Timing of appearance of versiconal hemiacetal acetate esterase and versiconal cyclase activity in cultures ofAspergillus parasiticus

The sequence of steps versiconal hemiacetal acetate (VHA) to versiconal (VL) catalyzed by an esterase and VL to versicolorin B (VB) catalyzed by VL cyclase has been previously demonstrated in cell-free systems fromAspergillus parasiticus. VHA esterase and VL cyclase activities were estimated by determining the amounts of VL and VB after incubation of VHA in cell-free extracts from mycelia that were either synthesizing or not synthesizing aflatoxins. VHA esterase activity but not VL cyclase activity was present in extracts from cells grown in a nonaflatoxin-producing medium. VHA esterase activity was present in extracts from mycelia grown in aflatoxin-producing medium harvested after one to six days of incubation. VL cyclase activity was absent at one day, low at two days, maximal at three to five days, and lower at six days. VL cyclase activity appears in the later part of the growth period which is also the period of aflatoxin biosynthesis. This supports a role for VL cyclase in the aflatoxin biosynthetic pathway.     

A versiconal hemiacetal acetate converting enzyme in aflatoxin biosynthesis

Conversion of the aflatoxin biosynthetic intermediate versiconal hemiacetal acetate (VHA) in a cell free extract ofAspergillus parasiticus ATCC 15517 is investigated. The enzymatic reaction is monitored by a method using high performance liquid chromatography (HPLC). The major product of the enzymatic reaction is a water soluble compound not chloroform-extractable at pH 7.5. The product becomes chloroform extractable upon acidification of the reaction medium and is separated and quantitated by reversed-phase HPLC. It is tentatively identified as versiconal hemiacetal alcohol, which is converted to versicolorin C (VC) upon acid treatment.     

Conversion of versiconal acetate to versiconal and versicolorin C in extracts from Aspergillus parasiticus

The primary product of hydrolysis of versiconal acetate catalyzed by porcine liver esterase and the 35–70% ammonium sulfate fraction from a soluble extract from mycelia of Aspergillus parasiticus was versiconal. Versiconal was stable at neutral pH for several hours and was rapidly converted to versi-colorin C by treatment with 0.4 M HCl. The addition of NADPH to the 35–70% ammonium sulfate fraction resulted in conversion of versiconal acetate to both versiconal and versicolorin C. The conversion of versiconal acetate to versicolorin C in the cell-free system is proposed to involve an esterase and an NADPH-dependent cyclase.     

Influence of trace elements and nitrogen sources on versicolorin production by a mutant strain of Aspergillus parasiticus

A mutant strain of Aspergillus parasiticus blocked in aflatoxin biosynthesis accumulates versicolorin A and versicolorin C. The effect of trace elements on the growth and versicolorin production by this strain was studied in a defined medium. The omission of manganese was slightly stimulatory to versicolorin production; when zinc was omitted from the medium, no detectable versicolorins were produced. Experiments on nitrogen sources in a highsucrose medium indicated that fourfold to fivefold increases in versicolorin yields could be obtained by substituting 3 ml/l corn steep liquor or 0.1 M NH₄NO₃ for the 0.023 M (NH₄)₂SO₃ used previously as the nitrogen source in studies on versicolorin production by this strain. These improved yields will facilitate attempts to accumulate enough versicolorin A and versicolorin C for toxicity and carcinogenicity testing. Chromatographic profiles of mycelial extracts of cultures grown in a defined medium with 0.1 M NH₄NO₃ as the nitrogen source revealed 2 previously unrecognized compounds. The accumulation of these new metabolites in a mutant blocked in aflatoxin production may indicate that they are biosynthetically related to aflatoxin.     

Effect of peanut tannin extracts on growth of Aspergillus parasiticus and aflatoxin production

Twenty-three peanut (Arachis hypogaea L.) genotypes were evaluated for kernel resistance to Aspergillus parasiticus Spear. colonization and aflatoxin contamination when incubated under high relative humidity. Also, tannin-containing extracts from kernel coats (testae) and cotyledons of these genotypes were prepared and tested for their effect on A. parasiticus growth and aflatoxin production in vitro. The lowest degree of colonization, less than 30% was noted in kernels from the genotypes, Toalson x UF 73-4022 (selections TX-798731 and TX-798736), A72118, SN 55-437, PI337409, and Florunner. Genotypes with low levels of colonization also had the lowest aflatoxin contamination. The coefficient of correlation between infection frequency and aflatoxin contamination was 0.66. Higher levels of tannins were detected in the testae (23.9–97.2 mg g tissue) compared to the cotyledons (0.17–0.82 mg g tissue). Some of the methanol-extracted and water-soluble tannin extracts from testae and cotyledons, when incorporated in yeast extract sucrose liquid medium (100 mg l), significantly inhibited A. parasiticus growth and reduced the levels of aflatoxin produced. There was no overall correlation between the peanut genotypes and the influence of tannin extracts on A. parasiticus growth and aflatoxin production. However, correlations were higher for specific genotypes. For example, the coefficient of correlation between the ability of tannin extracts from testae of genotypes PI337409 and TX-798736 to inhibit aflatoxin production was 0.93 and 0.85 respectively.     

