Biological control of Fusarium moniliforme in maize.
(49/310)
Fusarium moniliforme Sheldon, a biological species of the mating populations within the (italic)Gibberella fujikuroi species complex, i.e., population A [= G. moniliformis (Sheld.) Wineland], is an example of a facultative fungal endophyte. During the biotrophic endophytic association with maize, as well as during saprophytic growth, F. moniliforme produces the fumonisins. The fungus is transmitted vertically and horizontally to the next generation of plants via clonal infection of seeds and plant debris. Horizontal infection is the manner by which this fungus is spread contagiously and through which infection occurs from the outside that can be reduced by application of certain fungicides. The endophytic phase is vertically transmitted. This type infection is important because it is not controlled by seed applications of fungicides, and it remains the reservoir from which infection and toxin biosynthesis takes place in each generation of plants. Thus, vertical transmission of this fungus is just as important as horizontal transmission. A biological control system using an endophytic bacterium, Bacillus subtilis, has been developed that shows great promise for reducing mycotoxin accumulation during the endophytic (vertical transmission) growth phase. Because this bacterium occupies the identical ecological niche within the plant, it is considered an ecological homologue to F. moniliforme, and the inhibitory mechanism, regardless of the mode of action, operates on the competitive exclusion principle. In addition to this bacterium, an isolate of a species of the fungus Trichoderma shows promise in the postharvest control of the growth and toxin accumulation from F. moniliforme on corn in storage. (+info)
Control of fumonisin: effects of processing.
(50/310)
Of about 10 billion bushels of corn that are grown each year in the United States, less than 2% is processed directly into food products, and about 18% is processed into intermediates such as high-fructose corn syrup, ethanol, and cornstarch. The vast majority of the annual crop is used domestically for animal feed (60%), and about 16% is exported. Thus, any program for controlling residues of fumonisin (FB) in food must recognize that most of the crop is grown for something other than food. Studies on the effects of wet milling on FB residues found these residues nondetectable in cornstarch, the starting material for high-fructose corn syrup and most other wet-milled food ingredients. Similar effects are noted for the dry-milling process. FB residues were nondetectable or quite low in dry flaking grits and corn flour, higher in corn germ, and highest in corn bran. Extrusion of dry-milled products reduces FB concentrations by 30-90% for mixing-type extruders and 20-50% for nonmixing extruders. Cooking and canning generally have little effect on FB content. In the masa process measurable FB is reduced following the cooking, soaking, and washing steps, with little conversion of FB to the hydrolyzed form. Sheeting, baking, and frying at commercial times and temperatures generally have no effect. In summary, all available studies on the effects of processing corn into food and food ingredients consistently demonstrate substantial reductions in measurable FB. No studies have shown a concentration in FB residues in food products or ingredients. (+info)
Prospects for reducing fumonisin contamination of maize through genetic modification.
(51/310)
Fumonisins (FB) are mycotoxins found in (italic)Fusarium verticillioides-infected maize grain worldwide. Attention has focused on FBs because of their widespread occurrence, acute toxicity to certain livestock, and their potential carcinogenicity. FBs are present at low levels in most field-grown maize but may spike to high levels depending on both the environment and genetics of the host plant. Among the strategies for reducing risk of FB contamination in maize supplied to the market, development and deployment of Fusarium ear mold-resistant maize germplasm is a high priority. Breeding for increased ear mold tolerance and reduced mycotoxin levels is being practiced today in both commercial and public programs, but the amount of resistance achievable may be limited due to complicated genetics and/or linkage to undesirable agronomic traits. Molecular markers can be employed to speed up the incorporation of chromosomal regions that have a quantitative effect on resistance (quantitative trait loci). Transgenic approaches to ear mold/mycotoxin resistance are now feasible as well. These potentially include genetically enhanced resistance to insect feeding, increased fungal resistance, and detoxification/prevention of mycotoxins in the grain. An example of the first of these approaches is already on the market, namely transgenic maize expressing Bacillus thuringiensis (Bt) toxin, targeted to the European corn borer. Some Bt maize hybrids have the potential to reduce FB levels in field-harvested grain, presumably through reduced feeding of Bt-susceptible insects in ear tissues. However, improved ear mold resistance per se is still an important goal, as the plant will still be vulnerable to noninsect routes of entry to (italic)Fusarium. A second approach, transgene-mediated control of the ability of Fusarium to infect and colonize the ear, could potentially be achieved through overexpression of specific antifungal proteins and metabolites, or enhancement of the plant's own defense systems in kernel tissues. This has not yet been accomplished in maize, although promising results have been obtained recently in other monocots versus other fungal and bacterial pathogens. Achieving reproducible and stable enhanced ear mold resistance under field conditions will be immensely challenging for biotechnologists. A third approach, transgene strategies aimed at preventing mycotoxin biosynthesis, or detoxifying mycotoxins in planta, could provide further protection for the grower in environments where FBs present a risk to the crop even when the maize is relatively resistant to Fusarium mold. In one example of such a strategy, enzymes that degrade FBs have been identified in a filamentous saprophytic fungus isolated from maize, and corresponding genes have been cloned and are currently being tested in transgenic maize. (+info)
Rat kidney pathology induced by chronic exposure to fumonisin B1 includes rare variants of renal tubule tumor.
