GSK-3ß inhibitor TDZD-8 reduces neonatal hypoxic-ischemic brain injury in mice.

AIMS: Glycogen synthase kinase 3ß (GSK-3ß) is activated following hypoxic-ischemic (HI) brain injury. TDZD-8 is a specific GSK-3ß inhibitor. Currently, the impact of inhibiting GSK-3ß in neonatal HI injury is unknown. We aimed to investigate the effect of TDZD-8 following neonatal HI brain injury. METHODS: Unilateral common carotid artery ligation followed by hypoxia was used to induce HI injury in postnatal day 7 mouse pups pretreated with TDZD-8 or vehicle. The infarct volume, whole-brain imaging, Nissl staining, and behavioral tests were used to evaluate the protective effect of TDZD-8 on the neonatal brain and assess functional recovery after injury. Western blot was used to evaluate protein levels of phosphorylated protein kinase B (Akt), GSK-3ß, and cleaved caspase-3. Protein levels of cleaved caspase-3, neuronal marker, and glial fibrillary acidic protein were detected through immunohistochemistry. RESULTS: Pretreatment with TDZD-8 significantly reduced brain damage and improved neurobehavioral outcomes following HI injury. TDZD-8 reversed the reduction of phosphorylated Akt and GSK-3ß, and the activation of caspase-3 induced by hypoxia-ischemia. In addition, TDZD-8 suppressed apoptotic cell death and reduced reactive astrogliosis. CONCLUSION: TDZD-8 has the therapeutic potential for hypoxic-ischemic brain injury in neonates. The neuroprotective effect of TDZD-8 appears to be mediated through its antiapoptotic activity and by reducing astrogliosis.

[Effects of simulated nitrogen deposition on growth and phosphorus efficiency of Pinus massoniana under low phosphorus stress].

Atmospheric nitrogen (N) deposition dramatically raised in recent decades, resulting in increases of soil N availability and N/P ratio, which would impact plant growth and P efficiency under low P stress. Taking breeding population of Pinus massoniana as test materials, a pot experiment was conducted to simulate two P conditions, i. e., homogeneous low P availability vs. heterogeneous low P among soil layers, in combination with two N deposition levels on growth traits and P absorption and utilization efficiency of P. massoniana. Under the homogeneous low phosphorus condition, growth traits and P efficiency of P. massoniana were not significantly improved by simulated nitrogen deposition, but significant nitrogen x family interaction effect was detected, with the biomass of family 40x44 and 71x20 being increased, 36x29 and 73x23 being decreased. Under the heterogeneous low P condition, significant N effects on the seedling height, biomass and P absorption efficiency were observed, due to promoted root length and root distribution ratio of topsoil. In addition, the effects of simulated N deposition on growth and P efficiency of P. massoniana were relevant to the N/P ratio. Under the homogeneous low P condition, the N/P ratio of P. massoniana plant was 13.8, plants exhibited a low sensitivity to simulated N deposition, root secreted APase activity was increased but the plant growth was not promoted. In comparison, the plant N/P ratio was 9.7 under the heterogeneous low P condition, and the plant growth and P efficiency were significantly promoted, while no obvious change occurred in root secreted APase activity.

Phosphatidylinositol 4,5-biphosphate (PIP2) modulates syntaxin-1A binding to sulfonylurea receptor 2A to regulate cardiac ATP-sensitive potassium (KATP) channels.

Cardiac sarcolemmal syntaxin (Syn)-1A interacts with sulfonylurea receptor (SUR) 2A to inhibit ATP-sensitive potassium (KATP) channels. Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous endogenous inositol phospholipid, known to bind Kir6.2 subunit to open KATP channels, has recently been shown to directly bind Syn-1A in plasma membrane to form Syn-1A clusters. Here, we sought to determine whether the interaction between Syn-1A and PIP2 interferes with the ability of Syn-1A to bind SUR2A and inhibit KATP channel activity. We found that PIP2 dose-dependently reduced SUR2A binding to GST-Syn-1A by in vitro pulldown assays. FRET studies in intact cells using TIRFM revealed that increasing endogenous PIP2 levels led to increased Syn-1A (-EGFP) cluster formation and a severe reduction in availability of Syn-1A molecules to interact with SUR2A (-mCherry) molecules outside the Syn-1A clusters. Correspondingly, electrophysiological studies employing SUR2A/Kir6.2-expressing HEK cells showed that increasing endogenous or exogenous PIP2 diminished the inhibitory effect of Syn-1A on KATP currents. The physiological relevance of these findings was confirmed by ability of exogenous PIP2 to block exogenous Syn-1A inhibition of cardiac KATP currents in inside-out patches of mouse ventricular myocytes. The effect of PIP2 on physical and functional interactions between Syn-1A and KATP channels is specific and not observed with physiologic concentrations of other phospholipids. To unequivocally demonstrate the specificity of PIP2 interaction with Syn-1A and its impact on KATP channel modulation by Syn-1A, we employed a PIP2-insensitive Syn-1A-5RK/A mutant. The Syn-1A-5RK/A mutant retains the ability to interact with SUR2A in both in vitro binding and in vivo FRET assays, although as expected the interaction is no longer disrupted by PIP2. Interestingly, at physiological PIP2 concentrations, Syn-1A-5RK/A inhibited KATP currents to a greater extent than Syn-1A-WT, indicating that the inhibitory effect of Syn-1A on KATP channels is not due to direct competition between Syn-1A and Kir6.2 for PIP2 binding. At high-dose PIP2, however, inhibition of KATP currents by Syn-1A-5RK/A was greatly reduced, likely overridden by the direct activating effect of PIP2 on KATP channels. Finally, depleting endogenous PIP2 with polyphosphoinositide phosphatase synaptojanin-1 known to disperse Syn-1A clusters, freed Syn-1A from Syn-1A clusters to bind SUR2A, causing optimal inhibition of KATP channels. These results taken together led us to conclude that PIP2 affects cardiac KATP channels not only by its actions on the channel directly but also by multi-modal effects of dynamically modulating Syn-1A mobility from Syn-1A clusters and thereby the availability of Syn-1A to inhibit KATP channels via interaction with SUR2A on the plasma membrane.

