Uptake of aflatoxin B1 and T-2 toxin by two mycotoxin bioassay microorganisms: Kluyveromyces marxianus and Bacillus megaterium.

Uptake of aflatoxin B1 (AFB1) and trichothecene T-2 toxin from growth medium by mycotoxin bioassay strains of Klutyveomyces marxianus and Bacillus megaterium was assessed by incubating, washing, and sonicating the cells, extracting samples with chloroform, and analysing the extracts by a combination of high-performance thin-layer chromatography (HPTLC) and fluorescence densitometry. Using cultures of K. marxianus, the entire AFB1 dose was recovered and no AFB1 metabolites were detected. Less than 1% of the AFB1 was recovered from the cells, and AFB1 did not inhibit growth. Methanol in the incubation medium had no significant effect on the levels of AFB1 associated with K. marxianus cells. The entire dose of T-2 toxin was also recovered from K. marxianus cultures, and no metabolites were detected; again, less than 1% of T-2 toxin was cell-associated, but growth was completely inhibited. AFB1 partially inhibited the growth of B. megaterium; approximately 12% of the dose could not be recovered, and no AFB1-related metabolites were detected. Methanol increased the levels of recoverable AFB1 associated with B. megaterium cells. In the case of T-2 toxin, around 8% of the dose was not recovered, and no metabolites were detected; growth of B. megaterium was stimulated. These results suggest irreversible binding of both toxins, or derivatives of them, to the cells of B. megaterium.

A novel colorimetric yeast bioassay for detecting trichothecene mycotoxins.

A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of beta-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 microl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the beta-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

A colorimetric technique for detecting trichothecenes and assessing relative potencies.

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.

Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning, sequencing and chromosomal localization of its TRP1 gene.

The genome of the amylolytic yeast strain Lipomyces starkeyi NCYC 1436 was analysed using contour-clamped homogeneous electric field gel electrophoresis (CHEF). The banding pattern under a variety of running conditions indicating the presence of 11 different chromosome-sized DNA molecules. The sizes of these chromosome bands were determined by comparison with chromosomes from standard strains of Schizosaccharomyces pombe and Saccharomyces cerevisiae. The chromosomal bands were estimated to be within the range 0.7-2.8 Mb, with the genome (excluding mitochondrial DNA) estimated at 15 Mb. The molecular cloning of the TRP1 gene, isolated from a genomic library of this strain, is also reported: the gene was present on a 6.5-kb Sau3A DNA fragment, and complemented the trpC gene of E. coli. The DNA sequence was determined (EMBL accession No. Z68292) and compared to other tryptophan biosynthetic genes encoding N-(5'-phosphoribosyl) anthranilate isomerase (PRAI) activity. The gene was also used as a probe in hybridization studies, and by this means, its chromosomal location was identified.

Cloning of the HIS3 gene of Yarrowia lipolytica.

The HIS3 gene of the yeast Yarrowia lipolytica has been cloned from a genomic library by complementation of the his3 mutation of Saccharomyces cerevisiae. The gene was subsequently subcloned in Escherichia coli and characterized by restriction enzyme mapping.

Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis.

The chromosomal locations of four glucoamylase-specifying genes in the yeast Saccharomyces cerevisiae have been determined. Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelled STA2 and marker DNA from specific yeast chromosomes. The three genes encoding extracellular glucoamylases, STA1 (DEX2), STA2 (DEX1) and STA3 (DEX3) are located on chromosomes IV, II and XIV, respectively. SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.

A genetic analysis of glucoamylase activity in the diastatic yeast Saccharomyces cerevisiae NCYC 625.

The wild diastatic yeast Saccharomyces cerevisiae NCYC 625 has been shown to be homozygous for the glucoamylase-specifying gene STA2. spoII-1-mapping has positioned STA2 on chromosome II. Expression of STA2 is suppressed in some but not all diploids capable of sporulation, and is also inhibited by unlinked nuclear suppressor genes (SGL) found in some S. cerevisiae tester strains. EMS-induced glucoamylase-negative mutants often contain STA2-suppressor mutations. Depending on the allelic status of GEP1, a nuclear gene which also appears able to antagonise SGL-mediated suppression, STA2 expression can be blocked in petite mutants.

Vanadate inhibition of mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae.

The effects of vanadate on mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae were studied. A 50% inhibition of oxygen uptake in isolated mitochondria was produced by 4.4 mM-V2O5. Activity of H+ ATPase in whole mitochondria was inhibited by 50% by 5.5 microM-V2O5, in submitochondrial particles by 55 microM-V2O5; and in the chloroform-released H+ ATPase by 0.5 mM-V2O5. Vanadate was also found to relieve growth inhibition caused by the mitochondrial H+ ATPase inhibitors NN'-decyclohexylcarbodiimide and oligomycin. These results imply that vanadate could affect mitochondrial respiration by interacting with the H+ ATPase in S. cerevisiae.

