TheAspergillus parasiticus polyketide synthase genepksA,a homolog ofAspergillus nidulans wA,is required for aflatoxin B1 biosynthesis

Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced byAspergillus parasiticus andAspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway inA. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipientA. parasiticus genome at a specific locus, designatedpksA. Sequence analysis suggest thatpksA is a homolog of theAspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conservedβ-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of thepksA product. Noβ-ketoacyl reductase and enoyl reductase domains were found, suggesting thatpksA does not encode catalytic activities for processingβ-carbon similar to those required for long chain fatty acid synthesis. ThepksA gene is located in the aflatoxin pathway gene cluster and is linked to thenor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose thatpksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.     

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 M tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]_i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 M) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 M) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO₄ (400 M) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine. These results indicate that free radical-induced irreversible cell injury results from a loss of protein thiols. Changes in the cellular energy charge and lipid peroxidation do not bear a simple relationship to the survival of cardiac myocytes under oxidative stress.     

Effects of Neem Leaf Volatiles on Submerged Cultures of Aflatoxigenic Aspergillus parasiticus

Microbe-free compressed air was passed continuously for a 3-day test period through an enclosed system containing fresh neem leaves; the resultant emitted volatiles were passed over the surface of submerged liquid cultures of a wild-type aflatoxigenic isolate of Aspergillus parasiticus. Aflatoxin determinations for the fungal culture that received neem-derived volatiles, after a 3-day incubation period, resulted in a 90% overall reduction in aflatoxin production and a 51% reduction in fungal biomass when compared with cultures that did not receive neem volatiles. In a separate experiment but in a similarly enclosed system, volatiles from fresh neem leaves were collected on a small Tenax column and were thermally desorbed and cryogenically focused on a capillary gas chromatography column. The neem volatiles were subsequently separated and identified by gas chromatography-mass spectrometry. Sixty-eight compounds were identified by comparison of retention times and mass spectra with either authentic compounds or spectra from a computer-assisted library database of mass spectra. It was found that 10% of the total headspace volatiles were composed of C₃ to C₉ alkenals, which are toxic to aflatoxigenic Aspergillus spp., which could explain the bioactivity that resulted in reduced biomass in the neem-treated cultures.     

Subject: Endocrinology and chronobiology

Research Notes on Avian Biology 1994: Selected Contributions from the 21st International Ornithological CongressMorphology and Physiology: Endocrinology

Subject: Endocrinology and chronobiology     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi.

The Aspergillus parasiticus aflR gene, a gene that may be involved in the regulation of aflatoxin biosynthesis, encodes a putative zinc finger DNA-binding protein. PCR and sequencing were used to examine the presence of aflR homologs in other members of Aspergillus Section Flavi. The predicted amino acid sequences indicated that the same zinc finger domain, CTSCASSKVRCTKEKPACARCIERGLAC, was present in all of the Aspergillus sojae, Aspergillus flavus, and Aspergillus parasiticus isolates examined and in some of the Aspergillus oryzae isolates examined. Unique base substitutions and a specific base deletion were found in the 5' untranslated and zinc finger region; these differences provided distinct fingerprints. A. oryzae and A. flavus had the T-G-A-A-X-C fingerprint, whereas A. parasiticus and A sojae had the C-C-C-C-C-T fingerprint at the corresponding positions. Specific nucleotides at positions -90 (C or T) and -132 (G or A) further distinguished A. flavus from A. oryzae and A. parasiticus from A. sojae, respectively. A sojae ATCC 9362, which was previously designated A. oryzae NRRL 1988, was determined to be a A. sojae strain on the basis of the presence of the characteristic fingerprint, A-C-C-C-C-C-C-T. The DNAs of other members of Aspergillus Section Flavi, such as Aspergillus nomius and Aspergillus tamarii, and some isolates of A. oryzae appeared to exhibit low levels of similarity to the A. parasiticus aflR gene since low amounts of PCR products or no PCR products were obtained when DNAs from these strains were used.     