[Root architecture and phosphorus efficiency of different provenance Pinus massoniana under low phosphorous stress].

Taking four representative provenances of Pinus massoniana from Chun' an of Zhejiang, Wuping of Fujian, Cenxi of Guangxi, and Xinyi of Guangdong in East and South China as test materials, a pot experiment was conducted to study their variations in root architecture and phosphorus (P) efficiency under heterogeneous and homogeneous low P stress. Large variations were detected in the major growth traits and the indices of P efficiency among the P. massoniana provenances under both heterogeneous and homogeneous low P stress. Under heterogeneous low P stress, the provenances from Xinyi of Guangdong and Wuping of Fujian exhibited higher P efficiency and greater dry matter accumulation, and their root architecture presented large adaptive changes, with the values of root parameters in P-rich soil surface layer being significantly higher than those of the P inefficient provenances from Chun'an of Zhejiang and Cenxi of Guangxi, which should be responsible for the higher P absorption efficiency and high P efficiency of the provenances from Xinyi and Wuping under heterogeneous low P condition. The root architecture parameters in P-rich soil surface layer and the plant dry matter accumulation of different provenance P. massoniana under heterogeneous low P stress had a correlation coefficient >0.95. Under homogeneous low P stress, the provenances with higher P efficiency had a significantly higher P uptake efficiency than the provenances with lower P efficiency, but the root parametres of the provenances with higher P efficiency had no significant correlation with the P efficiency of the provenances. There existed differences in the adaptive mechanism for the heterogeneous and homogeneous low P stress among the the P. massoniana provenances, and thus, different strategies should be adopted in the selection of P-efficient provenances for different forest stands.

A genetic screen for dihydropyridine (DHP)-resistant worms reveals new residues required for DHP-blockage of mammalian calcium channels.

Dihydropyridines (DHPs) are L-type calcium channel (Ca(v)1) blockers prescribed to treat several diseases including hypertension. Ca(v)1 channels normally exist in three states: a resting closed state, an open state that is triggered by membrane depolarization, followed by a non-conducting inactivated state that is triggered by the influx of calcium ions, and a rapid change in voltage. DHP binding is thought to alter the conformation of the channel, possibly by engaging a mechanism similar to voltage dependent inactivation, and locking a calcium ion in the pore, thereby blocking channel conductance. As a Ca(v)1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Ca(v)1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Ca(v)1 residues important for DHP-sensitivity, we screened 440,000 randomly mutated Caenorhabditis elegans genomes for worms resistant to DHP-induced growth defects. We identified 30 missense mutations in the worm Ca(v)1 pore-forming (alpha(1)) subunit, including eleven in conserved residues known to be necessary for DHP-binding. The remaining polymorphisms are in eight conserved residues not previously associated with DHP-sensitivity. Intriguingly, all of the worm mutants that we analyzed phenotypically exhibited increased channel activity. We also created orthologous mutations in the rat alpha(1C) subunit and examined the DHP-block of current through the mutant channels in culture. Six of the seven mutant channels examined either decreased the DHP-sensitivity of the channel and/or exhibited significant residual current at DHP concentrations sufficient to block wild-type channels. Our results further support the idea that DHP-block is intimately associated with voltage dependent inactivation and underscores the utility of C. elegans as a screening tool to identify residues important for DHP interaction with mammalian Ca(v)1 channels.

Molecular determinants of cysteine string protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Galpha subunits but not Galphabeta subunits whereas the C terminal binding site of CSP associates with either free Galphabeta subunits or Galphabeta in complex with Galpha. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.