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The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.
Trichothecene toxins cause serious hazard towards human health and economical crops. However, there are no sufficient molecular strategies to reduce the hazard of trichothecene toxins. Thus it is urgent to exploit novel approaches to control the hazard of trichothecenes. In this study, four trichothecene toxin-resistance genes including mfs1, GNAT1, TRP1 and tri12 in Paramyrothecium roridum were excavated based on genome sequencing results, and then expressed in toxin-sensitive Saccharomyces cerevisiae BJ5464, the toxin resistance genes pdr5, pdr10 and pdr15 of which were firstly knocked out simultaneously by the introduction of TAA stop codon employing CRISPR/Cas9 system. Therefore, three novel hazardous toxin-resistance genes mfs1, GNAT1, TRP1 in P. roridum were firstly excavated by the co-incubation of DON toxin and toxin resistant genes-containing BJ5464 strains. The in vitro function and properties of novel toxin-resistance genes coding proteins including GNAT1, MFS1 and TRP1 were identified by heterologous expression and cellular location analysis as well as in vitro biochemical reaction. The excavation of novel trichothecene toxin-resistance genes provide novel molecular clues for controlling the harm of trichothecenes, meanwhile, this study will also pave a new way for the yield improvement of trichothecenes by heterologous expression to facilitate the development of trichothecenes as anti-tumor lead compounds.
NX and its acetylated form 3ANX are two new type A trichothecenes produced by Fusarium graminearum whose toxicity is poorly documented. The aim of this study was to obtain a general view of the intestinal toxicity of these toxins. Deoxynivalenol (DON), which differs from NX by the keto group at C8, served as a benchmark. The viability of human intestinal Caco-2 cells decreased after 24 h of exposure to 3 μM NX (-21.4%), 3 μM DON (-20.2%) or 10 μM 3ANX (-17.4%). Histological observations of porcine jejunal explants exposed for 4 h to 10 μM of the different toxins showed interstitial edema and cellular debris. Explants exposed to NX also displayed cell vacuolization, a broken epithelial barrier and high loss of villi. Whole transcriptome profiling revealed that NX, DON and 3ANX modulated 369, 146 and 55 genes, respectively. Functional analyses indicated that the three toxins regulate the same gene networks and signaling pathways mainly; cell proliferation, differentiation, apoptosis and growth, and particularly immune and pro-inflammatory responses. Greater transcriptional impacts were observed with NX than with DON. In conclusion, our data revealed that the three toxins have similar impacts on the intestine but of different magnitude: NX > DON ≫ 3ANX. NX and 3ANX should consequently be included in overall risk analysis linked to the presence of trichothecenes in our diet.
Eight trichothecenes, including four new compounds 1-4 and four known entities 5-8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1-9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 μM, respectively. Compounds 1-2, 4-6, and 7-8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.
Fusarium graminearum can cause Giberella Ear Rot (GER) and seedling blight in maize, resulting in major yield losses. Besides GER, the infected grains are consequently contaminated with multiple mycotoxins of F. graminearum. Zearalenone and trichothecenes, such as deoxynivalenol and its acetylated forms, are among the major mycotoxins associated with F. graminearum infection in maize. In the current work, we explored the effect of the endophytic fungal genera of Epicoccum and Sordaria, to control F. graminearum infection in comparative trials with Piriformospora spp., an elusive endophytic genus. Furthermore, we investigated the effect of these endophytes on zearalenone, deoxynivalenol, and 15-acetyldeoxynivalenol levels using in vitro and in planta assays. As plants are endowed with several detoxification mechanisms comprising e.g., glucosylation of trichothecenes, the effect of the isolated fungal endophytes on the deoxynivalenol-3-glucoside level was also assessed. In general, results showed a considerable variability in the antifungal activity, both among species and among isolates within one species. Additionally, the effect on mycotoxin levels was variable, and not necessarily related to the antifungal activity except for zearalenone levels which were consistently reduced by the endophytes. These results highlight the great potential of certain endophytic fungal strains as new biocontrol agents in agricultural science.
