Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts.

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.

Ethylene modulates development and toxin biosynthesis in aspergillus possibly via an ethylene sensor-mediated signaling pathway.

Ethylene, a biologically active natural compound, inhibited aflatoxin accumulation by Aspergillus parasiticus on a solid growth medium in a dose-dependent manner at concentrations of 0.1 to 150 ppm. The activity of the nor-1 promoter (an early aflatoxin gene) was reduced to nondetectable levels by similar quantities of ethylene, suggesting that the inhibitory effect on toxin synthesis occurred, at least in part, at the level of transcription. The inhibitory effect of ethylene on aflatoxin accumulation was also observed when A. parasiticus was grown on raw peanuts. Under similar growth conditions and doses, ethylene strongly inhibited development of asci and ascospores in Aspergillus nidulans, with no detectable effect on Hülle cell formation, conidiation, or sterigmatocystin accumulation. During early growth, A. parasiticus and A. nidulans produced ethylene with approximately twofold higher quantities measured in continuous light than in the dark. 1-Methylcyclopropene (an inhibitor of ethylene receptors in plants), light, CO2, temperature, and growth medium composition altered the effect of ethylene on A. nidulans and A. parasiticus. These observations are consistent with the existence of an ethylene sensor molecule that mediates the function of an ethylene-responsive signaling pathway(s) in Aspergillus.

Evidence that MRas1 and MRas3 proteins are associated with distinct cellular functions during growth and morphogenesis in the fungus Mucor racemosus.

The filamentous fungus Mucor racemosus provides a simple and unique model system for defining the function of individual ras genes in a gene family which is closely related to mammalian ras genes. The current study was designed to investigate the role of Mras1 and Mras3 in different stages of fungal morphogenesis, including sporangiospore germination, sporulation, and dimorphic transitions. The overall patterns of Mras1 and Mras3 transcript and protein accumulation were markedly different but, in general, transcripts and proteins were present at low levels during spherical growth and their accumulated level increased severalfold during polar growth (germ tube emergence and elongation). In contrast to Mras1, relatively high levels of Mras3 transcript accumulated during sporulation and MRas3 protein accumulated in sporangiospores. Transformation of M. racemosus with an activated allele of Mras3 reduced growth rate during aerobic sporangiospore germination, while a dominant-negative allele of Mras3 caused a 40% decrease in viable asexual spores. An activated allele of Mras1 increased growth rate during sporangiospore germination but neither activated nor dominant-negative alleles of Mras1 affected total number of asexual spores. Expression of MRas3 and MRas1 proteins appear to be subject to different regulatory mechanisms: exogenous dibutyryl-cAMP and fusidienol caused a strong repression of the level of MRas3 protein (but not MRas1) concurrent with the inhibition of polar growth. Differential posttranslational modification and intracellular localization of MRas1 and MRas3 proteins were also observed. The data strongly suggest that Mras3 and Mras1 play different roles in regulation of cell growth and morphogenesis in Mucor.

Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus.

The filamentous dimorphic fungus Mucor racemosus possesses three ras genes, Mras1, 2, and 3, whose expression is correlated to morphogenesis of the fungus. Lovastatin, an indirect inhibitor of protein prenylation, altered the processing of MRas1 protein, blocked the accumulation of MRas3 protein, and caused the MRas1/p20 protein complex to disappear in M. racemosus. Concurrently it arrested sporangiospore germination, decreased growth rate, caused a loss of cell viability accompanied by cell shrinkage, increased cell density and cytoplasm condensation, and triggered DNA fragmentation, resulting in nucleosomes and nucleosome multimers. The specific morphological and biochemical events seen in Mucor cell death, particularly DNA fragmentation, resemble the best known characteristics of classical apoptosis in mammalian cells and prompted us to classify lovastatin-induced cell death as an apoptosis-like process. Lovastatin did not cause cell death in a leucine auxotroph of Mucor grown in YNB minimal medium, conditions which support only spherical growth during spore germination. Exogenous dibutyryl-cAMP initiated morphogenesis from hyphal (polar) growth to yeast-like (spherical) growth during spore germination and strongly prevented cell death which resulted from lovastatin treatment. Wortmannin added together with dibutyryl-cAMP showed a synergistic effect in the prevention of fungal cell death. These data suggest that the regulation of lovastatin-induced cell death in Mucor requires a signal transduction pathway(s) involving cAMP whose function is specific to a particular developmental stage.

[Disruption of the regulation of glycogen phosphorylation by biogenic amines]. / O narushenii reguliatsii fosforoliza glikogena biogennymi aminami.

In rats with experimental cardiomyopathy the content of liver glycogen was considerably higher than in controls and the biogenic amines had no activating effect on glycogen phosphorolysis. The activity of monoamine oxidase was lower in experimental animals than in controls. After injections of monoamine oxidase inhibitors to normal rats no activation of phosphorolysis by biogenic amines was observed.

[Disruption of hydrolytic decomposition of glycogens in the liver of rats with experimental autoimmune cardiomyopathies]. / Narushenie gidroliticheskogo rasshchepleniia glikogena v pecheni krys s éksperimental'noi autoimmunnoi kardiomiopatiei.

Rate of glycogen hydrolytic destruction, catalyzed by cytoplasmic enzymes, namely by alpha-amylase, was decreased in liver tissue of rats with experimental autoimmune cardiomyopathy. These findings are discussed in relation to an increase of the glycogen content in liver tissue of the impaired animals.

