Y. Nakajima, T. Tokai, Kazuyuki Maeda, A. Tanaka, N. Takahashi-Ando, K. Kanamaru, Tetsuo Kobayashi, M. Kimura
Abstract 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.
{"title":"A set of heterologous promoters useful for investigating gene functions in Fusarium graminearum","authors":"Y. Nakajima, T. Tokai, Kazuyuki Maeda, A. Tanaka, N. Takahashi-Ando, K. Kanamaru, Tetsuo Kobayashi, M. Kimura","doi":"10.2520/MYCO.64.147","DOIUrl":"https://doi.org/10.2520/MYCO.64.147","url":null,"abstract":"Abstract 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.","PeriodicalId":19069,"journal":{"name":"Mycotoxins","volume":"56 1","pages":"147-152"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80460844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
sex-determining factors including Sry (sex determining region Y) and sex hormones play a crucial role in onset of male HCC and its conversion into malignant tumor via cancer stem cell (CSC). These findings may confer the useful information for development of promising chemoprevention and anti-cancer drugs against male HCC.
{"title":"Implication of sex-determing factors for gender disparity in liver cancer","authors":"S. Murakami, F. Tashiro","doi":"10.2520/MYCO.64.95","DOIUrl":"https://doi.org/10.2520/MYCO.64.95","url":null,"abstract":"sex-determining factors including Sry (sex determining region Y) and sex hormones play a crucial role in onset of male HCC and its conversion into malignant tumor via cancer stem cell (CSC). These findings may confer the useful information for development of promising chemoprevention and anti-cancer drugs against male HCC.","PeriodicalId":19069,"journal":{"name":"Mycotoxins","volume":"1 1","pages":"95-103"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76026100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Penicillium expansum was cultured in apple juice media prepared from six different commercial apple juices. The patulin production was profoundly affected by the differences in apple juices, whereas fungal growth was generally not. The maximum concentration of patulin was 7 .3-fold of that in the media containing minimum concentration of patulin. The six apple juices were concentrated by evaporation and reconstructed to the original volumes by adding Milli-Q water. P. expansum was cultured in the media prepared from reconstructed apple juices, and the patulin concentration and fungal growth were determined. Evaporation of apple juice tended to decrease patulin production and to increase fungal growth, suggesting that the volatile compounds promote patulin production and inhibit fungal growth. The volatile compounds in the apple juice were then analyzed by GC-MS. The 13 compounds of which concentrations were largely decreased by evaporation were selected for evaluation of their stimulation of patulin production. Seven out of the 13 compounds, 2-methylpropyl acetate, ethyl butyrate, ethyl 2-methylbutanoate, 3-methyl-1-butanol, hexyl acetate, 1-hexanol, and 2-methylbutanoic acid, increased the patulin production of P. expansum concentration-dependently; 2-methylbutanoic acid and its ethyl ester were highly effective. Our results indicated that the composition of volatile compounds in apple juice media largely affects the patulin production and the growth of P. expansum.
