Elena Sheveleva

Increased Salt and Drought Tolerance by D-Ononitol Production in Transgenic Nicotiana tabacum L

A cDNA encoding myo-inositol O-methyltransferase (IMT1) has been transferred into Nicotiana tabacum cultivar SR1. During drought and salt stress, transformants (I5A) accumulated the methylated inositol D-ononitol in amounts exceeding 35 [mu]mol g-1 fresh weight In I5A, photosynthetic CO2 fixation was inhibited less during salt stress and drought, and the plants recovered faster than wild type. One day after rewatering drought-stressed plants, I5A photosynthesis had recovered 75% versus 57% recovery with cultivar SR1 plants. After 2.5 weeks of 250 mM NaCl in hydroponic solution, I5A fixed 4.9 [plus or minus] 1.4 [mu]mol CO2 m-2 s-1, whereas SR1 fixed 2.5 [plus or minus] 0.6 [mu]mol CO2 m-2 s-1. myo-Inositol, the substrate for IMT1, increases in tobacco under stress. Preconditioning of I5A plants in 50 mM NaCl increased D-ononitol amounts and resulted in increased protection when the plants were stressed subsequently with 150 mM NaCl. Pro, Suc, Fru, and Glc showed substantial diurnal fluctuations in amounts, but D-ononitol did not. Plant transformation resulting in stress-inducible, stable solute accumulation appears to provide better protection under drought and salt-stress conditions than strategies using osmotic adjustment by metabolites that are constitutively present.

Plant stress adaptations--making metabolism move.

Persistently sub-optimal environmental conditions constitute stress. Perception and signaling lead to protein expression changes, the activation of new biochemical pathways, and repression of others which are characteristic of the unstressed state. Protective metabolic adaptations alter physiological reactions of the whole plant. Paramount among the mechanisms are oxygen radical scavenging, maintenance of ion uptake and water balance, and reactions altering carbon and nitrogen allocation, such that reducing power is defused. Elements of the stress signaling pathways and proteins that lead to stress protection have recently become known.

Translational Control of Nrf2 Protein in Activation of Antioxidant Response by Oxidants

Nf-E2 related factor-2 (Nrf2) is a basic leucine zipper transcription factor that binds and activates the antioxidant response element (ARE) in the promoters of many antioxidant and detoxification genes. We found that H2O2 treatment caused a rapid increase in endogenous Nrf2 protein level in rat cardiomyocytes. Semiquantitative or real-time reverse transcription-polymerase chain reaction failed to show an increase of Nrf2 mRNA level by H2O2 treatment. Measurements of Nrf2 protein stability excluded the possibility of Nrf2 protein stabilization. Although inhibiting protein synthesis with cycloheximide prevented H2O2 from elevating Nrf2 protein level, RNA synthesis inhibition with actinomycin D failed to do so. Measurements of new protein synthesis with [35S]methionine incorporation confirmed that H2O2 increased the translation of Nrf2 protein. Inhibitors of phosphoinositide 3-kinase were able to abolish the induction of Nrf2 protein by H2O2. Although H2O2 increased phosphorylation of p70 S6 kinase, rapamycin failed to inhibit H2O2 from elevating Nrf2 protein. H2O2 also induced phosphorylation of eukaryotic translation initiation factor (eIF) 4E and eIF2α within 30 and 10 min, respectively. Inhibiting eIF4E with small interfering siRNA or increasing eIF2α phosphorylation with salubrinal did not affect Nrf2 elevation by H2O2. Our data present a novel phenomenon of quick onset of the antioxidant/detoxification response via increased translation of Nrf2 by oxidants. The mechanism underlying such stress-induced de novo protein translation may involve multiple components of translational machinery.

Corticosteroids induce COX-2 expression in cardiomyocytes: role of glucocorticoid receptor and C/EBP-β

Psychological stress increases the level of glucocorticoids in the circulating system. We found that dexamethasone administration in adult mice elevates the expression of COX-2 in the myocardium. With isolated neonatal cardiomyocytes, corticosterone (CT) at physiologically relevant doses (0.01-1 microM) induces the expression of COX-2 gene. The induction first appeared at 4 h and remained for at least 24 h with 1 microM CT treatment. This response is likely cardiomyocyte cell type specific since CT did not induce COX-2 expression in cardiac fibroblasts and glucocorticoids are known to suppress the expression of COX-2 in lymphocytes and several organs. Corticosteroids, but not estrogen or progesterone, induce COX-2 expression. The glucocorticoid receptor (GR) antagonist mifepristone (MF) prevented CT from inducing COX-2 gene, suggesting a GR-dependent induction in cardiomyocytes. COX-2 gene promoter deletion and mutation studies indicate a role of CCAAT/enhancer binding protein-beta (C/EBP-beta) in CT-induced COX-2 gene expression. Chromatin immunoprecipitation assays revealed that CT caused the binding of both GR and C/EBP-beta to COX-2 promoter, while MF pretreatment blocked such binding. Coimmunoprecipitation experiments demonstrated that CT treatment induced the interaction of GR with C/EBP-beta. Small interfering RNA against C/EBP-beta prevented CT from activating COX-2 promoter or elevating COX-2 protein. Our data suggest that the interaction between GR and C/EBP-beta contributes to elevated COX-2 gene transcription by CT in cardiomyocytes.

