Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene f...Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.展开更多
To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the test...To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry. RESULTSAmong the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC<sub>50</sub> = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G<sub>1</sub> DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphorylation of Akt and Erk 1/2. Activation of p38 MAPK was also confirmed using Western blot analysis. This analysis also revealed down-regulation of NF-κB1 (p50) and RelA (p65) proteins and the loss of their anti-apoptotic activity. CONCLUSIONIn our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.展开更多
Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically re...Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.展开更多
The phytoalexin elicitor β-(1→3)-branched β-(1→6)-linked glucohexatose has been regie- and stereospeciflcally synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of m...The phytoalexin elicitor β-(1→3)-branched β-(1→6)-linked glucohexatose has been regie- and stereospeciflcally synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of multiol 3,6-branched gluco-trisac-charides 13 which consists of free 5,6'-OH trisaccharide, free 5,2' ,6'-OH trisaccharide, free 5,3' ,6'-OH trisaccharide and so on. The compounds 10 and 13 were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 2, 3, 4, 6-tetra-O-ben-zoyl-α-D-glucopyranosyl trichloroacetimidate, and 2,3,4,6-te-tra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate through re-gio- and stereoselective manners.展开更多
文摘Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.
文摘To investigate the mechanism of the antiproliferative effect of synthetic indole phytoalexin derivatives on human colorectal cancer cell lines. METHODSChanges in cell proliferation and the cytotoxic effect of the tested compounds on human colorectal cancer cell lines and human fibroblasts were evaluated using MTS and BrdU assay, allowing us to choose the most potent substance. Cell cycle alterations were analyzed using flow cytometric analysis. The apoptosis-inducing effect of compound K-453 on the HCT116 cell line was examined with annexin V/PI double staining using flow cytometry, as well as acridine orange/propidium iodide (AO/PI) staining. The flow cytometry method also allowed us to measure changes in levels or activation states of other factors associated with apoptosis, such as poly (ADP-ribose) polymerase (PARP), caspase-3 and -9, cytochrome c, Bcl-2 family proteins, and also the integrity of the mitochondrial membrane. To evaluate activity of the transcription factors and proteins involved in signaling pathways we used Western blot analysis together with flow cytometry. RESULTSAmong the ten tested compounds, compound K-453 {(±)-trans-1,2-dimethoxy-2’-(3,5-bis-trifluoromethylphenylamino)spiro{indoline-3,5’[4’,5’]dihydrothiazol} exhibited the most potent activity with IC<sub>50</sub> = 32.22 ± 1.14 μmol/L in human colorectal HCT116 cells and was thus selected for further studies. Flow cytometric analysis revealed a K-453-induced increase in the population of cells with sub-G<sub>1</sub> DNA content, which is considered as a marker of apoptotic cell death. The apoptosis-inducing effect of compound K453 was also confirmed by annexin V/PI double staining and AO/PI staining. The apoptosis was associated with the loss of mitochondrial membrane potential, PARP cleavage, caspase-3 and caspase-9 activation, release of cytochrome c, as well as changes in the levels of Bcl-2 family members. Moreover, flow cytometry showed that compound K-453 stimulates phosphorylation of p38 MAPK but decreases phosphorylation of Akt and Erk 1/2. Activation of p38 MAPK was also confirmed using Western blot analysis. This analysis also revealed down-regulation of NF-κB1 (p50) and RelA (p65) proteins and the loss of their anti-apoptotic activity. CONCLUSIONIn our study compound K-453 exhibited an antiproliferative effect by induction of intrinsic apoptosis as well as modulation of several signaling pathways.
基金TI 800/7-1 and TI 800/7-2(SPP 2125 DECRyPT)from the Deutsche Forschungsgemeinschaft,Germany,to A.T.We also acknowledge support from the Cluster of Excellence on Plant Sciences(CEPLAS)the Deutsche Forschungsgemeinschaft under Germany's ExcellenceStrategy-EXC 2048/1-Project ID:390686111and the grant ZU263/11-2(SPP 2125DECRyPT)to A.Z.
文摘Plant immunity is a multilayered process that includes recognition of patterns or effectors from pathogens to elicit defense responses.These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence.Here,we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana(Bs)and Fusarium graminearum(Fg)at the metabolite level.We identify hordedanes,a previously undescribed set of labdane-related diterpenoids with antimicrobial properties,as critical players in these interactions.Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 60o-kb gene cluster.Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes,including one of the most functionally decorated products 19-β-hydroxy-hordetrienoic acid(19-OH-HTA).Barley mutants in the diterpene synthase genes of this cluster are unable to produce hordedanes but,unexpectedly,show reduced Bs colonization.By contrast,colonization by Fusarium graminearum,another fungal pathogen of barley and wheat,is 4-fold higher in the mutants completely lacking hordedanes.Accordingly,19-OH-HTA enhances both germination and growth of Bs,whereas it inhibits other pathogenic fungi,including Fg.Analysis of microscopy and transcriptomics data suggest that hordedanes delay the necrotrophic phase of Bs.Taken together,these results show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.
基金Project supported by the Beijing Natural Science Foundation(No.6021004)and the Ministry of Science and Technology(No.2001AA246014).
文摘The phytoalexin elicitor β-(1→3)-branched β-(1→6)-linked glucohexatose has been regie- and stereospeciflcally synthesized by coupling of the 3, 6-branched gluco-trisaccharide Schmidt reagent 10 with a mixture of multiol 3,6-branched gluco-trisac-charides 13 which consists of free 5,6'-OH trisaccharide, free 5,2' ,6'-OH trisaccharide, free 5,3' ,6'-OH trisaccharide and so on. The compounds 10 and 13 were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose, 2, 3, 4, 6-tetra-O-ben-zoyl-α-D-glucopyranosyl trichloroacetimidate, and 2,3,4,6-te-tra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate through re-gio- and stereoselective manners.
