Jong Bok Seo

Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice

BackgroundThe rice roots are highly salt-sensitive organ and primary root growth is rapidly suppressed by salt stress. Sucrose nonfermenting 1-related protein kinase2 (SnRK2) family is one of the key regulator of hyper-osmotic stress signalling in various plant cells. To understand early salt response of rice roots and identify SnRK2 signaling components, proteome changes of transgenic rice roots over-expressing OSRK1, a rice SnRK2 kinase were investigated.ResultsProteomes were analyzed by two-dimensional electrophoresis and protein spots were identified by LC-MS/MS from wild type and OSRK1 transgenic rice roots exposed to 150 mM NaCl for either 3 h or 7 h. Fifty two early salt -responsive protein spots were identified from wild type rice roots. The major up-regulated proteins were enzymes related to energy regulation, amino acid metabolism, methylglyoxal detoxification, redox regulation and protein turnover. It is noted that enzymes known to be involved in GA-induced root growth such as fructose bisphosphate aldolase and methylmalonate semialdehyde dehydrogenase were clearly down-regulated. In contrast to wild type rice roots, only a few proteins were changed by salt stress in OSRK1 transgenic rice roots. A comparative quantitative analysis of the proteome level indicated that forty three early salt-responsive proteins were magnified in transgenic rice roots at unstressed condition. These proteins contain single or multiple potential SnRK2 recognition motives. In vitro kinase assay revealed that one of the identified proteome, calreticulin is a good substrate of OSRK1.ConclusionsOur present data implicate that rice roots rapidly changed broad spectrum of energy metabolism upon challenging salt stress, and suppression of GA signaling by salt stress may be responsible for the rapid arrest of root growth and development. The broad spectrum of functional categories of proteins affected by over-expression of OSRK1 indicates that OSRK1 is an upstream regulator of stress signaling in rice roots. Enzymes involved in glycolysis, branched amino acid catabolism, dnaK-type molecular chaperone, calcium binding protein, Sal T and glyoxalase are potential targets of OSRK1 in rice roots under salt stress that need to be further investigated.

Nontargeted Metabolite Profiling in Compatible Pathogen-Inoculated Tobacco (Nicotiana tabacum L. cv. Wisconsin 38) Using UPLC-Q-TOF/MS

A biphasic reactive oxygen species (ROS) production has previously been observed in tobacco at 1 and 48 h after inoculation with the hemibiotrophic compatible pathogen, Phytophthora parasitica var. nicotianae (Ppn). To characterize the response of tobacco to biphasically produced ROS concerning the propagation of Ppn, ultraperformance liquid chromatography-quadrupole-time of flight/ mass spectrometry (UPLC-Q-TOF/MS) based metabolic profiling combined with multivariate statistical analysis was performed. Among the nonredundant 355 mass ions in ESI+ mode and 345 mass ions in ESI- mode that were selected as significantly changed by Ppn inoculation (|p(corr)| > 0.6 on S-plot of orthogonal partial least-squares discriminant analysis (OPLS-DA), fold-change > 2, and p < 0.05 in the independent two-sample t test), 76 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Phenolic amino acids, phenylpropanoids, hydroxycinnamic acid amides, linoleic acid, linolenic acid, lysophospholipids, glycoglycerolipids, and trioxidized phospholipids were identified as having changed after Ppn inoculation. On the basis of their quantitative changes, the metabolic responses occurring at each phase of ROS production after Ppn inoculation were investigated in this study.

Anti-inflammatory effect of Rhus verniviflua Stokes by suppression of iNOS-mediated Akt and ERK pathways: in-vitro and in-vivo studies.

Objectives  Rhus verniciflua Stokes (RVS), which has valuable medicinal properties, has for many years been prescribed for inflammation in east Asian medicine. Recent studies suggest that RVS has potent antioxidative, antitumor and anti‐inflammatory properties.

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two-dimensional electrophoresis.

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH4HCO3 + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to d-(+)-raffinose and N-acetyllactosamine were much higher than that to l-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model

BackgroundTo investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders.ResultsBy performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models.ConclusionsUPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis.

