Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings

The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.     

The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis

Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4-dichlorophenoxyacetic acid (2,4-D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative-to-embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical-inducible activation-tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high-frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol-inducible XVE-WUS transgene can phenocopy pga6-1 and pga6-2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.     

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

Role of abscissic acid in water stress-induced antioxidant defense in leaves of maize seedlings

Roles of abscisic acid (ABA) in water stress-induced oxidative stress were investigated in leaves of maize ( Zea mays L.) seedlings exposed to water stress induced by polyethylene glycol (PEG 6000). Treatment with PEG at -0.7 MPa for 12 and 24 h led to a reduction in leaf relative water content (RWC) by 7.8 and 14.1%, respectively. Duration of the osmotic treatments is considered as mild and moderate water stress. The mild water stress caused significant increases in the generation of superoxide radical ( O 2 - ) and hydrogen peroxide (H 2 O 2 ), the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the contents of ascorbate (ASC), reduced glutathione (GSH). The moderate water stress failed to further enhance the capacity of antioxidant defense systems, as compared to the mild water stress. The contents of catalytic Fe, which is critical for H 2 O 2 -dependent hydroxyl radical ( •OH) production, and the oxidized forms of ascorbate and glutathione pools, dehydroascorbate (DHA) and oxidized glutathione (GSSG), markedly increased, a significant oxidative damage to lipids and proteins took place under the moderate water stress. Pretreatment with ABA caused an obvious reduction in the content of catalytic Fe and significant increases in the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants, and then significantly reduced the contents of DHA and GSSG and the degrees of oxidative damage in leaves exposed to the moderate water stress. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA induced by water stress, reduced the enhancement in the capacity of antioxidant defense systems, and resulted in an increase in catalytic Fe, DHA and GSSG, and oxidative damage in the water-stressed leaves. These effects were completely prevented by addition of ABA, which raised the internal ABA content. Our data indicate that ABA plays an important role in water stress-induced antioxidant defense against oxidative stress.     

Expression profiles of 109 apoptosis pathway-related genes in 82 mouse tissues and experimental conditions

Apoptosis is an important process in development and tissue homeostasis. To understand the similarities and differences in the apoptosis machinery in different normal, developmental, and diseased tissues, the expression profiles of 109 apoptosis-pathway-related genes in 82 mouse tissues and experimental conditions were examined using Incyte Mouse GEMI cDNA arrays. It has been found that the compositions of the apoptotic machinery vary among different tissues, developmental stages, and disease states, with subsets of apoptotic genes co-ordinately expressed in the 82 tissues and experimental conditions. Additional genes whose expression profiles resemble selected genes from the 109 apoptotic gene list were also identified. This study provides valuable information on possible molecular mechanisms of differential apoptotic responses to developmental signals, environmental stimuli, and therapeutic treatments in tissue-specific manner.     

Mechanism of action of RNase T. I. Identification of residues required for catalysis,substrate binding,and dimerization

Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.     

Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.     

A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis

Two-dimensional electrophoresis is a critical technique for proteome research, but currently available methods are not capable of resolving the >10,000 protein components in most eukaryotic proteomes. We have developed and demonstrated the utility of a novel solution isoelectrofocusing device and method that can reproducibly prefractionate cell extracts into well-defined pools prior to 2D PAGE on a scale directly compatible with the high sensitivity of proteome studies. A prototype device was used to separate metabolically radiolabeled Escherichia coli extracts in method optimization and proof-of-principle experiments. Samples were loaded into separation chambers divided by thin polyacrylamide gels containing immobilines at specific pH values and isoelectrically focused for several hours, which resulted in well-resolved fractions. Total recoveries in the fractionated samples were greater than 80% and most protein spots in the original sample were recovered after this prefractionation step. Nonideal behavior (precipitation/aggregation), typically encountered when unfractionated samples at high protein loads were applied directly to either narrow- or broad-range IPG gels, was dramatically reduced. Hence this approach allows increases in overall protein loads, resolution, and dynamic detection range compared with either alternative prefractionation methods or direct use of parallel narrow pH range gels without sample prefractionation. The pH ranges and number of fractions can be readily adapted to the requirements of specific types of samples and projects. This method should allow quantitative comparisons of at least 10,000 protein components on a series of narrow pH range gels, and protein detection limits are estimated to be 1000 molecules per cell when mammalian proteomes are fractionated into five or more pools.