The Heat Shock Factor A4A Confers Salt Tolerance and Is Regulated by Oxidative Stress and the Mitogen-Activated Protein Kinases MPK3 and MPK6

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.Peer reviewe

Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically

We tested the efficacy of treatment with talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in pre-symptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its efficacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective

Identification of genes preferentially expressed in wheat egg cells and zygotes

Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction-dependent manner

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1-RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways

Modelling Blood Flow and Metabolism in the Preclinical Neonatal Brain during and Following Hypoxic-Ischaemia

Hypoxia-ischaemia (HI) is a major cause of neonatal brain injury, often leading to long-term damage or death. In order to improve understanding and test new treatments, piglets are used as preclinical models for human neonates. We have extended an earlier computational model of piglet cerebral physiology for application to multimodal experimental data recorded during episodes of induced HI. The data include monitoring with near-infrared spectroscopy (NIRS) and magnetic resonance spectroscopy (MRS), and the model simulates the circulatory and metabolic processes that give rise to the measured signals. Model extensions include simulation of the carotid arterial occlusion used to induce HI, inclusion of cytoplasmic pH, and loss of metabolic function due to cell death. Model behaviour is compared to data from two piglets, one of which recovered following HI while the other did not. Behaviourally-important model parameters are identified via sensitivity analysis, and these are optimised to simulate the experimental data. For the non-recovering piglet, we investigate several state changes that might explain why some MRS and NIRS signals do not return to their baseline values following the HI insult. We discover that the model can explain this failure better when we include, among other factors such as mitochondrial uncoupling and poor cerebral blood flow restoration, the death of around 40% of the brain tissue. Copyright

Cyclooxygenase-2 inhibitor NS398 preserves neuronal function after hypoxia/ischemia in piglets

Anoxic stress attenuates NMDA-induced pial arteriolar dilation via a mechanism involving actions of cyclooxygenase (COX)-derived reactive oxygen species (ROS). We examined whether the selective COX-2 inhibitor NS398 would protect neuronal function after global hypoxia/ischemia (H/I) in piglets. Pial arteriolar responses to NMDA (10-100 mu mol/l) were determined using intravital microscopy in anesthetized piglets before and 1 h after H/I. Study groups received vehicle, 0.3, 1, or 5 mg/kg NS398, or 0.3 mg/kg indomethacin (n = 7, 6, 6, 5 and 8, respectively) i.v. 20 min prior to H/I. H/I reduced NMDA-induced dilation to. 44 +/- 6% (100 mu mol/l NMIDA, mean +/- s.e.m.) of the pre-ischemic response in vehicle animals (p<0.05). However NS398 dose-dependently protected arteriolar dilation to NMIDA (77 +/- 8, 81 +/- 16, and 102 +/- 10% preservation at 0.3, 1 and 5 mg/kg, respectively). Indomethacin caused similar preservation. However, indomethacin but not NS398 reduced serum thromboxane B-2 levels to undetectable values. In conclusion, COX-2 appears to be a major source of ROS in the piglet cerebral cortex after H/I