(52/310)
The carcinogenicity of fumonisin B1 (FB1), a worldwide contaminant of corn produced by Fusaria species of fungi, has been tested recently in 2-year feeding studies in Fischer F344 rats and B6C3F1 mice. Inclusion of FB1 at 50 and 80 ppm in the diet induced liver tumors in female mice, and at 50 and 150 ppm induced renal tumors in male rats (22). In the present study, the kidneys from the rat bioassay were examined to characterize the various histopathological changes associated with renal tumor induction. In all high-dose (150 ppm) and mid-dose (50 ppm) male rats, and to a lesser extent in high-dose (100 ppm) female rats, there was evidence of sustained nephrotoxicity manifested as basophilia, apoptosis, cell regeneration, and simple tubule hyperplasia, affecting proximal convoluted tubules in the deep cortex, extending into the outer region of the outer stripe of outer medulla. A further alteration consisted of sporadic areas of interstitial hyalinization in deep cortex, suggestive of expanded basement membrane, coupled with tubule atrophy. The continued presence of nephrotoxicity throughout chronic exposure to FB1 suggested that renal tumor development may have been an outcome of sustained cell loss and compensatory regeneration. In some cases, preneoplastic tubules or incipient renal tumors presented an immature or fetal form in association with interstitial hyalinization. The renal tubule tumors induced by FB1 were typified by a rare, highly malignant, anaplastic variant capable of growth by infiltration. Of the 10 renal tubule tumors diagnosed in the mid-dose males, and the 16 in the high-dose males, 8 and 10, respectively, were graded as carcinomas. Anaplastic variants represented 50% of the mid-dose carcinomas and 80% of the high-dose carcinomas. One of the anaplastic carcinomas in a mid-dose male was a true sarcomatoid phenotype not previously recorded in the rodent. Metastatic invasion of the lung occurred with 25% of the mid-dose carcinomas and 50% of the high-dose carcinomas. It was speculated that FB1 may have been influencing the growth characteristics of the induced renal tumors via its inhibitory action on the synthesis of sphingolipids, which in turn, participate in regulating cell contact, growth, and differentiation, or alternatively by affecting cell adhesion molecules. (+info)
FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1.
(53/310)
The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins. (+info)
Assessing chronic exposure to fumonisin mycotoxins: the use of hair as a suitable noninvasive matrix.
(54/310)
This study describes for the first time the accumulation of measurable levels of fumonisin mycotoxins in the hair of nonhuman primates (vervet monkeys, Cercopithecus aethiops) and rats exposed to contaminated feed. Hair was subjected to reflux with methanol, and the resulting extract was cleaned up on strong anion exchange (SAX) and C18 solid-phase sorbents. Fumonisins FB1, FB2, and FB3 as well as their hydrolysis products commonly known as aminopolyols, AP1 and AP2, were detected in monkey hair using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS). Despite matrix interferences, the two-stage mass spectrometric process (MS-MS) yielded product ion mass spectra, which served as diagnostic indicators thus providing unequivocal identification of FB1, FB2, and FB3 as well as AP1 and AP2. In vervet monkeys, the levels of exposure related well to the levels of toxin detected in hair, and levels as high as 5.98 mg FB1, 33.77 mg FB1, and 65.93 mg FB1/kg of hair were found in monkeys receiving control, low-dose, and high-dose contaminated diets, respectively. Hair was also analyzed from rats given either single gavage doses of 1 and 10 mg FB1/kg body weight or contaminated feed (50 mg FB1/kg), resulting in an exposure of approximately 4.25 mg FB1/kg body weight/day based on the measured daily feed intake. Analysis of rat hair over a four-week period indicated that mean levels up to 34.50 mg/kg and 42.20 mg/kg were detectable by the fourth week in the rats treated by gavage (10 mg FB1/kg body weight) and those receiving contaminated feed, respectively. This relationship indicates that hair can provide an easily applicable non-invasive matrix for assessing chronic exposure to fumonisin mycotoxins. (+info)
Role of ceramide synthase in oxidant injury to renal tubular epithelial cells.
(55/310)
Ceramide has been implicated to play an important role in the cell signaling pathway involved in apoptosis. Most studies that have used the apoptotic model of cellular injury have suggested that enhanced ceramide generation is the result of the breakdown of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation in response to oxidant stress has not been previously examined in any tissue. Hydrogen peroxide (H(2)O(2)) (1 mM) resulted in a rapid increase in ceramide generation (as measured by in vitro diacylglycerol kinase assay) in LLC-PK1 cells. The intracellular ceramide level was significantly increased at 5 min after exposure of cells to H(2)O(2) and thereafter continuously increased up to 60 min. H(2)O(2) also resulted in a rapid increase (within 5 min) in ceramide synthase activity (as measured by incorporation of [(14)C] from the labeled palmytoyl-CoA into dihydroceramide) in microsomes. In contrast, the exposure of cells to H(2)O(2) did not result in any significant change in sphingomyelin content or acid or neutral sphingomyelinase activity. An increase in ceramide production induced by H(2)O(2) preceded any evidence of DNA damage and cell death. The specific inhibitor of ceramide synthase, fumonisin B1 (50 microM), was able to suppress H(2)O(2)-induced ceramide generation and provided a marked protection against H(2)O(2)-induced DNA strand breaks, DNA fragmentation, and cell death. Taken together, these data provide the first evidence that H(2)O(2) is a regulator of ceramide synthase rather than sphingomyelinases and that ceramide synthase-dependent ceramide generation plays a key role in DNA damage and cell death in oxidant stress to renal tubular epithelial cells. (+info)
Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae.
(56/310)
Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. (+info)