[Histological, electron microscopic and biochemical study of the rat liver in experimental cardiomyopathy]. / Gistologischeskoe, élektronno-mikroskopicheskoe i biokhimicheskoe izuchenie pecheni krys s éksperimental'noi kardiomiopatiei.

Morphological, electron microscopic, and biochemical studies of the livers of rats with experimental autoimmune cardiomyopathy were carried out. Morphological alterations of the liver included changes in hepatocytes, significant deposition of glycogen in liver cells, fatty dystrophy, signs of necrobiosis and necrosis in some parts of the liver. Electron microscopic studies of intracellular localization of glycogen revealed its accumulation in the cytoplasm, glycogen granules being represented mostly by alpha-particles. Lysosomes filled with glycogen were also demonstrated. These data are in good accord with the results of biochemical studies. The experimental results suggest possible causes of increased content of glycogen in the cytoplasm and lysosomes of liver cells in animals with experimental autoimmune cardiomyopathy, as compared with normal animals. The relationship between disorders in glycogen and lipid metabolism in this disease is discussed.

[Characteristics of glycogen metabolic regulation in rats with experimental autoimmune cardiomyopathy]. / Osobennosti reguliatsii obmena glikogena u krys s éksperimental'noi autoimmunnoi kardiomiopatiei.

Alterations in glycogen metabolism and in its regulation were found in heart muscle and liver tissue, but not in sceletal muscle, of rats with experimental autoimmune cardiomyopathy. In development of the pathological process content of glycogen was unaltered in heart muscle as compared with the normal state; at the same time, activities of glycogen synthetase, phosphorylase and acid alpha-glucosidase were increased. After administration of adrenaline, noradrenaline and serotonin into animals with the cardiomyopathy, the phosphorylase from heart muscle was not activated, contrary to the respone of the enzyme under normal conditions. Content of glycogen was drastically increased in liver tissue of rats with experimental cardiomyopathy, as compared with the normal state. Aministration of biogenic amines into the animals led to a decrease in concentration of glycogen in liver tissue and to increase in activity of acid alpha-glucosidase, but did not affect the activities of phosphorylase A and glycogen synthetase. A possibility is considered for a decrease of the abnormally high content of glycogen in liver tissue of the impaired rats using treatment with biogenic amines.

[Effect of noradrenaline and serotonin on glycogen metabolism in rat tissues]. / Vliianie noradrenalina i serotonina na obmen glikogena v tkaniakh krysy.

Effects of biogenic amines--noradrenaline and serotonin--on phosphorylase, acid alpha-glucosidase (gamma-amilase), glycogen synthetase as well as on content of glycogen in rat liver tissue, heart and skeletal muscles were studied in vivo. Administration of noradrenaline and serotonin led to activation of phosphorylase in liver tissue, heart and skeletal muscles. Noradrenaline, administered into rats, caused a decrease in activity of acid alpha-glucosidase in heart and liver tissues and did not affect the enzymatic activity in skeletal muscles. Serotonin did not cause any effect on the activity of acid alpha-glucosidase in all the tissues studied. After administration of both amines inhibition of glycogen synthetase occurred in heart muscle, whereas the enzymatic activity was unaltered in skeletal muscles and liver tissue. Content of glycogen was decreased in heart muscle of the rats in which noradrenaline was administered. Content of glycogen was increased after serotonin administration in similar experiments. In liver tissue both amines caused a decrease in glycogen concentration and did not affect its content in skeletal muscles. Possible interrelationship is discussed between phosphorolysis and hydrolysis of glycogen under conditions of myocardial hypoxia, caused by noradrenaline administration.

[Effect of biogenic amines on phosphorolysis and gamma-amylolysis of glycogen in the cardiac muscle of rats under anesthesia]. / Vliianie biogennykh aminov na fosforoliz i gamma-amiloliz glikogena v serdechnoi myshtse krysy pri anestezii.

Adrenaline, poradrenaline, serotonin, triptamine and 3-hydroxytyramine activated glycogen phosphorolysis in heart of nonanesthetized rats due to increase in the phosphorylase A activity. Anesthesia with nembutal and ether prevented the stimulating effect of biogenic amines (excluding serotonin) on phosphorolysis. Adrenaline, administered into animals anesthetized with nembutal, inhibited the glycogen phosphorolysis. Noradrenaline caused a decrease in gamma-amylolysis of glycogen in anesthetized and untreated rats. The inhibitory effect of adrenaline on glycogen gamma-amylolysis occurred in rat heart muscle only under the conditions of anesthesia.

[Immobilization of phosphorylase B and study of its enzymatic and immunological properties]. / Immobilizatsiia fosforilazy b i izuchenie EE fermentativnykh i immunologicheskikh svoistv.

The properties of phosphorylase B (PhB) immobilized on an agar derivative were studied. It was shown that the enzyme activity makes up to 15-20% as compared to that of the soluble enzyme, the Km value for glucose-1-phosphate is increased 1.5-fold and the pH optimum remains unchanged, whereas the thermostability of enzyme shows a considerable increase. PhB immobilized on a highly activated sorbent completely losses its enzymatic activity but retains its antigenic properties and binds 1.6-2 mol antibodies (per monomer). Using immunosorbents, purified antibodies homogeneous during electrophoresis in polyacrylamide gel were isolated. The immunosorbent capacity is 500-800 mg of antibodies per 1 g of dry weight. The purified antibodies are characterized by a lower inhibitory power upon interaction with soluble PhB. The type of inhibition of both immobilized and soluble enzyme is similar. It is assumed that immobilization produces conformational changes only at the active site of enzyme, which is spatially separated from the antibody binding site.