Isodicentric Y chromosome: cytogenetic,molecular and clinical studies and review of the literature

Dicentrics are among the most common structural abnormalities of the human Y chromosome. Predicting the phenotypic consequences of different duplications and deletions of dicentric Y chromosomes is usually complicated by varying degrees of mosaicism (45,X cell lines), which may, in some cases, remain undetected. Molecular studies in patients with dicentric Y chromosomes have been few, and only two studies have attempted to determine the presence of SRY (the putative testis-determining factor gene). We report an 18-year-old female with short stature, amenorrhea, hirsutism, hypoplastic labia minora, and clitoromegaly who has a 45,X/46,X,idic(Y)(p11.32)/47,X,idic(Y)(p11.32),idic(Y) (p11.32) karyotype. Southern analysis using Y-specific probes (Y97, 2D6, 1F5, pY3.4) and polymerase chain reaction (PCR) analysis using primers for ZFY and SRY were positive for all loci tested, indicating that almost all of the Y chromosome was present. Our findings and an extensive review of the literature emphasize the importance of molecular analyses of abnormal Y chromosomes before any general conclusions can be reached concerning the relative effects of the Y-chromosome abnormality and mosaicism on sexual differentiation.     

Cyclic AMP transport across membrane vesicles of ultra-violet light irradiatedEscherichia coli

Transport of cyclic AMP acrossEscherichia coli membrane was studied using membrane vesicles. Uptake of cyclic AMP was measured using normally oriented vesicles, whereas uptake in everted vesicles was taken as a measure of the efflux of cyclic AMP. Ultra-violet irradiation of the cells led to an inhibition of both uptake and efflux of cyclic AMP across the membrane. The presence of cyclic AMP in the growth medium prior to ultra-violet irradiation caused an enhancement of the uptake and efflux. The uptake and efflux of cyclic AMP were less in vesicles from glucose grown cells as compared to the uptake and efflux by the vesicles prepared from glycerol grown cells. Similarly both uptake and efflux of cyclic AMP were more in vesicles prepared from cells grown on glycerol or glucose in the presence of cyclic AMP than in vesicles from cells grown in absence of cyclic AMP. It is suggested that the number of cyclic AMP carrier molecules were reduced in cells under catabolite repression by glucose as well as by ultra-violet irradiation     

Comparison of Assimilatory Organic Nitrogen, Sulfur, and Carbon Sources for Growth of Methanobacterium Species

Experiments document the ability of two species of autotrophic methanogens to assimilate and utilize organic substrates as the nutrient sulfur or nitrogen source and as a carbon source during growth on H₂-CO₂. Methanobacterium thermoautotrophicum strain ΔH and the mesophilic species Methanobacterium sp. strain Ivanov grew with glutamine as the nitrogen source or cysteine as the sulfur source. M. thermoautotrophicum also utilized urea as the nitrogen source and as a carbon precursor for methane and cell synthesis. Methanobacterium sp. strain Ivanov grew with methionine as the sulfur source. The growth rate of two different Methanobacterium species was lower on an organic N or S source than on ammonium or sulfide. ³⁵S and ¹⁴C tracer studies demonstrated that amino acid or urea assimilation correlated with time and amount of growth. The rate of [³⁵S]cysteine incorporation was similar in strain ΔH (34 nmol h⁻¹ mg of cells⁻¹) and strain Ivanov (23 nmol h⁻¹ mg of cells⁻¹). However, the rate of [¹⁴C]acetate incorporation was dramatically different (17 versus 208 nmol h⁻¹ mg of cells⁻¹ in strains ΔH and Ivanov, respectively). [¹⁴C]acetate accounted for 1.3 and 21.2% of the total cell carbon synthesized by strains ΔH and Ivanov, respectively. Amino acids and urea were mainly assimilated into the cell protein fraction, but accounted for less than 2.0% of the total cell carbon synthesized. The data suggest that a biochemical-genetic approach to understanding cell carbon synthesis in methanogens is feasible; mutants that are auxotrophic for either acetate, glutamine, cysteine, or methionine are suggested as future targets for genetic studies.     

Inhibition of glycolysis by furfural in Saccharomyces cerevisiae

Summary Furfural, a Maillard reaction product, was found to inhibit growth and alcohol production by Saccharomyces cerevisiae. Furfural concentrations above 1 mg ml^–1 significantly decreased CO₂ evolution by resuspended yeast cells. Important glycolytic enzymes such as hexokinase, phosphofructokinase, triosephosphate dehydrogenase, aldolase and alcohol dehydrogenase were assayed in presence of furfural. Dehydrogenases appeared to be the most sensitive enzymes and are probably responsible for the observed inhibition of alcohol production and growth.