Effect on aflatoxin production of competition between wild-type and mutant strains of Aspergillus parasiticus

Co-cultivation of a strain of Aspergillus parasiticus, capable of making aflatoxins, with blocked mutant strains, capable of producing none or only a low level of aflatoxins, reduced the net yield of aflatoxins more than that expected based on spore recovery. Yields of aflatoxins were 8-fold less for a norsolorinic acid-producing strain, 14-fold less for an averantin-producing strain, 6-fold less for an averufin-producing strain, and 21-fold less for a versicolorin A-producing strain when co-cultured in equal amounts with a wild-type strain of Aspergillus parasiticus. Even when the wild-type strain was initially present in 100-fold excess, with two of the mutant strains, reduced aflatoxin production was still observed.     

Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus

Analysis of fadA and pkaA mutants in the filamentous fungus Aspergillus nidulans demonstrated that FadA (Galpha) stimulates cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity resulting, at least in part, in inhibition of conidiation and sterigmatocystin (ST) biosynthesis. In contrast, cAMP added to the growth medium stimulates aflatoxin (AF) synthesis in Aspergillus parasiticus. Our goal was to explain these conflicting reports and to provide mechanistic detail on the role of FadA, cAMP, and PKA in regulation of AF synthesis and conidiation in A. parasiticus. cAMP or dibutyryl-cAMP (DcAMP) were added to a solid growth medium and intracellular cyclic nucleotide levels, PKA activity, and nor-1 promoter activity were measured in A. parasiticus D8D3 (nor1::GUS reporter) and TJYP1-22 (fadAGA2R, activated allele). Similar to Tice and Buchanan [34], cAMP or DcAMP stimulated AF synthesis (and conidiation) associated with an AflR-dependent increase in nor-1 promoter activity. However, treatment resulted in a 100-fold increase in intracellular cAMP/DcAMP accompanied by a 40 to 80 fold decrease in total PKA activity. ThefadAG42R allele in TJYP1-22 decreased AF synthesis and conidiation, increased basal PKA activity 10 fold, and decreased total PKA activity 2 fold. In TJYP1-22, intracellular cAMP increased 2 fold without cAMP or DcAMP treatment; treatment did not stimulate conidiation or AF synthesis. Based on these data, we conclude that: (1) FadA/PKA regulate toxin synthesis and conidiation via similar mechanisms in Aspergillus spp.; and (2) intracellular cAMP levels, at least in part, mediate a PKA-dependent regulatory influence on conidiation and AF synthesis.     

Impact on growth and aflatoxin B1 accumulation by Kluyveromyces isolates at different water activity conditions

This study showed the impact on germination, mycelial growth and aflatoxin B₁ accumulation when interacting Aspergillus aflatoxigenic strains with Kluyveromyces isolates and the effect of water activity on this relationship. Isolates Y₁₄ and Y₁₆ reduced the percentage of germination of all Aspergillus strains and decrease germ tube elongation rate at majority of water activity assayed. Similarly they produced an increase of germination lag phase and lag phase of growth beside decreased growth rate of all Aspergillus strains. At water activities 0.994, 0.982, 0.955 and 0.937, no aflatoxins were produced in paired cultures with isolates Y_25, Y₂₂, Y₁₆, and Y₁₄, and Kluyveromyces isolates Y₁₄ and Y₁₆ impact both growth and aflatoxin accumulation at wide range of water activity.     

Aflatoxins in sunflower seeds: Effect of zinc in aflatoxin production by two strains of Aspergillus parasiticus

Growth and aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus parasiticus RC 12 were studied both in sunflower seed and a synthetic culture medium (with and without zinc enrichment).On a synthetic culture medium the strains behaved in different ways according to the zinc concentration.In sunflower seed medium the influence of zinc was not so evident. Thus the results show that the influence of zinc is not the same for different strains and substrates.     

Aspergillus nomius,a new aflatoxin-producing species related to Aspergillus flavus and Aspergillus tamarii

Aspergillus nomius is described and represents a new aflatoxigenic species phenotypically similar to A. flavus. Strains examined were isolated from insects and agricultural commodities. Separation from A. flavus is based on the presence of indeterminate sclerotia and a lower growth temperature. Comparisons of DNA relatedness show A. nomius to have only relatively recently evolved from A. flavus and A. tamarii.     

