Instability of N-acetylated fumonisin B1 (FA1) and the impact on inhibition of ceramide synthase in rat liver slices.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides. It inhibits ceramide synthase, which is a proposed underlying mechanism responsible for the myriad of toxic endpoints observed. We previously reported that N-acetylation of FB1 prevents ceramide synthase inhibition, but cautioned that impure preparations of FA1 can contain a contaminant with the ability to inhibit ceramide synthase. We now report that FA1 spontaneously rearranges to O-acetylated analogs. These rearrangement products are putative inhibitors of ceramide synthase. Rat liver slices exposed to impure FA1 containing O-acetylated FB1 had sphinganine/sphingosine (Sa:So) ratios of 1.15-1.64. Control slices had Sa:So ratios of 0.07-0.24. Clean-up to remove the O-acetylated FB1 yielded purified FA1, which produced Sa:So ratios in liver slices of 0.08-0.18. After storage for approximately 1 year as either a dry powder in a desiccator, or as a dried film at 4 degrees C, the purified FA1 again contained O-acetylated FB1, and was capable of ceramide synthase inhibition. FA1 was most stable in neutral solution, but in acidic solution the equilibrium shifted towards the O-acetylated forms. FA1 in solid form also rearranged, but more slowly than in acid solution. As FA1 is considerably less cytotoxic than FB1, these results provide additional support for the conclusion that a primary amino group is necessary for both ceramide synthase inhibition and toxicity.

Fate of fumonisins during the production of fried tortilla chips.

The fate of fumonisin B(1) (FB(1)), a mycotoxin found in corn, during the commercial manufacture of fried tortilla chips was studied. FB(1) and hydrolyzed FB(1) (HFB(1)) concentrations in four lots of corn and in the masa, other intermediates, liquid and waste byproducts, and fried chips were determined by HPLC. FB(1) concentrations in the masa and chips were reduced significantly, up to 80% in the fried chips, compared to that in the raw corn. HFB(1) was also found in the masa and chips, but at low concentrations compared to FB(1). LC-MS analyses corroborated HPLC findings and further showed the presence of partially hydrolyzed FB(1) (PHFB(1)), which, like HFB(1), was formed during the nixtamalization (cooking/steeping the corn in alkaline water to make masa) step and found predominantly in the cooking/steeping liquid and solid waste. No significant amounts of N-(carboxymethyl)-FB(1) or N-(1-deoxy-D-fructos-1-yl)-FB(1), indicative of fumonisin-sugar adduct formation, were found. Thus, FB(1) is removed from corn and diverted into liquid and waste byproducts during the commercial production of fried tortilla chips. Nixtamalization and rinsing are the critical steps, whereas grinding, sheeting, baking, and frying the masa had little effect.

Fusarium species from nepalese rice and production of mycotoxins and gibberellic acid by selected species.

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.

Rapid purification of fumonisins and their hydrolysis products with solid-phase extraction columns.

Fumonisins B(3) and B(4) (FB(3) and FB(4)) were recovered from the 50:50 acetonitrile/water extract of corn cultures of a strain of Fusarium moniliforme that does not make FB(1) or FB(2) by stirring the extract with IRA-68, a weak anion-exchange resin. The fumonisins were desorbed with 5% acetic acid in the same solvent. After dilution with water, the desorbed fumonisins were separated into FB(3) (FB(3) and FA(3)) and FB(4) (FB(4), FC(4), and FA(4)) fractions with a tC(18) solid-phase extraction (SPE) cartridge. The FB(3) fraction was then separated into FB(3) and FA(3) by using an NH(2) SPE cartridge and eluting with 5% acetic acid and increasing amounts of acetonitrile in water. Finally, FB(1) and FA(3) were hydrolyzed with calcium hydroxide. After recovery from the reaction mixture using a tC(18) cartridge, the hydrolyzed and partially hydrolyzed analogues were separated and the unreacted fumonisins recovered by using an NH(2) cartridge, initially in the normal-phase mode with increasing amounts of water in acetonitrile and then in the reversed-phase mode after the addition of 5% acetic acid to the solvent and eluting in the reverse order.

