Baicalin protects neonatal rat brains against hypoxicischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway

Baicalin is a flavonoid compound extracted from Scutellaria baicalensis root.Recent evidence indicates that baicalin is neuroprotective in models of ischemic stroke.Here,we investigate the neuroprotective effect of baicalin in a neonatal rat model of hypoxic-ischemic encephalopathy.Seven-day-old pups underwent left common carotid artery ligation followed by hypoxia（8% oxygen at 37°C） for 2 hours,before being injected with baicalin（120 mg/kg intraperitoneally） and examined 24 hours later.Baicalin effectively reduced cerebral infarct volume and neuronal loss,inhibited apoptosis,and upregulated the expression of p-Akt and glutamate transporter 1.Intracerebroventricular injection of the phosphoinositide 3-kinase/protein kinase B（PI3 K/Akt） inhibitor LY294002 30 minutes before injury blocked the effect of baicalin on p-Akt and glutamate transporter 1,and weakened the associated neuroprotective effect.Our findings provide the first evidence,to our knowledge that baicalin can protect neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the PI3 K/Akt signaling pathway.

Defense-Related Calcium Signaling Mutants Uncovered via a Quantitative High-Throughput Screen in Arabidopsis thaliana

Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs （Microbe-Associated Molecular Patterns）, such as fig22 and elf18 that are derived from bacterial flagellin and elongation factor Tu, respectively. Here, Arabidopsis thaliana mutants with changed calcium elevation （cce） in response to fig22 treatment were isolated and characterized. Besides novel mutant alleles of the fig22 receptor, FLS2 （Flagellin-Sensitive 2）, and the receptor-associated kinase, BAK1 （Brassinosteroid receptor 1-Associated Kinase 1）, the new cce mutants can be categorized into two main groups--those with a reduced or an enhanced calcium elevation. Moreover, cce mutants from both groups show differ- ential phenotypes to different sets of MAMPs. Thus, these mutants will facilitate the discovery of novel components in early MAMP signaling and bridge the gaps in current knowledge of calcium signaling during plant-microbe interactions. Last but not least, the screening method is optimized for speed （covering 384 plants in 3 or 10 h） and can be adapted to genetically dissect any other stimuli that induce a change in calcium levels.

Loss of GSNOR1 Function Leads to Compromised Auxin Signaling and Polar Auxin Transport

Cross talk between phytohormones, nitric oxide （NO）, and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study, we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnorl-3, a loss-of-function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnorl-3 mutant as revealed by significantly reduced DR5-GUS/ DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin-mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compro- mised in gsnorl-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a manner independent of transcription and 26S proteasome degradation. Our results suggest that S-nitrosylation and GSNORl-mediated de-nitrosylation contribute to auxin physiology, and impaired auxin signaling and compromised auxin transport are responsible for the auxin-related morpho- logical phenotypes displayed by the gsnorl-3 mutant.

Cytosolic Ca^2＋ Signals Enhance the Vacuolar Ion Conductivity of Bulging Arabidopsis Root Hair Cells

Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.

Delays for Both Pedestrians Classified and Vehicles at a Signalized Crosswalk

Delays of both pedestrians,who are classified according to whether complying with traffic law,and vehicles at a signalized crosswalk are analyzed in this paper.The truncated Adams' model is applied to generate the probability and mean of delay of pedestrians non-complying with traffic law.Using the section-based traffic queuing-theory and the stochastic decomposition property of M/G/1vacation system with exhaustive service,the mean delay of vehicles is formulated.A multi-objective optimization model simultaneously minimizing the delays of pedestrians and vehicles during a signal period is proposed.The effects,which several model parameters have on the delays and the optimal solution of the model,are illustrated by numerical examples.

Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

Calcium-dependent protein kinases （CDPKs） comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wildtype, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants

Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca^2＋ ion. Calmodulin （CAM） is the predominant Ca^2＋ sensor and plays a crucial role in decoding the Ca^2＋ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca^2＋ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca^2＋ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.