An intermediary role for the tremorgenic mycotoxin TR-2 in the biosynthesis of verruculogen.

14C-Labelled compound TR-2, a tremorgenic mycotoxin, was administered to Penicillium raistrickii in submerged fermentation. Half of the added radiolabel was taken up by the fungus during the 60 h incubation period and the secondary metabolites subsequently isolated, principally verruculogen but also fumitremorgin B, were found to be radiolabelled. The efficiency of biosynthetic incorporation of TR-2 into verruculogen within the mycelium was at least 35%, demonstrating for the first time an intermediary role for TR-2. Fumitremorgin B was also TR-2-derived but may not be an important intermediate in verruculogen biosynthesis.     

The intermediary role of a steroid 8,14-dien-3β-ol system in ergosterol biosynthesis

1. A mechanism for the removal of the 14alpha-methyl group in ergosterol biosynthesis that involves the intermediacy of an 8,14-diene system is outlined. 2. In accordance with the requirements of this scheme, it is shown that 5alpha-ergosta-8,14-dien-3beta-ol is converted into ergosterol by Saccharomyces cerevisiae.     

The role of cis-carotenoids in abscisic acid biosynthesis

Evidence has been obtained which is consistent with 9-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography R_ts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15–20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D₂O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PA phaseic acid - t trans - Xan xanthoxin - flc flacca - not notabilis - sit sitiens The authors would like to thank the following for their help and advice: G. Britton (Department of Biochemistry, University of Liverpool, UK), B.H. Davies (Department of Biochemistry, University of Wales, Aberystwyth), P. Molnar, J. Szabolcs, D.C. Walton (Department of Biology, Suny, Syracuse, N.Y., USA), and Mr. J.K. Heald for his expert operation of the mass spectrometer. A.D.P. was supported initially by a Science and Engineering Research Council CASE award with Shell Biosciences, Sittingbourne, Kent, UK, and later by a Agricultural and Food Research Council (AFRC) grant. M.J.B. received a NATO fellowship. The mass spectrometer and HPLC-photodiode-array detector were purchased with funds provided by the AFRC.     

The role of succinic acid in the biosynthesis of levorin

The influence of succinic acid as a component of media for biosynthesis of levorin, a polyenic antibiotic was studied. It was shown that with the use of the soybean-corn medium supplemented with succinic acid (0.05-0.4 per cent) the antibiotic content in the fermentation broth was higher than that in the control. The highest stimulating effect (135 per cent) was observed with addition of 0.1 per cent of succinic acid. For providing optimal antibiotic production in the synthetic medium supplemented with succinic acid (0.4 per cent) addition of acetic acid (0.05 per cent) was required. Studies with the soybean-corn medium with and without succinic acid revealed differences in the level of p-aminoacetophenone, an aromatic fragment of the levorin molecule. Under the conditions of the medium with succinic acid the content of p-aminoacetophenone in the mycelium was higher by 10 to 18 per cent as compared to that in the control and depended on the fermentation period. The role of succinic acid in biosynthesis of levorin is discussed.     

Mode of action of melinacidin, an inhibitor of nicotinic acid biosynthesis

Melinacidin, a new antibacterial agent, blocked the synthesis of nicotinic acid and its amide in Bacillus subtilis cells. The inhibitory activity of the agent was reversed by nicotinic acid, its amide, or nicotinamide adenine dinucleotides, but not by l-kynurenine, l-3-hydroxykynurenine, l-hydroxyanthranilic acid, or quinolinic acid. These properties indicated that the antibiotic interferes with the conversion of quinolinic acid to nicotinate ribonucleotide by the enzyme quinolinate phosphoribosyl-transferase. However, the activity of a purified preparation of this enzyme derived from a Pseudomonas strain was not impaired by the antibiotic. This suggested that, in B. subtilis, melinacidin interferes with a reaction which occurs before the formation of quinolinic acid in the biosynthetic pathway leading to nicotinic acid. Failure of quinolinic acid to reverse melinacidin inhibition in B. subtilis cultures might be due to insufficient penetration of the cell membranes by quinolinate.     

On the possible role of qinghao acid in the biosynthesis of artemisinin

Artemisinin (qinghaosu), a seco-sesquiterpene peroxide, is the clinically established antimalarial principle isolated from the leaves of the Chinese medicinal herb, Artemisia annua. Recent studies have suggested that arteannuin B, another metabolite of this plant, could serve as a precursor for artemisinin. In the present study, qinghao acid, the major sesquiterpene constituent of A. annua, was converted to arteannuin B by singlet oxygen (¹O₂) generated by sensitized photo-oxygenation. The formation of this compound was monitored by high-pressure liquid chromatographic analysis, and the identity of the isolated material was established by direct comparison. Since ¹O₂ is known to play a role in biogenetic reactions, it appears that qinghao acid can serve as a biogenetic precursor for artemisinin.     

Recent advances in the role and biosynthesis of ascorbic acid in plants

The past few years have provided many advances in the role and biosynthesis of L -ascorbic acid (AsA) in plants. There is an increasing body of evidence confirming that AsA plays an important role in the detoxification of reactive oxygen species. The role of AsA in photoprotection has been confirmed in vivo with the use of Arabidopsis mutants. A player in the defence against reactive oxygen species, AsA peroxidase, has been extensively studied at the molecular level, and regulation of this key enzymatic activity appears to occur at several levels. As a cofactor in the hydroxylation of prolyl and lysl-residues by peptidyl-prolyl and -lysyl hydroxylases, AsA plays a part in cell wall synthesis, defence, and possibly cell division. The maintenance of reduced levels of AsA appears to be highly regulated, involving the interplay of both monodehydroascorbate and dehydroascorbate reductases and possibly auxin. A major breakthrough in plant AsA biosynthesis has been made recently, and strong biochemical and genetic evidence suggest that GDP-mannose and L -galactose are key substrates. In addition, evidence for an alternative AsA biosynthetic pathway(s) exists and awaits additional scrutiny. Finally, newly described Arabidopsis mutants deficient in AsA will further increase our understanding of AsA biosynthesis     

Messenger-specific role for nicotinic acid adenine dinucleotide phosphate in neuronal differentiation

Cells possess several Ca2+-mobilizing messengers, which couple stimulation at the cell surface by a multitude of extracellular cues to the regulation of intracellular Ca2+-sensitive targets. Recent studies suggest that agonists differentially select from this molecular palette to generate their characteristic Ca2+ signals but it is still unclear whether different messengers mediate different functions or whether they act in a redundant fashion. In this study, we compared the effects of nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing messenger, with that of the prototypical messenger inositol trisphosphate on cytosolic Ca2+ levels and differentiation status of PC12 cells. We demonstrate that liposomal delivery of NAADP mediated release of Ca2+ from acidic Ca2+ stores and that this stimulus was sufficient to drive differentiation of the cells to a neuronal-like phenotype. In sharp contrast, cell fate was unaffected by more transient Ca2+ signals generated by inositol trisphosphate-evoked release of endoplasmic reticulum Ca2+ stores. Our data establish for the first time (i) the presence of novel NAADP-sensitive Ca2+ stores in PC12 cells, (ii) a role for NAADP in differentiation, and (iii) that Ca2+-dependent function can be messenger-specific. Thus, differential recruitment of intracellular Ca2+-mobilizing messengers and their target Ca2+ stores may represent a robust means of maintaining stimulus fidelity in the control of Ca2+-dependent cell function.