Suppression of spore germination and aflatoxin biosynthesis in Aspergillus parasiticus during and after exposure to high levels of phosphine

Agar cultures of toxigenic Aspergillus parasiticus NRRL 2999 were exposed to phosphine (PH3), in levels ranging from 0 to 2000 ppm (vol/vol). It was found that with PH3 concentrations of 400 ppm or higher the growth of the fungus was totally arrested. When PH3 was vented and the agar plates were exposed to open air, 100% of the initial CFU developed into fully grown colonies after PH3 levels below 300 ppm, but at higher PH3 concentrations only 50% of the colonies developed. The same strain of A. parasiticus was inoculated into high moisture corn under conditions highly favorable for aflatoxin production, and it was exposed to a range of PH3 levels. After exposure to 500 ppm PH3, both fungal growth and aflatoxin synthesis resumed shortly after elimination of the toxic gas, but after exposure to PH3 levels of 1000 ppm and higher, the physical appearance of the contaminated corn was remarkably changed, showing reduced mycelial growth and almost complete absence of green pigmentation. In addition, aflatoxin synthesis was totally absent for the remainder of the experiment (20 days). These results strongly suggest that exposure to PH3 levels of 1000 ppm or higher could bring about persistent metabolic changes in surviving Aspergillus organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Development of a homologous transformation system for Aspergillus parasiticus with the gene encoding nitrate reductase

Summary The nitrate reductase structural gene (niaD) and an niaD mutant strain were isolated from Aspergillus parasiticus and used to develop a homologous transformation system. A transformation frequency of 110 to 120 transformants per microgram linear DNA was obtained with the 10.9 kb plasmid pSL82, which contained the niaD gene of A. parasiticus. Plasmid pSL82 was also capable of complementing Aspergillus nidulans FGSC A691, a niaD mutant, though at lower frequencies. Southern hybridization analyses of A. parasiticus niaD transformants showed that the niaD gene of pSL82 had integrated into the fungal genome. In addition, vector (bacterial plasmid) sequences were also present in one of the niaD transformants.Authors with primary and equal contribution in the research project     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermal stability of aflatoxin B1 and ochratoxin A

Within this research project, the LCI (Lebensmittelchemisches Institut des Bundesverbandes der Deutschen Süßwarenindustrie e. V.) conducted systematic studies to determine the thermal stability of the mycotoxins ochratoxin A (OTA) and aflatoxin B₁, as the available literature provides contradictory data. Firstly, the said mycotoxins in pure form were subjected to thermal treament. Secondly, tests were conducted to determine the influence of certain matrix substances, including carbohydrates and proteins, on the thermal decomposition behaviour of the mycotoxins. As a result it can be said that OTA seems to be stable up to 180 °C; however aflatoxin B₁ was almost completely degraded at heating temperatures of 160 °C and above. In several model assays it could further be shown that the degradation of mycotoxins is improved by the existence of certain matrix compounds.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts

A 2-year study was carried out to determine the effect of applying nontoxigenic strains of Aspergillus flavus and A. parasiticus to soil separately and in combination on preharvest aflatoxin contamination of peanuts. A naturally occurring, nontoxigenic strain of A. flavus and a UV-induced mutant of A. parasiticus were applied to peanut soils during the middle of each of two growing seasons using a formulation of conidia-coated hulled barley. In addition to an untreated control, treatments included soil inoculated with nontoxigenic A. flavus only, soil inoculated with nontoxigenic A. parasiticus only, and soil inoculated with a mixture of the two nontoxigenic strains. Plants were exposed to late-season drought conditions that were optimal for aflatoxin contamination. Results from year one showed that significant displacement (70%) of toxigenic A. flavus occurred only in peanuts from plots treated with nontoxigenic A. flavus alone; however, displacement did not result in a statistically significant reduction in the mean aflatoxin concentration in peanuts. In year two, soils were re-inoculated as in year one and all treatments resulted in significant reductions in aflatoxin, averaging 91.6%. Regression analyses showed strong correlations between the presence of nontoxigenic strains in peanuts and aflatoxin reduction. It is concluded that treatment with the nontoxigenic A. flavus strain alone is more effective than the A. parasiticus strain alone and equally as effective as the mixture. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.     

Structural characterization andin vivo immunosuppressive activity of neuroblastoma GD2

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, G_D2. G_D2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of G_D2 in vivo were then determined in an established murine model. G_D2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus G_D2-treated mice;p<0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2×10⁶ cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as G_D2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.     

Induced synchrony in Cryptococcus neoformans after release from G2-arrest

Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.