The secondary metabolites of Fusarium sporotrichioides, an endophytic fungus with anti-tumor activity isolated from Rauvolfia yunnanensis Tsiang, were investigated. Five trichothecenes, including one previously undescribed metabolite, were isolated and identified. Their structures were elucidated by means of extensive spectroscopic methods; the absolute configuration of compound 1 was determined by the ECD method. Surprisingly, 8-n-butyrylneosolaniol (3) exhibited stronger anti-tumor activity than T-2 toxin against Huh-7 cell line, with an IC50 value of 265.9 nM. 8-n-butyrylneosolaniol (3) promoted apoptosis induction in Huh-7 cells. Moreover, cell cycle analysis showed that cell cycle arrest caused by 8-n-butyrylneosolaniol (3) at the G2/M phase resulted in cell proliferation inhibition and pro-apoptotic activity. Further studies showed a significant decrease in mitochondrial membrane permeabilization and a significant increase in ROS generation, which led to the activation of caspase cascades and subsequent cleavage of PARP fragments. In conclusion, 8-n-butyrylneosolaniol (3) induced cell apoptosis in Huh-7 cells via the mitochondria-mediated apoptotic signaling pathway, which could be a leading compound for anti-tumor agents.
Type B 8-keto-trichothecenes are muco-active mycotoxins that exist as inevitable contaminants in cereal-based foodstuffs. Gut-associated inflammation is an early frontline response during human and animal exposure to these mycotoxins. Despite various tools for chemical identification, optimized biomonitoring of sentinel response-associated biomarkers is required to assess the specific proinflammatory actions of 8-keto-trichothecenes in the gut epithelial barrier. In the present study, intoxication with 8-keto-trichothecenes in human intestinal epithelial cells was found to trigger early response gene 1 product (EGR-1) that plays crucial roles in proinflammatory chemokine induction. In contrast, epithelial exposure to 8-keto-trichothecenes resulted in downregulated expression of nuclear factor NF-kappa-B p65 protein, a key transcription factor, during general inflammatory responses in the gut. Based on the early molecular patterns of expression, the inflammation-inducing activity of 8-keto-trichothecenes was quantified using intestinal epithelial cells with dual reporters for EGR-1 and p65 proteins. EGR-1-responsive elements were linked to luciferase reporter while p65 promoter was bound to secretory alkaline phosphatase (SEAP) reporter. In response to conventional inflammagens such as endotoxins and cytokines such as TNF-α, both luciferase and SEAP activity were elevated in a dose-dependent manner. However, as expected from the mechanistic evaluation, 8-keto-trichothecene-exposed dual reporters of luciferase and SEAP displayed contrasting expression patterns. Furthermore, 8-keto-trichothecene-elevated EGR-1-responsive luciferase activity was improved by deficiency of PSMA3, an α-type subunit of the 20S proteasome core complex for ubiquitin-dependent EGR-1 degradation. This molecular event-based dual biomonitoring in epithelial cells is a promising supplementary tool for detecting typical molecular inflammatory pathways in response to 8-keto-trichothecenes in the food matrix.
Trichothecenes are sesquiterpenoid metabolites produced by several fungal strains that impair human and animal health. Since sphingolipids were connected with fungal toxicity the aim of the present study was to test the influence of fungal metabolites on sphingolipid metabolism in neural cells. The crude extract of fungal strain Spicellum roseum induced accumulation of glucosylceramide (GlcCer), and simultaneous reduction of the formation of lactosylceramide (LacCer) and complex gangliosides in primary cultured neurons. Following a bioassay-guided fractionation of the respective fungal extract we could demonstrate that the two isolated trichothecene derivatives, 8-deoxy-trichothecin (8-dT) and trichodermol (Td-ol) were responsible for this effect. Thus, incubation of primary cultured neurons as well as of neuroblastoma B104 cells for 24 h with 30 microM of either of the two fungal metabolites resulted in uncoupling of sphingolipid biosynthesis at the level of LacCer. For the observed reduction of LacCer synthase activity by about 90% cell integrity was crucial in both cell types. In neuroblastoma cells the amount of LacCer synthase mRNA was reduced in the presence of trichothecenes, whereas in primary cultured neurons this was not the case, suggesting a post-transcriptional mechanism of action in the latter cell type. The data also show that the compounds did not interfere with the translocation of GlcCer in neuroblastoma cells. Collectively, our results demonstrate that trichodermol and 8-deoxy-trichothecin inhibit LacCer synthase activity in a cell-type-specific manner.