Analytical determination of fumonisins and other metabolites produced by Fusarium moniliforme and related species on corn.

Fumonisins, secondary metabolites of the fungus Fusarium moniliforme are potent toxins that can be found in fungal contaminated corn. The detection and measurement of these toxins by HPLC with detection by an evaporative light scattering detector and by electrospray MS is reported. The light scattering detector had enough sensitivity to analyze culture materials, however, clean-up was necessary to detect fumonisins at sub-ppm levels in naturally contaminated corn extracts. The detection limit for FB1 with the light scattering detector was in the low ng range (10-50) while the detection limit of less than 1 ng injected was observed for the electrospray detector. Several previously unreported fumonisin isomers were observed in electrospray chromatograms of culture extracts. Two of these compounds, FA3 and FA4 were isolated and their proposed structure confirmed by NMR experiments.

Acylated flavonoids in callus cultures of Citrus aurantifolia.

Two new acylated flavonol glycosides were isolated along with kaempferol 3-O-beta-rutinoside from 10-year-old callus cultures of Mexican lime. The structures of these new compounds are kaempferol 3-O-beta-D-glucopyranoside-6"-(3-hydroxy-3-methyl glutarate) and kaempferol 3-O-beta-D-glucopyranoside-6"-(3-hydroxy-3-methyl glutarate)-7-O-beta-D-glucopyranoside.

Identification of endogenous gibberellins in navel orange shoots.

Eight gibberellins (GAs) were identified from vegetative shoots of navel orange trees (Citrus sinensis L. Osbeck cv Washington) after sequential purification by reverse-phase C(18) high performance liquid chromatography, Nucleosil 5N(CH(3))(2) high performance liquid chromatography, and capillary gas chromatography-mass spectrometry. GA(1), GA(17), GA(19), GA(20), GA(29), and iso-GA(3) were identified based on the full scan mass spectra and Kovats retention indices. GA(8) was tentatively identified based on the comparison of the full scan mass spectra with the published spectra. GA(44) was tentatively identified from the characteristic masses at the correct Kovats retention index.

Paclobutrazol Inhibits Abscisic Acid Biosynthesis in Cercospora rosicola.

Three plant growth regulators, paclobutrazol, ancymidol, and decylimidazole, which are putative inhibitors of gibberellin (GA) biosynthesis, were studied to determine their effect on abscisic acid (ABA) biosynthesis in the fungus Cercospora rosicola. All three compounds inhibited ABA biosynthesis, and paclobutrazol was the most effective, inhibiting ABA 33% at 0.1 micromolar concentrations. In studies using (E,E,)-[1-(14)C] farnesyl pyrophosphate, it was shown that ancymidol blocked biosynthesis prior to farnesyl pyrophosphate (FPP), whereas paclobutrazol and decylimidazole acted after FPP. The three inhibitors did not prevent 4'-oxidation of (2Z,4E)-alpha-ionylideneacetic acid. C. rosiciola metabolized ancymidol by demethylation to alpha-cyclopropyl-alpha-(p-hydroxyphenyl)-5-pyrimidine methyl alcohol. Paclobutrazol was not metabolized by the fungus. Information that these plant growth regulators inhibit ABA as well as GA biosynthesis should prove useful in determining the full range of action of these compounds.

Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants.

The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds.

Extracellular siderophores of rapidly growing Aspergillus nidulans and Penicillium chrysogenum.

The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).

Chemical bioinduction of rubber in guayule plant.

The treatment of young guayule plants with 2-(3,4-dichlorophenoxy)-triethylamine stimulated the accumulation of polyisoprenoid rubber in the stem and root tissues. This finding suggests that rubber productivity can be improved by the use of chemical agents on guayule and other rubber-forming plants.