[Objective] This study was to establish a method for the rapid and accurate detection of the coupling ratio of artificial antigen. [Method] Artificial synthetic antigen of citrinin （CIT） was first conjugated with ve...[Objective] This study was to establish a method for the rapid and accurate detection of the coupling ratio of artificial antigen. [Method] Artificial synthetic antigen of citrinin （CIT） was first conjugated with vector protein bovine serum albumin （BSA） and then identified by SDS-PAGE electrophoresis,and its coupling ratio was measured by UV absorption and mass spectrometry. [Result] The identification result showed that artificial antigen of CIT was successfully coupled,and the coupling ratio was 8.4 by UV absorption while 6.0 by mass spectrometry. [Conclusion] The comparison experiment showed that mass spectrometry could rapidly identify the artificial antigen and accurately detect its coupling ratio. This study provided basis for the preparation of CIT antigen as well as the establishment of an for enzyme-linked immunosorbent assay （ELISA） for citrinin determination.展开更多
Objective To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin （IgY） to build an enzyme-linked immunosorbent assay （ELISA） for citrinin （CTN）. Methods CTN was conjugated with bovine s...Objective To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin （IgY） to build an enzyme-linked immunosorbent assay （ELISA） for citrinin （CTN）. Methods CTN was conjugated with bovine serum albumin （BSA）, ovalbumin （OVA） with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet （UV） spectrometry and Infrared （IR） spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied. Results UV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng·mL^-1, with a good linearity ranging 20-640 ng·mL^-1. Conclusion Artificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.展开更多
Objective:To Isolate,purify,characterize,and evaluate the bioaclive compounds from the sponge-derived fungus Penicillium sp.FF001 and to elucidate its structure.Methods:The fungal strain FF001 with an interesting bioa...Objective:To Isolate,purify,characterize,and evaluate the bioaclive compounds from the sponge-derived fungus Penicillium sp.FF001 and to elucidate its structure.Methods:The fungal strain FF001 with an interesting bioactivity profile was isolated from a marine Fijian sponge Melophlus sp.Based on conidiophores aggregation,conidia development and mycelia morphological characteristics,the isolate FF001 was classically identified as a Penicillium sp.The bioactive compound was identified using various spectral analysis of UV,high resolution electrospray ionization mass spectra,1H and 13C NMR spectral data.Further minimum inhibitory concentrations(MICs)assay and brine shrimp cytotoxicity assay were also carried out to evaluate the biological properties of the purified compound.Results:Bioassay guided fractionation of the EtOAc extract of a static culture of this Penicillium sp.by different chromatographic methods led the isolation of an antibacterial,anticryptococcal and cytotoxic active compound,which was identified as citrinin(1).Further,citrinin(1)is reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus(S.aureus),rifampicin-resistant 5.aureus,wild type S.aureus and vancomycin-resistant Enterococcus faecium showed MICs of 3.90,0.97,1.95 and7.81μg/mL,respectively.Further citrinin(1)displayed significant activity against the pathogenic yeast Cryptococcus neoformans(MIC 3.90μg/mL),and exhibited cytotoxicity against brine shrimp larvae LD_(50)of 96μg/mL.Conclusions:Citrinin(1)is reported from sponge associated Penicillium sp.from this study and for its strong antibacterial activity against multi-drug resistant human pathogens including cytotoxicity against brine shrimp larvae,which indicated that sponge associated Penicillium spp.are promising sources of natural bioactive metabolites.展开更多
Objective To obtain the full-length cDNA of a novel gene （named yp05） associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3＇ and 5＇ cDNA end of...Objective To obtain the full-length cDNA of a novel gene （named yp05） associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3＇ and 5＇ cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene （named yp05） was obtained from the electronic assembly of 3＇-RACE and 5＇- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame （ORF） and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription ofyp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citfinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.展开更多
To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. Methods Total RNA was extracted from the mycelium, cDN...To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. Methods Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method. Results Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaⅢ enzyme in inserts. There were seven repeated clones. Conclusion With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags≥10) and 143 unique tags to moderately expressed genes (repeat tags≥2).展开更多