Contamination by fungal and bacterial species and their metabolites can affect grain quality and health of wheat consumers. In this study, sequence analyses of conserved DNA regions of fungi and bacteria combined with determination of trichothecenes and aflatoxins revealed the microbiome and mycotoxins of wheat from different silo positions (top, middle, and bottom) and storage times (3, 6, 9, and 12 months). The fungal community in wheat on the first day of storage (T₀) included 105 classified species (81 genera) and 41 unclassified species. Four species had over 10% of the relative abundance: Alternaria alternata (12%), Filobasidium floriforme (27%), Fusarium graminearum (12%), and Wallemia sebi (12%). Fungal diversity and relative abundance of Fusarium in wheat from top silo positions were significantly lower than at other silo positions during storage. Nivalenol and deoxynivalenol in wheat were 13⁻34% higher in all positions at 3 months compared to T₀, and mycotoxins in wheat from middle and bottom positions at 6 to 12 months were 24⁻57% higher than at T₀. The relative abundance of toxigenic Aspergillus and aflatoxins were low at T₀ and during storage. This study provides information on implementation and design of fungus and mycotoxin management strategies as well as prediction models.
Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces trichothecenes including deoxynivalenol (DON), nivalenol (NIV), and 3,7,15-trihydroxy-12,13-epoxytrichothec-9-ene (NX-3). These toxins contaminate grains and cause profound health problems in humans and animals. To explore exploiting a fungal self-protection mechanism in plants, we examined the ability of F. graminearum trichothecene 3-O-acetyltransferase (FgTri101) to detoxify several key trichothecenes produced by F. graminearum: DON, 15-ADON, NX-3, and NIV. FgTri101 was cloned from F. graminearum and expressed in Arabidopsis plants. We compared the phytotoxic effects of purified DON, NIV, and NX-3 on the root growth of transgenic Arabidopsis expressing FgTri101. Compared to wild type and GUS controls, FgTri101 transgenic Arabidopsis plants displayed significantly longer root length on media containing DON and NX-3. Furthermore, we confirmed that the FgTri101 transgenic plants acetylated DON to 3-ADON, 15-ADON to 3,15-diADON, and NX-3 to NX-2, but did not acetylate NIV. Approximately 90% of the converted toxins were excreted into the media. Our study indicates that transgenic Arabidopsis expressing FgTri101 can provide plant protection by detoxifying trichothecenes and excreting the acetylated toxins out of plant cells. Characterization of plant transporters involved in trichothecene efflux will provide novel targets to reduce FHB and mycotoxin contamination in economically important plant crops.
Trichothecene mycotoxins are common contaminants in cereal grains and negatively impact human and animal health. Although anorexia is a common hallmark of type B trichothecenes-induced toxicity, less is known about the anorectic potencies of type A trichothecenes. The purpose of this study was to compare the anorectic potencies of four type A trichothecenes (T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), and neosolaniol (NEO)) in mice. Following oral exposure to T-2, HT-2, DAS, and NEO, the no observed adverse effect levels (NOAELs) and lowest observed adverse effect levels (LOAELs) were 0.01, 0.01, 0.1, and 0.01 mg/kg body weight (BW), and 0.1, 0.1, 0.5, and 0.1 mg/kg BW, respectively. Following intraperitoneal (IP) exposure to T-2, HT-2, DAS, and NEO, the NOAELs were 0.01 mg/kg BW, except for DAS (less than 0.01 mg/kg BW), and the LOAELs were 0.1, 0.1, 0.01, and 0.1 mg/kg BW, respectively. Taken together, the results suggest that (1) type A trichothecenes could dose-dependently elicit anorectic responses following both oral gavage and IP exposure in mice; (2) the anorectic responses follow an approximate rank order of T-2 = HT-2 = NEO > DAS for oral exposure, and DAS > T-2 = HT-2 = NEO for IP administration; (3) IP exposure to T-2, HT-2, DAS, and NEO evoked stronger anorectic effects than oral exposure. From a public health perspective, comparative anorectic potency data should be useful for establishing toxic equivalency factors for type A trichothecenes.