The effects of vanadate on the yeast Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on non-fermentable medium was more sensitive to inhibition by vanadate than growth on fermentable medium. The frequency of petite mutants increased in cultures grown for 18 hours in fermentable medium containing vanadate. However, oxygen uptake markedly increased in yeast cultures grown in the presence of vanadate, a similar effect being produced by phosphate. It was also found that oligomycin toxicity was relieved by vanadate. These results suggest that vanadate may interact with the mitochondria of S. cerevisiae.

Purification and properties of an extracellular glucoamylase from a diastatic strain of Saccharomyces cerevisiae.

The extracellular glucoamylase from certain strains of Saccharomyces cerevisiae can be purified from culture medium by a simple chromatographic procedure. The native enzyme is heavily glycosylated and has an Mr of about 250,000, but gel filtration indicates the existence of oligomers of larger size. Dissociation yields a form of Mr about 70,000. The glucoamylase is rich in serine and threonine and in aspartic acid plus asparagine, and has a pI of 4.62 and a pH optimum of 4.5-6.5. The thermostability and resistance to denaturants of the yeast enzyme is compared with those of two other fungal glucoamylases. Kinetic data for the yeast enzyme and a variety of substrates is presented; the enzyme is particularly ineffective in cleaving alpha-(1----6)-glycosidic bonds.

Mitochondrial control of cell surface characteristics in Saccharomyces cerevisiae.

Defects in the inner mitochondrial membrane of petite mutants of yeast resulted not only in respiratory deficiency, but also in changes in cell surface characteristics. These were (1) concanavalin A agglutinability, (2) cell movement in a biphasic polymer system, (3) cell adhesion. Genetic analysis indicated that the control exerted by the mitochondria was on nuclear genes or on the products of these genes which were presumably specifying cell surface components. These findings ascribe a new role to mitochondria but also have implications for neoplastic transformation.

Primary antimitochondrial activity of the cancer drug methylglyoxal bis(guanylhydrazone) in yeast cells.

Primary action of methylglyoxal bis(guanylhydrazone) (MGBG) on the yeast mitochondrial system was demonstrated by (1) selective inhibition of cell growth in non-fermentable medium, (2) blockage of mitochondrial synthesis of cytochromes aa3 and b and (3) ultrastructural aberration. The drug caused extensive deletions in mitochondrial DNA detected by an increase in the frequency of the mitochondrial mutant petite but had little or no effect on cell viability. Growth inhibition by MGBG had any effect on growth inhibition by ethidium bromide. Strains showed no cross-correlation in their tolerance to MGBG and ethidium bromide.

The fractionation of suspensions of isolated hepatocytes by rate zonal centrifugation.

Parenchymal cells, isolated from rat liver by a simple non-enzymic technique, were fractioned according to ploidy class by rate zonal centrifugation on sucrose density gradients. This method of fractionation applied to liver cells prelabelled in vivo with tritiated thymidine separated different size classes of cells synthesising DNA.

Erythropoiesis and iron metabolism in Hodgkin's disease.

Recently developed techniques for the investigation of iron kinetics were used to study the disturbance of iron metabolism in 19 untreated patients with Hodgkin's diseases (HD). The erythroid abnormality in newly diagnosed HD appears to be confined to those patients with systemic symptoms of weight loss, night sweats and fever, and consists of depression of marrow erythroid activity. These patients had a significnatly lower haemoglobin and serum iron concentration and a higher serum ferritin concentration, both when compared to normal subjects and to those patients with HD who lacked systemic symptoms. Ineffective erythropoiesis and red-cell destruction were not significantly increased. The present findings, confirm that HD patients with systemic symptoms have a depression of erythropoiesis, and that in these patients the marrow fails to respond to the stimulus of mild anaemia.

Toxic and mutagenic effects of carcinogens on the mitochondria of Saccharomyces cerevisiae.

Nineteen haploid yeast (Saccharomyces cerevisiae) strains were used to assess the relative growth inhibitory potencies on fermentable vs. non-fermentable media of a collection of carcinogenic and non-carcinogenic chemicals. The majority of carcinogens were distinctly more potent on the non-fermentable (glycerol) medium, where mitochrondrial function is required for growth, than on the fermentable medium, where it is not. The anti-mitochondrial selectivity indicated by these growth tests was much slighter for the non-carcinogens. Similarly most carcinogens induced the cytoplasmic petite mutation whereas the non-carcinogens did not. Five carcinogens which were tested impaired the development of cytochromes aa3 and b in glucose cultures. Six carcinogens, when tested, inhibited growth on three fermentable sugars, the utilisation of which requires mitochondrial function. Out of five carcinogens which were examined, four suppressed the surface-dependent phenomenon of fluocculence in a flocculating strain of yeast, at concentrations primarily affecting the mitochondrial system; the fifth had a similar but less pronounced effect.

Cancer-specific density changes in lymphocytes after stimulation with phytohaemagglutinin.

A fluorescence polarisation technique ("S.C.M. test") for detecting responses to phytohaemagglutinin revealed that the responsive lymphocytes from patients with malignant disease had an abnormal distribution after centrifugation through lymphocyte separation medium. In a small blind series the technique accurately distinguished patients with histologically proven malignancies from those with nonmalignant disease and normal people.