Mechanism of Oxidative Burst in Tobacco Leaves and Cells Induced by Palmin from Phytophthora palmi

In order to reveal the signaling pathways triggered by elicitor in plant-microbe interactions, the mechanisms of hypersensitive necrosis responses in Nicotiana tabacum L. cv. Gexin III induced by palmin were studied at molecular and cellular level. The burst of superoxide, intercellular diffusion of hydrogen peroxide and process of cell death induced by palmin were investigated in tobacco plants by biochemical methods and Confocal microscopy. The results showed that a large amount of O-2(.-) was rapidly generated in tobacco cell elicited by palmin as a result of activation of NADPH oxidase, and the O-2(.-) was dismutated into H2O2 immediately by superoxide dismutase (SOD). Accumulation and intercellular diffusion of H2O2 were shown to be a trigger for hypersensitive cell death; and Ca2+ and some specific protein kinase were also shown to be involved in the activation of oxidative burst in tobacco cell induced by palmin.

Phagocytic and oxidative burst activity of neutrophils in the end stage of liver cirrhosis

AIM：To evaluate the phagocytic activity and neutrophil oxidative burst in liver cirrhosis.METHODS： In 45 patients with advanced postalcoholic liver cirrhosis （aged 45±14 years） and in 25 healthy volunteers （aged 38±5 years）, the percentage of phagocytizing cells after in vitro incubation with E. coli （Phagotest Kit）, phagocytic activity （mean intensity of fluorescence, MIF） and the percentage of neutrophil oxidative burst （Bursttest Kit）, and the level of free oxygen radical production （MIF of Rodamine 123） were analyzed by flow cytometry. The levels of soluble sICAM-1, sVCAM-1, sP-selectin, sE-selectin, sL-selectin, and TNF-α were determined in blood serum.RESISTS： The percentage of E. coli phagocytizing neutrophils in liver cirrhosis patients was comparable to that in healthy subjects. MIF of neutrophil - ingested E. coli was higher in patients with liver cirrhosis. The oxidative burst in E. coli phagocytizing neutrophils generated less amount of active oxygen compounds in liver cirrhosis pa- tients （MIF of R123：24.7±7.1 and 29.7±6.6 in healthy, P〈0.01）. Phorbol myristate acetate （PMA） - stimulated neutrophilsproduced less reactive oxidants in liver cirrhosis patients than in healthy subjects （MIF of R123： 42.7±14.6 vs 50.2±13.3, P〈0.01）. A negative correlation was observed between oxidative burst MIF of PMA-stimulated neutrophils and ALT and AST levels （r -0.35, P〈0.05; r-0.4, P〈0.03）. sVCAM-1, sICAM-1, sE-selectin concentrations correlated negatively with the oxygen free radical production （MIF of R123） in neutrophils after PMA stimulation in liver cirrhosis patients （r-0.45, P〈0.05, r-0.41, P〈0.05; r-0.39, P〈0.05, respectively）.CONCLUSION： Neutrophil metabolic activity diminishes together with the intensification of liver failure. The metabolic potential of phagocytizing neutrophils is significantly lower in liver cirrhosis patients, which can be one of the causes of immune mechanism damage. The evaluation of oxygen metabolism of E. coli-stimulated neutrophils reveals that the amount of released oxygen metabolites is smaller in liver cirrhosis patients than in healthy subjects.

Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

A rapid and concentration-dependent generation of superoxide anion （·O2^-）, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts （LaCl3 and CdCl3 ） were added to tobacco （ Nicotiana tabacum） cell suspension culture. Addition of superoxide dismutase （480 U·ml^-1） and Tiron （5 μmol·L^-1） to cell culture suspension decreases the level of lanthanide cation-induced ·O2^- generation, suggesting that ·O2^- generation is extra-cellular. Pretreatment of the cell culture suspension with diphenyleneiodonium （10 and 50 μmol·L^-1 ）, quinacrine （ 1 and 5 mmol· L^-1 ） and imidazol （ 10 mmol· L^-1 ）, inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase. In addition, addition of SHAM （1 and 5 mmol· L^-1）, azide （0.2 and 1 mmol· L^-1 ）, inhibitor of peroxidase, has no influence on ·O2^- generation.

Mycorrhiza improves cold tolerance of Satsuma orange by inducing antioxidant enzyme gene expression

A potted experiment was carried out to study the effect of an arbuscular mycorrhizal fungus(Diversispora versiformis)and arbuscular mycorrhizal like fungus(Piriformospora indica)on antioxidant enzyme defense system of Satsuma orange(Citrus sinensis cv.Oita 4)grafted on Poncirus trifoliata under favourable temperature(25°C)and cold temperature(0°C)for 12 h.Such short-term treatment of cold temperature did not cause any significant change in root fungal colonization and spore density in soil.Under cold stress,D.versiformis inoculation did not change the activity of superoxide dismutase(SOD),catalase(CAT),and peroxidase(POD)in leaves and roots,whereas P.indica inoculation significantly increased the activity of CAT in roots and POD in leaves only.In addition,inoculation of two mycorrhizal fungi under cold stress significantly increased the relative expression levels of PtPOD and PtF-SOD in leaves,P.indica up-regulated the expression levels of PtCu/Zn-SOD in leaves,and D.versiformis also induced the expression levels of PtMn-SOD and PtCAT1 in leaves.In addition,inoculated Oita 4 trees maintained significantly lower hydrogen peroxide levels and malondialdehyde contents in leaves and roots under cold temperature,suggesting lower oxidative damage.Therefore,we concluded that arbuscular mycorrhizal fungi(especially P.indica)mainly induced the expression of antioxidant enzyme genes,depending on the fungal species,and thus mitigated oxidative damage for higher cold resistance in inoculated plants.

Carcinoembryonic antigen inhibits neutrophil activation by N-formyl-methionyl-leucyl-phenylalanine

Carcinoembryonic antigen(CEA)is a surface glycoprotein expressed in human epithelial cells and is released from their surface,especially during colorectal cancer.Frequently,colorectal cancer is accompa­nied by inflammation,where tumor-infiltrating neutrophils play an important role.CEA was also found to be a strong chemotactic agent for neutrophils.The purpose of this study was to find out if CEA can enhance neu­trophil priming and activation.Primed neutrophils were activated by N-formyl-methionyl-leucyl-phenylalanine(formyl-MLP)and the resulting oxidative burst was measured luminometrically.Unexpectedly,in vitro priming of neutrophils by CEA,alone or preceded by LPS,inhibited subsequent activation of these cells by formyl-MLP.CEA may have anti-inflammatory properties in vivo.

Dihydrosphingosine-lnduced Programmed Cell Death in Tobacco BY-2 Cells Is Independent of H2O2 Production

Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.