Induction of Chlorosis,ROS Generation and Cell Death by a Toxin Isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.     

Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae

Laccase oxidation of phenolic azo dyes was examined with a commercially available laccase from Pyricularia oryzae as the model. Methyl-, methoxy-, chloro-, and nitro-substituted derivatives of 4-(4(prm1)-sulfophenylazo)-phenol were examined as substrates for this laccase. Only the substituents on the phenolic ring were changed. Among the dyes examined, only 2-methyl-, 2-methoxy-, 2,3-dimethyl-, 2,6-dimethyl-, 2,3-dimethoxy-, and 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol served as substrates. Preliminary kinetic studies suggest that 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol is the best substrate. Laccase oxidized the 2,6-dimethyl derivative of 4-(4(prm1)-sulfophenylazo)-phenol to 4-sulfophenylhydroperoxide (SPH) and 2,6-dimethyl-1,4-benzoquinone. The 2-methyl- and 2-methoxy-substituted dyes were oxidized to SPH and either 2-methyl- or 2-methoxy-benzoquinone. Six products were formed from laccase oxidation of the 2,6-dimethoxy-substituted dye. Three of them were identified as SPH, 4-hydroxybenzenesulfonic acid, and 2,6-dimethoxybenzoquinone. A mechanism for the formation of benzoquinone and SPH from laccase oxidation of phenolic azo dyes is proposed. This study suggests that laccase oxidation can result in the detoxification of azo dyes.     

Induction of cell death by adenoviruses

Adenoviruses have proved to be excellent tools for gaining insight into the regulation, and deregulation, of the mammalian cell cycle. With the widespread clinical use of gene therapy fast approaching, there comes a need for a better understanding of how the cell death process is regulated. A greater understanding will allow the development of therapeutic approaches that both maximise transgene expression while minimising cytotoxicity to the target cell. Consequently, much adenovirus research has centered on understanding the mechanisms governing adenovirus induced cell death or apoptosis. This review discusses recent advances in the field of adenovirus cell death regulation and evaluates the roles of implicated gene products and their respective data. The data suggest the existence of multiple virus gene products involved in cell death regulation and point towards several distinct, yet related, cell death pathways. A discussion of the shortcomings of current adenoviral research, along with a proposed model based upon the data is also given.     

Kitazin P,Inhibitor of Phosphatidylcholine Biosynthesis in Pyricularia oryzae

The effect of organophosphorus fungicide, Kitazin P (IBP, S-benzyl diisopropyl phosphorothiolate), on lipid biosynthesis of Pyricularia oryzae was investigated. Addition of IBP to the mycelial cells suspension of P. oryzae induced a striking decrease in incorporation of methionine-methyl-¹⁴C, S-adenosylmethionine-methyl-¹⁴C, and glycerol-1-¹⁴C into phosphatidylcholine, which is the most abundant phospholipid in P. oryzae, but incorporation of choline-methyl-¹⁴C into phosphatidylcholine and that of methionine-methyl-¹⁴C into simple lipids were not affected. Incorporation of methionine-methyl-¹⁴C into phosphatidylcholine is found to be directly proportional to mycelial cells growth of P. oryzae. Enzymes responsible for the biosynthesis from glycerol to phosphatidylcholine through Greenberg’s pathway, except phospholipid N-mefhyltransferase, were not inhibited by IBP. IBP concentration required for 50% inhibition of phospholipid N-methyltransferase was 40 ppm. IBP had no effect on activities of glycerokinase, glycerophosphate acyltransferase, phosphatidic acid cytidyltransferase, phosphatidylserine synthetase, and phosphatidylserine decarboxylase, respectively. Therefore, the specific inhibition of conversion from phosphatidylethanolamine to phosphatidylcholine by the transmethylation of S-adenosylmethionine might be regarded as one of the modes of action of IBP.     

Induction of apoptotic cell death by putrescine

The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high.     

Induction of mammalian cell death by a plant Bax inhibitor

Arabidopsis thaliana AtBI-1 is an orthologue of mammalian Bax inhibitor-1 capable of suppressing Bax-induced cell death in yeast as well as mammalian cells. Here we investigated whether or not AtBI-1 suppresses Bax-induced cell death using human fibrosarcoma HT1080 cells. Surprisingly, AtBI-1 did not block Bax-induced cell death, but it triggered apoptotic cell death in mammalian cells. The proapoptotic effect of AtBI-1 was blocked by the X-linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI-1 is similar to that caused by Bax.     

