Employing classical two-dimensional electrophoresis (2-DE), amino acid sequencing and immunoblot analysis, we examine for the first time the effect of ozone, a highly notorious environmental pollutant, on rice seedling proteins. Drastic visible necrotic damage to leaf by ozone and consequent increase in ascorbate peroxidase protein(s) was accompanied by rapid changes in the 2-DE protein profiles, over controls. Out of a total of 56 proteins investigated, which were reproducible in repeated experiments, 52 protein spots were visually identified as differentially expressed over controls. Six proteins were N-terminally blocked, and the sequence of 14 proteins could not be determined, whereas 36 proteins were N-terminally and one was internally sequenced. Ozone caused drastic reductions in the major leaf photosynthetic proteins, including the abundantly present ribulose-1, 5-bisphosphate carboxylase/oxygenase, and induction of various defense/stress related proteins. Most prominent change in leaves, within 24 h post-treatment with ozone, was the induced accumulation of a pathogenesis related (PR) class 5 protein, three PR 10 class proteins, ascorbate peroxidase(s), superoxide dismutase, calcium-binding protein, calreticulin, a novel ATP-dependent CLP protease, and an unknown protein. Present results demonstrate the highly damaging effect of ozone on rice seedlings at the level of the proteome.
Ozone (O(3)), a serious air pollutant, is known to significantly reduce photosynthesis, growth, and yield and to cause foliar injury and senescence. Here, integrated transcriptomics, proteomics, and metabolomics approaches were applied to investigate the molecular responses of O(3) in the leaves of 2-week-old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O(3) for a period of 24 h. On the basis of the morphological alteration of O(3)-exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12, and 24 h using rice DNA microarray chip. A total of 1535 nonredundant genes showed altered expression of more than 5-fold over the control, representing 8 main functional categories. Genes involved in information storage and processing (10%) and cellular processing and signaling categories (24%) were highly represented within 1 h of O(3) treatment; transcriptional factor and signal transduction, respectively, were the main subcategories. Genes categorized into information storage and processing (17, 23%), cellular processing and signaling (20, 16%) and metabolism (18, 19%) were mainly regulated at 12 and 24 h; their main subcategories were ribosomal protein, post-translational modification, and signal transduction and secondary metabolites biosynthesis, respectively. Two-dimensional gel electrophoresis-based proteomics analyses in combination with tandem mass spectrometer identified 23 differentially expressed protein spots (21 nonredundant proteins) in leaves exposed to O(3) for 24 h compared to respective control. Identified proteins were found to be involved in cellular processing and signaling (32%), photosynthesis (19%), and defense (14%). Capillary electrophoresis-mass spectrometry-based metabolomic profiling revealed accumulation of amino acids, gamma-aminobutyric acid, and glutathione in O(3) exposed leaves until 24 h over control. This systematic survey showed that O(3) triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice.
The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H(2)O(2).
Background:We have previously demonstrated that long-term inhibition of Rho-kinase ameliorates pulmonary arterial hypertension (PAH) in animal models. In the present study, we examined the clinical effects of mid-term oral treatment with an extended release formulation of AT-877 (fasudil hydrochloride), a specific Rho-kinase inhibitor (AT-877ER) on PAH. Methods and Results:23 PAH patients were treated with either placebo (10/2 females/males, 51±16 years, idiopathic PAH (IPAH) in 6, PAH associated with connective tissue disease (CTD-PAH) in 3, PAH with congenital heart disease (CHD-PAH) in 2, and portal PAH in 1) or AT-877ER (6/5 females/males, 47±14 years, IPAH in 2, CTD-PAH in 5, and CHD-PAH in 4); 3 patients were excluded. We performed a 6-min walk test and right heart catheterization in the remaining 20 patients, before and 3 months after the treatment (placebo n=11, AT-877ER n=9). Although there were no significant differences between the 2 groups for the 6-min walk distance, pulmonary hemodynamics tended to be improved in the AT-877ER group, especially the prevalence of improved cardiac index from baseline, which was significantly higher in the AT-877ER than in the placebo group. In the AT-877ER group, serum levels of hydroxyfasudil, an active metabolite of AT-877ER tended to correlate with improvements in the cardiac index and mean pulmonary artery pressure. Conclusions Study DesignFrom the viewpoint of feasibility, the sample size was planned as 30 patients in total (10 patients for WHO functional class I and 20 for functional classes II-III. The present study was designed as a 3-month, double-blind, randomized, placebocontrolled, multicenter trial in which 14 PAH centers in Japan participated. All patients were hospitalized 3-6 days before the first examination (day 1) and the last examination (week 12) ( Figure S1). Administration of the study drug was started and ended during the hospitalization periods. Patients received either AT-877ER or placebo capsule twice daily (Asahi Kasei Pharma Corporation, Tokyo, Japan) for 12 weeks in a blind manner ( Figure S1). The dosage of AT-877ER was increased every 3 days in a stepwise manner from 2 to 6 capsules/day ( Figure S1). All patients were administered 2 capsules/day until day 4, when the dose was increased by the investigator's decision to 4 capsules/day. Until day 7, 4 capsules/day were given and the next doses were decided by investigators on day 7. Before increasing the study drug on days 4, 7, and 10, investigators checked the safety of the treatment in each subject and determined the subsequent treatment plan as follows.Whenever it was difficult to follow the intended regimen because of adverse effects or other reasons, the situation was required to be judged as "continuation at the dose level at the time of occurrence of the adverse drug reaction", "continuation with reduced dosage", or "discontinuation of study treatment". The treatment was randomized according to the 6-min walk distance, with drugs prescribed at baseline as stratifying factors, and...
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