{"title":"Effects of volatile compounds in apple juices on fungal growth and patulin production of Penicillium expansum","authors":"T. Taguchi, A. Ishihara, H. Nakajima","doi":"10.2520/MYCO.64.1","DOIUrl":"https://doi.org/10.2520/MYCO.64.1","url":null,"abstract":"Penicillium expansum was cultured in apple juice media prepared from six different commercial apple juices. The patulin production was profoundly affected by the differences in apple juices, whereas fungal growth was generally not. The maximum concentration of patulin was 7 .3-fold of that in the media containing minimum concentration of patulin. The six apple juices were concentrated by evaporation and reconstructed to the original volumes by adding Milli-Q water. P. expansum was cultured in the media prepared from reconstructed apple juices, and the patulin concentration and fungal growth were determined. Evaporation of apple juice tended to decrease patulin production and to increase fungal growth, suggesting that the volatile compounds promote patulin production and inhibit fungal growth. The volatile compounds in the apple juice were then analyzed by GC-MS. The 13 compounds of which concentrations were largely decreased by evaporation were selected for evaluation of their stimulation of patulin production. Seven out of the 13 compounds, 2-methylpropyl acetate, ethyl butyrate, ethyl 2-methylbutanoate, 3-methyl-1-butanol, hexyl acetate, 1-hexanol, and 2-methylbutanoic acid, increased the patulin production of P. expansum concentration-dependently; 2-methylbutanoic acid and its ethyl ester were highly effective. Our results indicated that the composition of volatile compounds in apple juice media largely affects the patulin production and the growth of P. expansum.","PeriodicalId":19069,"journal":{"name":"Mycotoxins","volume":"11 1","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78821583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Takayuki Arazoe, S. Ohsato, Kazuyuki Maeda, T. Arie, S. Kuwata
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 agents̶methyl methanesulfonate, bleocin, and methyl viologen̶considerably 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. Somatic homologous recombination (HR) plays an important role in the DNA double-strand break (DSB) repair. However, HR can be mutagenic if the template for repair is similar, but not identical, to the broken sequence (ectopic HR) . In human cells, ectopic HR events drive genetic disorders through the genome rearrangement. In plants, the frequencies of somatic HR are increased by abiotic and biotic stress factors, and HR may be one of the mechanisms for introducing genetic variations that enable organisms to adapt and respond to the stress environment. Infection of food crops with toxigenic fungi can result in the contamination of infected grain with mycotoxins. The risk of contamination with mycotoxins has been recognized for decades. However, preventive measures remain costly and inadequate. We previously reported construction of a detection/ selection system for exploring ectopic HR at the somatic cell level of Pyricularia oryzae using nonfunctional yellow fluorescence protein (YFP) and blasticidin S deaminase (BSD) fusion genes. The system could detect ectopic HR events between two substrate genes by YFP fluorescence and blasticidin S (BS)-resistance via Note Corresponding Author * Graduate school of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan Tel: +81-44-934-7036; Fax: +81-44-934-7036; E-mail: kuwata@isc.meiji.ac.jp A full color PDF reprint of this article is available at the journal WEB site. 142 ARAZOE et al. JSM Mycotoxins restoration of functional YFP::BSD. Using this HR detection/selection system, we have shown that the onset of ectopic HR was stimulated by treatment with BS, an inhibitor of protein synthesis in both prokaryotes and eukaryotes. In the current study, the effects of various chemical agents on the frequency of ectopic HR were evaluated. We also discuss the applicability of the HR d
{"title":"The effect of chemicals on somatic homologous recombination in the rice blast fungus: its possible application for detection of mycotoxins","authors":"Takayuki Arazoe, S. Ohsato, Kazuyuki Maeda, T. Arie, S. Kuwata","doi":"10.2520/myco.64.141","DOIUrl":"https://doi.org/10.2520/myco.64.141","url":null,"abstract":"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 agents̶methyl methanesulfonate, bleocin, and methyl viologen̶considerably 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. Somatic homologous recombination (HR) plays an important role in the DNA double-strand break (DSB) repair. However, HR can be mutagenic if the template for repair is similar, but not identical, to the broken sequence (ectopic HR) . In human cells, ectopic HR events drive genetic disorders through the genome rearrangement. In plants, the frequencies of somatic HR are increased by abiotic and biotic stress factors, and HR may be one of the mechanisms for introducing genetic variations that enable organisms to adapt and respond to the stress environment. Infection of food crops with toxigenic fungi can result in the contamination of infected grain with mycotoxins. The risk of contamination with mycotoxins has been recognized for decades. However, preventive measures