon and Deutzia crenata. Intermediacy of deoxyloganic acid,loganin and iridodial glucoside

Administration of ²H-labelled compounds to Galium mollugo, G. spurium var. echinospermon and Deutzia crenata established that deoxyloganic acid is a precursor of asperuloside, geniposidic acid and secogalioside in G. mollugo as well as asperuloside in G. spurium, while iridodial glucoside is a precursor of deutzioside in D. crenata. Additionally, the intermediacy of loganic acid in the biosynthesis of the iridoid and secoiridoid glucosides in the Galium plants was reconfirmed.