The chemical investigation into the EtOAc extract of the deep-sea-derived fungus Penicillium citrinum W22 yielded three unprecedented citrinin dimers,neo-Dicitrinols A–C(1–3)and a known one,penicitrinone A(4).Their ...The chemical investigation into the EtOAc extract of the deep-sea-derived fungus Penicillium citrinum W22 yielded three unprecedented citrinin dimers,neo-Dicitrinols A–C(1–3)and a known one,penicitrinone A(4).Their structures were elucidated by extensive analysis of spectroscopic data,electronic circular dichroism(ECD)calculation,X-ray diffraction,and biogenetic consideration.neo-Dicitrinols A–C(1–3),bearing a tetramic acid unit,represent the first example of citrinin analogues as hybrid polyketide synthase-nonribosomal peptide synthase(PKS-NRPS)products.neo-Dicitrinol C(3)significantly inhibited renin-angiotensin system-selective lethal 3(RSL3)-induced ferroptosis with a half maximal effective concentration(EC50)value of 21.6μmol/L.展开更多
Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurr...Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurrence of both toxins was found in wheat grain and processed sunflower seeds. Citrinin levels exceeded those of ochratoxin A in the majority of co-contaminated feed samples, and the ratio of (1.1 - 10):1 proved to be the most frequent. A possible role of Aspergillus and Penicillium fungi in separate and simultaneous OTA and CIT occurrence in feeds is also discussed.展开更多
Citrinin(CIT)is one of by-products by Monascus fermentation with known nephrotoxicity.Except dihydro-citrinone,no other metabolites including the possible metabolic pathways have been found in vivo metabolism.In this ...Citrinin(CIT)is one of by-products by Monascus fermentation with known nephrotoxicity.Except dihydro-citrinone,no other metabolites including the possible metabolic pathways have been found in vivo metabolism.In this study,we applied ultra-performance liquid chromatography-mass spectrometry(UPLC-MS)to analyze and identify potential CIT metabolites in the human cytochrome 450(P450)subtype system.Four new metabolites with liver microsomes incubation were first found,namely 3-hydroxy-citrinin(M1),4,5-ene-citrinin(M2),5-hydroxymethyl-citrinin(M3),and 7-hydroxy-citrinin(M4).We also found the distinguished levels of the four metabolites with the incubation of various P450 subtypes.According to the results of molecular docking prediction,it showed the metabolic correlation of CYP3A4 with M1,CYP1A2,CYP2C9,and CYP2D6 with M2,M3,and M4.The maximum level of M1 metabolite was found in the incubation with liver microsome and P450 subtypes.Moreover,M1 and M4 metabolites were found in mice’s duodenum,jejunum,ileum,liver,kidney,and hippocampus,but not M2 and M3.These results indicated the mediation of citrinin metabolism by P450s.It also suggested the concentration differences of metabolites with the incubation of different P450 subtypes.展开更多
基金Supported by National High Technology Research and Development Program of China (863 Program) (2007AA10Z426-1)~~
文摘[Objective] This study was to establish a method for the rapid and accurate detection of the coupling ratio of artificial antigen. [Method] Artificial synthetic antigen of citrinin （CIT） was first conjugated with vector protein bovine serum albumin （BSA） and then identified by SDS-PAGE electrophoresis,and its coupling ratio was measured by UV absorption and mass spectrometry. [Result] The identification result showed that artificial antigen of CIT was successfully coupled,and the coupling ratio was 8.4 by UV absorption while 6.0 by mass spectrometry. [Conclusion] The comparison experiment showed that mass spectrometry could rapidly identify the artificial antigen and accurately detect its coupling ratio. This study provided basis for the preparation of CIT antigen as well as the establishment of an for enzyme-linked immunosorbent assay （ELISA） for citrinin determination.
基金supported by the National Natural Science Foundation of China (No. NSFC. 20872020)the Natural Science Foundation of Guangdong Province (No. 06012298 and No. 8251009001000005)
文摘Objective To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin （IgY） to build an enzyme-linked immunosorbent assay （ELISA） for citrinin （CTN）. Methods CTN was conjugated with bovine serum albumin （BSA）, ovalbumin （OVA） with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet （UV） spectrometry and Infrared （IR） spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied. Results UV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng·mL^-1, with a good linearity ranging 20-640 ng·mL^-1. Conclusion Artificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.