Corticosteroids Induce Cyclooxygenase 1 Expression in Cardiomyocytes: Role of Glucocorticoid Receptor and Sp3 Transcription Factor

Cyclooxygenase (COX) encodes a rate-limiting enzyme in the biosynthesis of prostanoids. Although COX-1 is constitutively expressed in many tissues, we found that glucocorticoids cause elevated expression of COX-1 gene in cardiomyocytes. Corticosterone (CT) at physiologically relevant doses (0.05-1 microm) induces transcriptional activation of COX-1 gene as shown by nuclear run-on and promoter reporter assays. An antagonist of glucocorticoid receptor (GR), mifepristone, prevented CT from inducing COX-1. COX-1 gene promoter deletion and mutation studies indicate a role of Sp transcription factors in CT-induced COX-1 gene. EMSAs or chromatin immunoprecipitation assays suggest that GR and Sp3 transcription factor bind to the promoter of COX-1 gene. Coimmunoprecipitation assays found an association of GR with Sp3. Silencing Sp3 protein with small interfering RNA suppressed CT-induced COX-1 promoter activation. Our data suggest that activated GR interacts with Sp3 transcription factor in binding to COX-1 promoter to enhance COX-1 gene expression in cardiomyocytes.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca(2+) concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

Imexon Induces an Oxidative Endoplasmic Reticulum Stress Response in Pancreatic Cancer Cells

Oxidative protein folding in the endoplasmic reticulum (ER) requires strict regulation of redox homeostasis. Disruption of the lumenal redox balance induces an integrated ER stress response that is associated with reduced protein translation, increased chaperone activity, and ultimately cell death. Imexon is a small-molecule chemotherapeutic agent that has been shown to bind glutathione (GSH) and induce oxidative stress in tumor cells; however, the mechanism of cytotoxicity is not well understood. In this report, we investigate the effects of imexon on the integrated ER stress response in pancreatic carcinoma cells. Acute exposure to imexon induces an ER stress response characterized by accumulation of the oxidized form of the oxidoreductase Ero1α, phosphorylation of eIF2α, and inhibition of protein synthesis. An RNA interference chemosensitization screen identified the eukaryotic translation initiation factor eIF2B5 as a target that enhanced imexon-induced growth inhibition of MiaPaCa-2 pancreatic cancer cells, but did not significantly augment the effects of imexon on protein synthesis. Concurrent reduction of intracellular thiols with N-acetyl cysteine reversed imexon activity, however cotreatment with superoxide scavengers had no effect, suggesting thiol binding may be a primary component of the oxidative effects of imexon. Moreover, the data suggest that disruption of the redox balance in the ER is a potential therapeutic target. Mol Cancer Res; 10(3); 392–400. ©2012 AACR.

Dopamine Transporter Neuroimaging as an Enrichment Biomarker in Early Parkinson's Disease Clinical Trials: A Disease Progression Modeling Analysis

Given the recognition that disease‐modifying therapies should focus on earlier Parkinsons disease stages, trial enrollment based purely on clinical criteria poses significant challenges. The goal herein was to determine the utility of dopamine transporter neuroimaging as an enrichment biomarker in early motor Parkinsons disease clinical trials. Patient‐level longitudinal data of 672 subjects with early‐stage Parkinsons disease in the Parkinsons Progression Markers Initiative (PPMI) observational study and the Parkinson Research Examination of CEP‐1347 Trial (PRECEPT) clinical trial were utilized in a linear mixed‐effects model analysis. The rate of worsening in the motor scores between subjects with or without a scan without evidence of dopamine transporter deficit was different both statistically and clinically. The average difference in the change from baseline of motor scores at 24 months between biomarker statuses was –3.16 (90% confidence interval [CI] = –0.96 to –5.42) points. Dopamine transporter imaging could identify subjects with a steeper worsening of the motor scores, allowing trial enrichment and 24% reduction of sample size.

Salt tolerance engineering - which are the essential mechanisms?

High soil salinity poses a problem to many agriculturally useable areas in the world and it can be expected that this problem becomes more severe due, mainly, to the need of keeping even marginal land under cultivation and as a consequence of irrigation in arid areas. These facts make it timely that the mechanisms are studied through which salt-tolerant plants can be productive under conditions under which glycophytic crop plants cannot grow. In our work over several years, we have attempted to find and describe mechanisms that have been suggested by physiological studies in the past. Once genes are identified which seem to control such mechanisms, we transfer them into salt-sensitive plants and study whether stress protection, even marginal, can be documented. From such work, and from the work of several other groups, we can deduce that a large number of genes are involved in plant stress tolerance and these fall into five groups that may define five mechanisms. These are (1) the synthesis of osmolytes, likely for osmotic adjustment, (2) the synthesis of enzymes and compounds which enhance the capacity of cells for scavenging of radical oxygen species, (3) the control over potassium uptake in the presence of high amounts of external sodium, (4) control over water uptake (rather, the avoidance of water loss in the presence of sodium), and (5) the acceleration of developmental processes that lead to flowering and seed formation. We will discuss what is known about these mechanisms.