Role of efflux-mediated toxin resistance to trichothecenes is known in trichothecene-producing species. However, the role of trichothecene efflux pump homologues in non-producing fusaria such as F. oxysporum and F. proliferatum was not investigated in detail. Analysis of the homologues of trichothecene efflux pump from multiple fungal species allowed us to uncover and catalogue functional gene copies of conserved structure. Putative Tri12 candidates in Fusarium oxysporum and F. proliferatum were characterised via expression profiling in response to different trigger compounds, providing supporting evidence for role of Tri12 homologues in the resistance to trichothecenes. Our analysis of Tri12 phylogeny also suggests that efflux-mediated trichothecene resistance is likely to predate the divergence of Trichoderma and Fusarium species. On the regulatory level, we posit that the increased tolerance of trichothecenes by F. oxysporum is possibly related to the decoupling of Tri12 homologue expression from pH, due to the deletion of PACC/RIM101 transcription factor binding site in its promoter region.
The current study investigated the fungal diversity in freshly harvested oat samples from the two largest production regions in Brazil, Paraná (PR) and Rio Grande do Sul (RS), focusing primarily on the Fusarium genus and the presence of type B trichothecenes. The majority of the isolates belonged to the Fusarium sambucinum species complex, and were identified as F. graminearum sensu stricto (s.s.), F. meridionale, and F. poae. In the RS region, F. poae was the most frequent fungus, while F. graminearum s.s. was the most frequent in the PR region. The F. graminearum s.s. isolates were 15-ADON genotype, while F. meridionale and F. poae were NIV genotype. Mycotoxin analysis revealed that 92% and 100% of the samples from PR and RS were contaminated with type B trichothecenes, respectively. Oat grains from PR were predominantly contaminated with DON, whereas NIV was predominant in oats from RS. Twenty-four percent of the samples were contaminated with DON at levels higher than Brazilian regulations. Co-contamination of DON, its derivatives, and NIV was observed in 84% and 57.7% of the samples from PR and RS, respectively. The results provide new information on Fusarium contamination in Brazilian oats, highlighting the importance of further studies on mycotoxins.
Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.
Deoxynivalenol is a food borne mycotoxin belonging to the trichothecenes family that may cause severe injuries in human and animals. The inhibition of protein synthesis via the interaction with the ribosome has been identified as a crucial mechanism underlying toxic action. However, it is not still fully understood how and to what extent compounds belonging to trichothecenes family affect human and animal health. In turn, this scenario causes delay in managing the related health risk. Aimed at supporting the hazard identification process, the in silico analysis may be a straightforward tool to investigate the structure-activity relationship of trichothecenes, finding out molecules of possible concern to carry forth in the risk assessment process. In this framework, this work investigated through a molecular modeling approach the structural basis underlying the interaction with the ribosome under a structure-activity relationship perspective. To identify further forms possibly involved in the total trichothecenes-dependent ribotoxic load, the model was challenged with a set of 16 trichothecene modified forms found in plants, fungi and animals, including also compounds never tested before for the capability to bind and inhibit the ribosome. Among them, only the regiospecific glycosylation in the position 3 of the sesquiterpenoid scaffold (i.e. T-2 toxin-3-glucuronide, α and β isomers of T-2 toxin-3-glucoside and deoxynivalenol-3-glucuronide) was found impairing the interaction with the ribosome, while the other compounds tested (i.e. neosolaniol, nivalenol, fusarenon-X, diacetoxyscirpenol, NT-1 toxin, HT-2 toxin, 19- and 20-hydroxy-T-2 toxin, T-2 toxin triol and tetraol, and 15-deacetyl-T-2 toxin), were found potentially able to inhibit the ribosome. Accordingly, they should be included with high priority in further risk assessment studies in order to better characterize the trichothecenes-related hazard.
In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 μM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 μM, respectively, which were higher than the values of 0.23 and 3.0 μM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.
