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.

Molecular and functional characterization of ferritin in abalone Haliotis diversicolor supertexta

Ferritin is an iron storage protein that plays a key role in the processes of physiology and pathology.In the present study,the authors reported the ferritin gene from abalone Haliotis diversicolor supertexta,which we named hds-ferritin.The full-length of hds-ferritin cDNA consisted of 879 bp with an ORF encoding a 171 amino acids.Amino acid sequence analysis revealed that hds-ferritin shared highly homology with other species.Real time PCR and western blot analysis showed that hds-ferritin was distributed ubiquitously in abalone tissues and had the highest expression level in digestive glands,but its transcripts are not modified remarkably by the stimulation with LPS.The recombinant protein was successfully expressed in Escherichia coli BL21 (DE3),and the titre of anti-ferritin antibody was about 1∶14000.The effects of ROS and RNS on ferritin were analyzed in the present study.The results showed that H2O2 played an important role in decreasing hds-ferritin,however NO cation appeared to have a protecting effect on H2O2-medied reduction of hds-ferritin.

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.