Expression and characterization of monocot rice cytosolic CuZnSOD protein in dicot Arabidopsis

Cytosolic CuZnSOD removes deleterious superoxides from plant cells. In order to understand its function better, we sought to express a monocot CuZnSODgene in transgenic Arabidopsis. We constructed a transgene usingthe CaMV 35S promoter to express a rice cytosolic CuZnSOD gene in Arabidopsis and generated over 200 transformants. A 16kD polypeptide, the same size as the native rice CuZnSOD polypeptide, was detected inthe transgenic Arabidopsis. Interestingly, two forms of riceCuZnSOD, rSODI and rSODII, having the same dimeric size, were detectedin the transgenic plants. rSODII protein was relatively abundant but hadlow specific activity. In contrast, rSODI protein was relatively rareand had high specific activity. Inter-conversion of rSODI and rSODIIcould be achieved by the addition and removal of copper ions into the purifiedrecombinant SOD and to the leaf extract of transgenic plants. Ouranalysis indicates that rSODI most likely corresponds to native riceCuZnSOD that has incorporated the Cu and Zn ions required for fullactivity, whereas the less active rSODII form may not have properlyincorporated the necessary copper ions.     

Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.     

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.     

Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-gamma

Th2 T cell immune-driven inflammation plays an important role in allergic asthma. We studied the effect of counterbalancing Th1 T cells in an asthma model in Brown Norway rats that favors Th2 responses. Rats received i.v. transfers of syngeneic allergen-specific Th1 or Th2 cells, 24 h before aerosol exposure to allergen, and were studied 18-24 h later. Adoptive transfer of OVA-specific Th2 cells, but not Th1 cells, and OVA, but not BSA exposure, induced bronchial hyperresponsiveness (BHR) to acetylcholine and eosinophilia in a cell number-dependent manner. Importantly, cotransfer of OVA-specific Th1 cells dose-dependently reversed BHR and bronchoalveolar lavage (BAL) eosinophilia, but not mucosal eosinophilia. OVA-specific Th1 cells transferred alone induced mucosal eosinophilia, but neither BHR nor BAL eosinophilia. Th1 suppression of BHR and BAL eosinophilia was allergen specific, since cotransfer of BSA-specific Th1 cells with the OVA-specific Th2 cells was not inhibitory when OVA aerosol alone was used, but was suppressive with OVA and BSA challenge. Furthermore, recipients of Th1 cells alone had increased gene expression for IFN-gamma in the lungs, while those receiving Th2 cells alone showed increased IL-4 mRNA. Importantly, induction of these Th2 cytokines was inhibited in recipients of combined Th1 and Th2 cells. Anti-IFN-gamma treatment attenuated the down-regulatory effect of Th1 cells. Allergen-specific Th1 cells down-regulate efferent Th2 cytokine-dependent BHR and BAL eosinophilia in an asthma model via mechanisms that depend on IFN-gamma. Therapy designed to control the efferent phase of established asthma by augmenting down-regulatory Th1 counterbalancing mechanisms should be effective.     

Signaling system in Porphyromonas gingivalis based on a LuxS protein

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.     

FLASH coordinates NF-kappa B activity via TRAF2

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.     

Antioxidative and antitumor promoting effects of [6]-paradol and its homologs

Benzo[a]pyrene diol epoxide, a metabolite of benzo[a]pyrene (BaP), and chlorohydrin, the reaction product of chloride and the epoxide, form in vitro the same trans- and cis-stereoisomeric DNA adducts, but in different proportions. In this study, we asked whether the DNA adduct concentration can be kept the same by applying the appropriate dose of (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)and (+/-)-7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-BPDCH) to rodent skin and whether the DNA adducts formed differ only in their trans- and cis-stereoisomerism. Skin from C57Bl6 mice, spontaneous hypertension rats (SHR) and Sprague-Dawley (SD) rats was treated ex vivo immediately after the death of the animals with anti-BPDE and its corresponding bay region chlorohydrin trans-BPDCH and the epidermis was analyzed for DNA adducts 1h after the application. We found that adduct formation at the exocyclic amino groups of deoxyguanosine and deoxyadenosine in epidermal DNA followed a linear dose-response within 6--100 nmol/cm(2) with both chemicals. In order to achieve the same adduct concentration in mouse, spontaneous hypertension rat (SHR), and Sprague-Dawley (SD) rat skin, respectively, a 37-, 23- and 10-fold lower dose of anti-BPDE than of trans-BPDCH had to be applied. The order of 2'-deoxyguanosine (dGuo) adduct concentration with anti-BPDE was similar to what has been reported, but the order with trans-BPDCH was (+)-cis-BPDE-N(2)-dGuo adduct>(+)-trans-BPDE-N(2)-dGuo=(-)-trans-BPDE-N(2)-dGuo>(-)-cis-BPDE-N(2)-dGuo in mouse skin. Irrespective of species or strain, a significantly higher proportion of cis-adducts was obtained after treatment with trans-BPDCH than after treatment with anti-BPDE. Therefore, DNA adduct concentration can be kept the same by applying the appropriate dose of anti-BPDE and trans-BPDCH to rodent skin and the DNA adducts formed differ only in their trans- and cis-stereoisomerism.