基金Supported by the U.S.National Institutes of Health's International Cooperative Biodiversity Groups program(Grant No.NIH ICBG U01-TW007401)
文摘Objective:To Isolate,purify,characterize,and evaluate the bioaclive compounds from the sponge-derived fungus Penicillium sp.FF001 and to elucidate its structure.Methods:The fungal strain FF001 with an interesting bioactivity profile was isolated from a marine Fijian sponge Melophlus sp.Based on conidiophores aggregation,conidia development and mycelia morphological characteristics,the isolate FF001 was classically identified as a Penicillium sp.The bioactive compound was identified using various spectral analysis of UV,high resolution electrospray ionization mass spectra,1H and 13C NMR spectral data.Further minimum inhibitory concentrations(MICs)assay and brine shrimp cytotoxicity assay were also carried out to evaluate the biological properties of the purified compound.Results:Bioassay guided fractionation of the EtOAc extract of a static culture of this Penicillium sp.by different chromatographic methods led the isolation of an antibacterial,anticryptococcal and cytotoxic active compound,which was identified as citrinin(1).Further,citrinin(1)is reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus(S.aureus),rifampicin-resistant 5.aureus,wild type S.aureus and vancomycin-resistant Enterococcus faecium showed MICs of 3.90,0.97,1.95 and7.81μg/mL,respectively.Further citrinin(1)displayed significant activity against the pathogenic yeast Cryptococcus neoformans(MIC 3.90μg/mL),and exhibited cytotoxicity against brine shrimp larvae LD_(50)of 96μg/mL.Conclusions:Citrinin(1)is reported from sponge associated Penicillium sp.from this study and for its strong antibacterial activity against multi-drug resistant human pathogens including cytotoxicity against brine shrimp larvae,which indicated that sponge associated Penicillium spp.are promising sources of natural bioactive metabolites.
基金This work was supported by National Natural Science Foundation of China (No. 30460006) Natural Science Foundation of Jiangxi Province (No. 0330040).
文摘Objective To obtain the full-length cDNA of a novel gene （named yp05） associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3＇ and 5＇ cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene （named yp05） was obtained from the electronic assembly of 3＇-RACE and 5＇- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame （ORF） and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription ofyp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citfinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.
基金This work was supported by Natural Science Foundation of Jiang Xi Province (No.0330040).
文摘To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. Methods Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method. Results Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaⅢ enzyme in inserts. There were seven repeated clones. Conclusion With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags≥10) and 143 unique tags to moderately expressed genes (repeat tags≥2).
基金financially supported by the Xiamen Southern Oceanographic Center(No.22GYY007HJ07)。
文摘The chemical investigation into the EtOAc extract of the deep-sea-derived fungus Penicillium citrinum W22 yielded three unprecedented citrinin dimers,neo-Dicitrinols A–C(1–3)and a known one,penicitrinone A(4).Their structures were elucidated by extensive analysis of spectroscopic data,electronic circular dichroism(ECD)calculation,X-ray diffraction,and biogenetic consideration.neo-Dicitrinols A–C(1–3),bearing a tetramic acid unit,represent the first example of citrinin analogues as hybrid polyketide synthase-nonribosomal peptide synthase(PKS-NRPS)products.neo-Dicitrinol C(3)significantly inhibited renin-angiotensin system-selective lethal 3(RSL3)-induced ferroptosis with a half maximal effective concentration(EC50)value of 21.6μmol/L.
文摘Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurrence of both toxins was found in wheat grain and processed sunflower seeds. Citrinin levels exceeded those of ochratoxin A in the majority of co-contaminated feed samples, and the ratio of (1.1 - 10):1 proved to be the most frequent. A possible role of Aspergillus and Penicillium fungi in separate and simultaneous OTA and CIT occurrence in feeds is also discussed.
基金supported by the National Natural Science Foundation of China(32125031)the Fundamental Research Funds for the Central Universities(JUSRP222001)the Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province,Jiangnan University.
文摘Citrinin(CIT)is one of by-products by Monascus fermentation with known nephrotoxicity.Except dihydro-citrinone,no other metabolites including the possible metabolic pathways have been found in vivo metabolism.In this study,we applied ultra-performance liquid chromatography-mass spectrometry(UPLC-MS)to analyze and identify potential CIT metabolites in the human cytochrome 450(P450)subtype system.Four new metabolites with liver microsomes incubation were first found,namely 3-hydroxy-citrinin(M1),4,5-ene-citrinin(M2),5-hydroxymethyl-citrinin(M3),and 7-hydroxy-citrinin(M4).We also found the distinguished levels of the four metabolites with the incubation of various P450 subtypes.According to the results of molecular docking prediction,it showed the metabolic correlation of CYP3A4 with M1,CYP1A2,CYP2C9,and CYP2D6 with M2,M3,and M4.The maximum level of M1 metabolite was found in the incubation with liver microsome and P450 subtypes.Moreover,M1 and M4 metabolites were found in mice’s duodenum,jejunum,ileum,liver,kidney,and hippocampus,but not M2 and M3.These results indicated the mediation of citrinin metabolism by P450s.It also suggested the concentration differences of metabolites with the incubation of different P450 subtypes.