Engleromyces goetzii is a traditional medicinal mushroom that is widely used to treat infection, inflammation and cancer in Tibet, Sichuan and Yunnan provinces of China. Two new trichothecenes, engleromycones A and B (1 and 2), one new cuparane-type sesquiterpenoid named infuscol F (11), eight known trichothecene analogs, sambucinol (3), 3-deoxysambucinol (4), trichothecolone (5), trichodermol (6), 8-deoxytrichothecin (7), trichothecin (8), trichothecinol B (9) and trichothecinol A (10), and one known cyclopentanoid sesquiterpene cyclonerodiol (12) were isolated from the cultures of E. goetzii. The new compounds were elucidated through spectroscopic analyses. The anticancer effects of trichothecenes 1-10 were examined in the HL-60, SMMC-7721, A549, MCF-7, and SW-480 human cancer cell lines using an MTT assay. Trichothecinol A (10) significantly inhibited the growth of MCF-7 cells, with an IC50 value of 0.006 µM, which was comparable to the cytotoxic activity of the positive control, paclitaxel, indicating that trichothecinol A (10) represents a potential anticancer agent.
Trichothecene mycotoxins, potent translational inhibitors that are associated with human food poisonings and damp-building illnesses, are of considerable concern to animal and human health. Food refusal is a hallmark of exposure of experimental animals to deoxynivalenol (DON) and other Type B trichothecenes but less is known about the anorectic effects of foodborne Type A trichothecenes (e.g., T-2 toxin, HT-2 toxin), airborne Type D trichothecenes (e.g. satratoxin G [SG]) or functionally analogous metabolites that impair protein synthesis. Here, we utilized a well-described mouse model of food intake to compare the anorectic potencies of T-2 toxin, HT-2 toxin, and SG to that of emetine, a medicinal alkaloid derived from ipecac that inhibits translation. Intraperitoneal (IP) administration with T-2 toxin, HT-2 toxin, emetine and SG evoked anorectic responses that occurred within 0.5 h that lasted up to 96, 96, 3 and 96 h, respectively, with lowest observed adverse effect levels (LOAELs) being 0.1, 0.1, 2.5 and 0.25 mg/kg BW, respectively. When delivered via natural routes of exposure, T-2 toxin, HT-2 toxin, emetine (oral) and SG (intranasal) induced anorectic responses that lasted up to 48, 48, 3 and 6 h, respectively with LOAELs being 0.1, 0.1, 0.25, and 0.5 mg/kg BW, respectively. All four compounds were generally much more potent than DON which was previously observed to have LOAELs of 1 and 2.5 mg/kg BW after IP and oral dosing, respectively. Taken together, these anorectic potency data will be valuable in discerning the relative risks from trichothecenes and other translational inhibitors of natural origin.
The food-borne trichothecene mycotoxins have been documented to cause human and animal food poisoning. Anorexia is a hallmark of the trichothecene mycotoxins-induced adverse effects. Type B trichothecenes have been previously demonstrated to elicit robust anorectic responses, and this response has been directly linked to secretion of the gut satiety hormones cholecystokinin (CCK) and glucagon-like peptide-17-36 amide (GLP-1). However, less is known about the anorectic effects and underlying mechanisms of the type A trichothecenes, including T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), neosolaniol (NEO). The purpose of this study was to relate type A trichothecenes T-2, HT-2, DAS and NEO-induced anorectic response to changes plasma concentrations of CCK and GLP-1. Following both oral gavage and intraperitoneal (IP) administration of 1mg/kg bw T-2, HT-2, DAS and NEO evoked robust anorectic response and secretion of CCK and GLP-1. Elevations of plasma CCK markedly corresponded to anorexia induction by T-2, HT-2, DAS and NEO. Following oral exposure, plasma CCK was peaked at 6h, 6h, 2h, 2h and lasted up to 24h, 24h, > 6h, > 6h for T-2, HT-2, DAS and NEO, respectively. IP exposed to four toxins all induced elevation of CCK with peak point and duration at 6h and >24h, respectively. In contrast to CCK, GLP-1 was moderately elevated by these toxins. Following both oral and IP exposure, T-2 and HT-2 evoked plasma GLP-1 elevation with peak point and duration at 2h and 6h, respectively. Plasma GLP-1 was peaked at 2h and still increased at 6h for IP and oral administration with DAS and NEO, respectively. In conclusion, CCK plays a contributory role in anorexia induction but GLP-1 might play a lesser role in this response.
Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.
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