The 17-kDa N-terminal region (NTR) of rat histone H10 (Thr 2 - Phe107 ) was generated during TAM-induced apoptosis and acted as a DNase, for which two histidine residues at 25 and 57 were essential. The 17-kDa H10NTR was generated by the restricted cleavage of H10 with the Ca2+-dependent protease calpain-2, which was activated by TAM-induced Ca2+ influx ([Ca2+]i). The forced expression of the 17-kDa H10NTR stimulated, while its downregulation suppressed TAM-induced apoptosis. These results imply the involvement of the 17-kDa H10NTR DNase downstream of the TAM-triggered cell death signaling pathway, suggest that TAM could be a promising chemoprevention and anticancer drug for AFB1 hepatocarcinogenesis, and also provide a new insight into the biological functions of H10.
We previously established a detection/selection system for somatic homologous recombination (HR), which is one of the genetic modification mechanisms in eukaryotes. Because HR is stimulated by the protein synthesis inhibitor blasticidin S, it is presumed that HR in Pyricularia oryzae can be induced by various chemical stresses. To evaluate the effects of chemical stresses on the frequency of HR, several chemical agents were applied to P. oryzae and HR were detected using our detection system. Three well-known DNA-damaging agents－methyl methanesulfonate, bleocin, and methyl viologen－considerably increased the frequency of HR. Adding the amino acid synthesis inhibitor bialaphos, or the protein synthesis inhibitor T-2 toxin, to the medium also significantly increased the frequency of HR. These results suggest that the increased frequency of HR caused by inhibitors of the primary metabolic pathway reflect destabilization of the genome by chemical stressors. Taken together, these findings suggest that the HR detection system may become one of the most useful biological assays for detecting mycotoxins.
The activities of four constitutive promoters from Aspergillus nidulans were compared in Fusarium graminearum by using β-glucuronidase (GUS) as a reporter gene. The promoter-GUS constructs were integrated into the Tri14 locus at the terminus of the trichothecene gene cluster and crude cell extracts were used for the reporter assay. The translation elongation factor 1-alpha (TEF1α) promoter yielded by far the strongest induction of GUS with little or no effect seen with the glyceraldehyde 3-phosphate dehydrogenase (GPD), polyubiquitin (UBI), and β-tubulin (TUB) gene promoters. The promoters of TUB and TEF1α, with or without an original trichothecene regulator gene (Tri6) opal codon, were connected to a transcriptional fusion of Tri6 and enhanced green fluorescent protein (EGFP) gene, and targeted to a locus downstream of the trichothecene 3-O-acetyltransferase gene (Tri101). Northern blot analysis revealed expression levels of these fusion genes to be proportional to the activities of the promoters as demonstrated by the GUS assay. In addition, analysis of trichothecene levels demonstrated drastically decreased activity of a translational fusion of TRI6 with EGFP (TRI6::EGFP) as a trichothecene transcription factor. These results indicate that the set of promoters reported in this study could be used to investigate biological functions of master genes by modulating their expression levels in F. graminearum.
Cereal grains such as corn and milo, most of which are imported, are primary livestock feeds in Japan. The climatic condition of the exporting countries, among other factors, may have impact on the concentrations of mycotoxins in these cereals, which may adversely affects health of both livestock and human. The Ministry of Agriculture Forestry, and Fisheries (MAFF) has established reference values for mycotoxins in feeds, and instructed manufacturers not to distribute feeds which contain mycotoxins above the respective reference values in order to protect the health of consumers who ingest foods of livestock origin such as milk and meat. In addition, the MAFF has conducted surveillance of the current occurrence of mycotoxins and monitoring the conformity to the reference values. This paper provides an overview of the risk management by the MAFF regarding mycotoxins in livestock feeds, including the method to establish reference values in line with the internationally adopted rule, and offering a real example on establishment of the reference value for aflatoxin (AF) B1 in compound feed for dairy cattle.
In recent years, international organizations such as the Codex Alimentarius Commission and European Union have set the maximum residue level (MRL) of aflatoxin M1 (AFM1) in milk according to evaluation of the risk assessment of AFM1 in milk and milk products. Under these circumstances, the Food Safety Commission (FSC) in Japan has evaluated the risk assessment of AFM1 in milk and reported it to the Ministry of Health, Labour and Welfare (MHLW) in 2013. Responding to the report, the MHLW moves into action to set the MRL for AFM1 in milk. The consultation for evaluation of methods for mycotoxin analysis organized under the MHLW has already validated the analytical method for AFM1 in milk and milk powder. In this paper, validated methods for AFM1 in milk and milk products will be mainly presented.
An international conference, International Society for Mycotoxicology (ISM) Beijing 2014 was held in Beijing (China) during 19th to 23rd May, 2014. The theme for the conference was “Perspectives on the Global Prevention and Control of Mycotoxin. More than 200 of topics including oral (91) and poster (115) presentations in addition to key note lecture (7) had been showed through out the conference. The participants shared their knowledge and exchange information on mycotoxin problem through active and fruitful discussions. This report is summary with an introducing some interesting presentations and suggestions from the conference.
I took part in the research for fungal metabolites for about 40 years. The metabolites of Emericella sp. were studied in the early 15 years. The macrocyclic mycotoxin, emestrin, was isolated from Emericella striata etc. and the histamine release inhibitors, emethallicins, were isolated from Emericella heterothallica. The yellow pigment related to the blue pigments of Aspergillus sp. was also isolated from Talaromyces derxii. Recently triterpenoidal glycosides (saponins), malbrancheosides, were isolated from Malbranchea filamentosa.
Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well-known producer of aﬂatoxin. This review highlights genetic bases for a difference in secondary metabolite production in two closely related species, some of which serve as safeguards against mycotoxin production in A. oryzae. In the course of the analysis of the cyclopiazonic acid biosynthetic gene cluster in A. oryzae, we found another genetic safeguard to ensure the safety of A. oryzae. The cpa cluster in A. oryzae contains genes that have been lost in A. flavus, one of which, cpaH, mediates the conversion of cyclopiazonic acid into the less toxic 2-oxo-cyclopiazonic acid. The detoxifying property of cpaH reﬂects the relationship of the two species.