E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response

Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

Identification and Characterization of Expansins from Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Ilsong Institute of Life Science, Hallym University, Anyang 431-060, Korea(Received on August 7, 2012; Accepted on August 16, 2012)We identified two novel expansin (EXP) genes in theexpressed sequence tag database of Bursaphelenchusxylophilus, designated as Bx-EXPB2 and -EXPB3.Novel Bx-EXPBs encoded 150 amino acids and theirsimilarities in coding sequence were 70.7−84.0% to thepreviously reported EXPB1 of B. xylophilus. Bx-EXPB2and Bx-EXPB3 were clustered with Bx-EXPB1 and Bm-EXPB1, respectively, forming the independent phylo-geny with other nematode EXPs. All identified Bx-EXPBscontained the signal peptide and were only expressedduring the propagative stage, suggesting that they aresecreted to facilitate nematode migration through hostsby loosening cell walls during infection. Quantitativereal-time PCR analysis showed that the relative ac-cumulation of Bx-EXPB3 mRNAs was the highestamong the three Bx-EXPs examined and the order ofmRNA accumulation was as follows: Bx-EXPB3 > Bx-EXPB2 >> Bx-EXPB1. Homology modeling of Bx-EXPBs showed that the structurally optimum templatewas EXLX1 protein of Bacillus subtilis, whichsharedresidues essential for catalytic activity with Bx-EXPB1and Bx-EXPB2 except for Bx-EXPB3. Taken together,Bx-EXPB1 and Bx-EXPB2 may be involved migrationthrough plant tissues and play a role in pathogenesis.Keywords : Bursaphelenchus xylophilus, infections and propagation in host trees. expansin, homo-logy protein modeling, pathogenicity, pine wilt disease The pinewood nematode, Bursaphelenchus xylophilus isthe known agent causative of pine wilt disease (Mamiya,1975; Lee et al., 2011). Bursaphelenchus xylphilus areknown to be transferred from infected pine trees to newones by the pine sawyer beetle, Monochamus alternates(Mamiya, 1975). To express their potent phytopathogeni-city that is associated with infection and maintenance inthe host trees, B. xylophilus must overcome the physicalbarriers such as plant cell walls, which are mainly com-posed of polysaccharides, lipids, and proteins. The architectof plant cell wall is determined by cellulose, made of re-peating glucose molecules joined by β-1,4 glycosidic bonds(McQueen-Mason and Cosgrove, 1994; van den Brink andde Vries, 2011). The cellulose framework of the wall isinterpenetrated by a cross-linked matrix of non-cellulosemolecules, hemicelluloses, or pectin. Even though pinewoodnematode infected trees usually wither and die within acouple of months (Mamiya, 1975), we still do not understandthe molecular and cellular pathophysiology underlying theabilities of pinewood nematodes to infection and propagatein the trees. However, the presence of diverse cell walldegrading enzymes such as endoglucanases (Kikuchi et al.,2004; Smant et al., 1998), pectate lyases (Kikuchi et al.,2006; Popeijus et al., 2000) and expansins (EXPs) (Kikuchiet al., 2009) that to play crucial roles in initiation andprogression of the plant diseases are present in B. xylophilusgenomes suggested that secreted cell wall degrading enzymesfrom B. xylophilus may be important factors involved withEXPs in plants play pivotal roles in a wide variety ofdevelopmental processes by loosening of their cell walls byweakening non-covalent interactions (McQueen-Mason andCosgrove, 1994; McQueen-Mason and Cosgrove, 1995;Whitney et al., 2000), which makes cell walls susceptible todegradation and remodeling by other cell wall degradingenzymes (Cosgrove, 2000; van den Brink and de Vries,2011). While most EXPs were identified from plants, recentfindings that plant-parasitic nematodes express functionalEXPs suggested that they could be one of the major factorsregulating plant-parasite interactions (Qin et al., 2004; vanden Brink and de Vries, 2011). However, compared withEXPs in plants, the understanding of in vivo functions of

Metabolic survey of defense responses to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae, in ethylene signaling-impaired tobacco.

Reactive oxygen species (ROS) and ethylene play an important role in determining the resistance or susceptibility of plants to pathogen attack. A previous study of the response of tobacco cultivar ( Nicotiana tabacum L. cv. Wisconsin 38) to a compatible hemibiotroph, Phytophthora parasitica var. nicotianae (Ppn) showed that biphasic bursts of ROS and ethylene are positively associated with disease severity. The levels of ethylene and ROS might influence the susceptibility of plants to pathogens, with changing levels of metabolite related to disease resistance or susceptibility. In this study, to obtain more detailed information on the interaction of ROS and ethylene signaling related to resistance and/or susceptibility of plants to pathogen, Ppn-induced metabolic profiles from wild type (WT) and ethylene signaling-impaired transgenic plants that expressed Ein3 antisense (Ein3-AS) were compared using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Nonredundant mass ions (576 in ESI+ mode and 336 in ESI- mode) were selected, and 56 mass ions were identified on the basis of their accurate mass ions and MS/MS spectra. Two-way hierarchical clustering analysis of the selected mass ions revealed that nicotine and phenylpropanoid-polyamine conjugates, such as caffeoyl-dihydrocaffeoyl-spermidine, dicaffeoyl-spermidine, caffeoyl-feruloyl-spermidine, and two bis(dihydrocaffeoyl)-spermine isomers, and their intermediates, such as arginine and putrecine, were present at lower levels in Ein3-AS transgenic plants during Ppn interaction than in WT, whereas galactolipid and oxidized free fatty acid levels were higher in Ein3-AS transgenic plants. Taken together, these results reveal a function for ethylene signaling in tobacco defense responses during Ppn interaction.

Drosophila Torsin Protein Regulates Motor Control and Stress Sensitivity and Forms a Complex with Fragile-X Mental Retardation Protein

We investigated unknown in vivo functions of Torsin by using Drosophila as a model. Downregulation of Drosophila Torsin (DTor) by DTor-specific inhibitory double-stranded RNA (RNAi) induced abnormal locomotor behavior and increased susceptibility to H2O2. In addition, altered expression of DTor significantly increased the numbers of synaptic boutons. One important biochemical consequence of DTor-RNAi expression in fly brains was upregulation of alcohol dehydrogenase (ADH). Altered expression of ADH has also been reported in Drosophila Fragile-X mental retardation protein (DFMRP) mutant flies. Interestingly, expression of DFMRP was altered in DTor mutant flies, and DTor and DFMRP were present in the same protein complexes. In addition, DTor and DFMRP immunoreactivities were partially colocalized in several cellular organelles in larval muscles. Furthermore, there were no significant differences between synaptic morphologies of dfmrp null mutants and dfmrp mutants expressing DTor-RNAi. Taken together, our evidences suggested that DTor and DFMRP might be present in the same signaling pathway regulating synaptic plasticity. In addition, we also found that human Torsin1A and human FMRP were present in the same protein complexes, suggesting that this phenomenon is evolutionarily conserved.