remain costly and inadequate. We previously reported construction of a detection/ selection system for exploring ectopic HR at the somatic cell level of Pyricularia oryzae using nonfunctional yellow fluorescence protein (YFP) and blasticidin S deaminase (BSD) fusion genes. The system could detect ectopic HR events between two substrate genes by YFP fluorescence and blasticidin S (BS)-resistance via Note Corresponding Author * Graduate school of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan Tel: +81-44-934-7036; Fax: +81-44-934-7036; E-mail: kuwata@isc.meiji.ac.jp A full color PDF reprint of this article is available at the journal WEB site. 142 ARAZOE et al. JSM Mycotoxins restoration of functional YFP::BSD. Using this HR detection/selection system, we have shown that the onset of ectopic HR was stimulated by treatment with BS, an inhibitor of protein synthesis in both prokaryotes and eukaryotes. In the current study, the effects of various chemical agents on the frequency of ectopic HR were evaluated. We also discuss the applicability of the HR d","PeriodicalId":19069,"journal":{"name":"Mycotoxins","volume":"43 1","pages":"141-146"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73402470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A simultaneous and multiple determination method for the major five Fusarium toxins (deoxynivalenol, nivalenol, T- 2 toxin, HT- 2 toxin, and zearalenone) contaminating wheat and barley was developed with liquid chromatography tandem-mass spectrometry (LC-MS/MS), and a harmonized collaborative validation of the method was conducted by the participants of 12 laboratories. Although there are many reports on the simultaneous and multiple determination of Fusarium toxins by instrumental analysis, only a few of them are developed to the harmonized collaborative validation. The variance in the extraction efficiency and the matrix effects on the ionization were occasionally innegli gible with the mycotoxin analysis by LC-MS/MS, however, the employment of internal standards with the present method apparently contributed to compensate for these effects. This is the first report of a simultaneous and multiple determination method for both type A and B trichothecenes along with ZEA by LC-MS/MS that was validated through the harmonized collaborative validation. On the other hand, the accurate mass and high-resolution measurement was carried out with LC-MS (LC-Orbitrap MS) for the screening of new masked mycotoxins. Consequently, mono-glucoside derivatives of type B trichothecenes (nivalenol and fusarenon-X) in wheat grains and those of type A trichothecenes (T- 2 toxin, HT- 2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol) in corn powder sample were detected, respectively. Di-glucoside derivatives were also found for T- 2 toxin and HT- 2 toxin. Our findings indicate that the presence of masked mycotoxins is not limited to some specific mycotoxins such as deoxynivalenol and zearalenone, but likely with the other Fusarium toxins.
{"title":"State of the art in the analyisis of Fusarium toxins by liquid chromatography-mass spectrometry (LC-MS)","authors":"H. Nakagawa","doi":"10.2520/MYCO.64.55","DOIUrl":"https://doi.org/10.2520/MYCO.64.55","url":null,"abstract":"A simultaneous and multiple determination method for the major five Fusarium toxins (deoxynivalenol, nivalenol, T- 2 toxin, HT- 2 toxin, and zearalenone) contaminating wheat and barley was developed with liquid chromatography tandem-mass spectrometry (LC-MS/MS), and a harmonized collaborative validation of the method was conducted by the participants of 12 laboratories. Although there are many reports on the simultaneous and multiple determination of Fusarium toxins by instrumental analysis, only a few of them are developed to the harmonized collaborative validation. The variance in the extraction efficiency and the matrix effects on the ionization were occasionally innegli gible with the mycotoxin analysis by LC-MS/MS, however, the employment of internal standards with the present method apparently contributed to compensate for these effects. This is the first report of a simultaneous and multiple determination method for both type A and B trichothecenes along with ZEA by LC-MS/MS that was validated through the harmonized collaborative validation. On the other hand, the accurate mass and high-resolution measurement was carried out with LC-MS (LC-Orbitrap MS) for the screening of new masked mycotoxins. Consequently, mono-glucoside derivatives of type B trichothecenes (nivalenol and fusarenon-X) in wheat grains and those of type A trichothecenes (T- 2 toxin, HT- 2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol) in corn powder sample were detected, respectively. Di-glucoside derivatives were also found for T- 2 toxin and HT- 2 toxin. Our findings indicate that the presence of masked mycotoxins is not limited to some specific mycotoxins such as deoxynivalenol and zearalenone, but likely with the other Fusarium toxins.","PeriodicalId":19069,"journal":{"name":"Mycotoxins","volume":"22 1","pages":"55-62"},"PeriodicalIF":0.0,"publicationDate":"2014-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90493302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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