Identification of Sternbin and Naringenin as Detoxified Metabolites from the Rice Flavanone Phytoalexin Sakuranetin by Pyricularia oryzae

Sakuranetin ( 1 ) is a flavanone phytoalexin that has been reported to play an important role in disease resistance in rice plants. The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) has been reported to metabolize 1 to lower its antifungal activity. Here, two flavanones, sternbin ( 2 ) and naringenin ( 3 ), were identified as metabolites of 1 in P. oryzae suspension culture by liquid chromatography tandem mass spectrometry (LC/MS/MS). The inhibition of 1 , 2 , and 3 on P. oryzae mycelial growth were 45%, 19%, and 19%, respectively, at a concentration of 100 μm . Thus, 2 and 3 are detoxified metabolites of 1 by P. oryzae.     

Participation of Oryza sativa leaf wax in appressorium formation by Pyricularia oryzae

Appressorium formation of Pyricularia oryzae P2 on cover-glass coated with each of the components of rice leaf wax was examined. Wax esters, aldehydes and alcohols, having polar groups and low contact angles, promoted appressorium formation, but alkanes, non-polar molecules having high contact angles, had no effects. Germination of conidia, however, was not aftected with those constituents.     

Cytolethal distending toxin generates cell death by inducing a bottleneck in the cell cycle

Cytolethal distending toxin (CDT) is a bacterial protein that is widely distributed among gram-negative bacteria including Escherichia coli, Campylobacter spp., enterohepatic Helicobacter spp., Actinobacillus actinomycetemcomitans and Haemophilus ducreyi. In vitro studies demonstrated that it is able to stop proliferation of various cell lines. The toxin is composed of three subunits designated CDTs A, B and C. The B subunit targets the eukaryotic DNA and triggers a signalling pathway involving different protein kinases which results in a cell block before entering into mitosis. To date, the individual role of the A and C subunits has not been totally elucidated. There are indications that the CDT is also produced in vivo. Its exact role in pathogenesis is not yet clear, but possible actions include inhibition of epithelial cell proliferation, apoptosis of immune cells and inhibition of a fibrotic response.     

茶多酚对稻瘟病菌的抑制作用及抑菌机理

用不同浓度茶多酚对稻瘟病菌进行抑菌和抑菌机理研究。结果表明,不同浓度的茶多酚对稻瘟病菌菌丝生长和分生孢子萌发具有很强的抑制作用。随着茶多酚浓度的增加,其抑制作用增强,其中5.00 mg/mL和10.00 mg/mL抑制效果最好,其抑制率高达100%,且分生孢子畸形,细胞破裂,原生质外溢。其作用机理主要是破坏菌体的细胞膜结构,抑制CAT、POD酶活,使其丧失细胞膜的屏障和酶系的保护功能,最终导致菌体生长受到抑制或死亡。     

Induction of autophagic cell death by a novel molecule is increased by hypoxia

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.     

Induction of cross-links between DNA and protein by PR toxin,a mycotoxin from Penicillium roqueforti

PR toxin, a mycotoxin from Penicillium roqueforti, induces DNA—protein cross-links in chromatin of both cultured cells and isolated rat-liver nuclei. The presence of the aldehyde group in the PRT molecule is required for the induction of cross-linking; methylene bridges between nucleic acid and protein are presumably involved in the complex formation. The role of other functional groups of PR toxin is discussed.     

Differentiation of Physiologic Races of the Rice Blast Fungus, Pyricularia oryzae Cav. by PAGE-electrophoresis

Eighty-three isolates of the rice blast fungus (Pyricularia oryzae) were tested with respect to genetic diversity and the possibility of race differentiation by electrophoresis. The fungus was genetically very heterogeneous. The isolates were differentiated into 6 races by pathogenicity on race differential varieties. There was little correlation between pathogenicity and zymogram types of one particular enzyme such as esterase, phosphatase or catalase. The isolates were divided into 14 groups by the combination of the zymogram types of the three enzymes. The isolates in the same group showed similar pathogenicity. A new method is proposed which differentiates the blast fungus races by the combination of zymogram type of enzymes. The details, are discussed.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.