In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH) and has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Abeta(1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro- and antiapoptotic factor proteins. The present study was undertaken to test the hypothesis that intraperitoneal injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitoneally (ip) with D609 and subsequently treated in vitro with the oxidants Fe(2+)/H(2)O(2) (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals), and AD-relevant amyloid beta-peptide 1-42 [Abeta(1-42)]. Brain mitochondria isolated from the gerbils previously injected ip with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal [a lipid peroxidation product], 3-nitrotyrosine, and cytochrome c release compared to oxidant-treated brain mitochondria isolated from saline-injected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These antiapoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.     

Exogenous Glycine Betaine Reduces Drought Damage by Mediating Osmotic Adjustment and Enhancing Antioxidant Defense in <i>Phoebe hunanensis</i>

Drought stress negatively impacts growth and physiological processes in plants. The foliar application of glycine betaine (GB) is an effective and low-cost approach to improve the drought tolerance of trees. This study examined the effect of exogenously applied GB on the cell membrane permeability, osmotic adjustment, and antioxidant enzyme activities of Phoebe hunanensis Hand.-Mazz under drought stress. Two levels (0 and 800 mL) of water irrigation were tested under different applied GB concentrations (0, 50, 100, and 200 mM). Drought stress decreased the relative water content by 58.5% while increased the electric conductivity, malondialdehyde, proline, soluble proteins, soluble sugars, and antioxidant enzyme activities (superoxide dismutase, catalase, peroxidase) by up to 62.9%, 42.4%, 87.0%, 19.1%, 60.5%, 68.3%, 71.7%, and 83.8%, respectively, on the 25^th day. The foliar application of GB, especially at 100 mM, increased the relative water content of P. hunanensis leaves under drought stress. The concentration of GB from 50 to 100 mM effectively alleviated the improvement of cell membrane permeability and inhibited the accumulation of membrane lipid peroxidation products. Under drought stress, the concentrations of proline, soluble proteins, and soluble sugars in the leaves of P. hunanensis increased as the applied GB concentration was increased and the water stress time was prolonged. Exogenously applied GB decreased oxidative stress and improved antioxidant enzyme activities as compared with treatments without GB application. Furthermore, the physiological and biochemical indexes of P. hunanensis showed a certain dose effect on exogenous GB concentration. These results suggest that GB helps maintain the drought tolerance of P. hunanensis.     

Regulation of Glycogen Content in Astrocytes via Cav-1/PTEN/AKT/GSK-3β Pathway by Three Anti-bipolar Drugs

Here we present the data indicating that chronic treatment with three antibipolar drugs, lithium, carbamazepine and valproic acid regulates Cav-1/PTEN/PI3K/AKT/GSK-3β signalling pathway and glycogen content in primary cultured astrocytes. All three drugs down-regulate gene expression of Caveoline 1 (Cav-1), decrease membrane content of phosphatase and tensin homolog (PTEN), increase activity of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and serine-threonine kinase (AKT), and elevate glycogen synthase kinase 3β (GSK-3β) phosphorylation thus suppressing its activity. As expected, treatment with any of these three drugs increases glycogen content in astrocytes. Our findings indicate that regulation of glycogen content via Cav-1/PTEN/AKT/GSK-3β pathway by the three anti-bipoar drugs may be responsible for therapeutic effects of these drugs, and Cav-1 is an important signal element that may contribute to pathogenesis of various CNS diseases and regulation of its gene expression may be one of the underlying mechanisms of drug action for antibipolar drugs and antidepressants currently in clinical use.     

Fibroblast growth and H-7 protein kinase inhibitor response monitored in microimpedance sensor arrays

Functional genomic studies and drug candidate testing both require high throughput, parallel experimentation strategies to screen for variable cellular behaviors. In this article we describe the use of an impedance sensing electrode array that is capable of sensing cell "presence" as well as the extent of cell (focal) attachment to the substrate. The signals provided by mouse fibroblasts on a sensing structure containing four different sized electrodes are reported. In the absence of cells, each electrode's impedance was found to depend as expected on electrode size and frequency. The impedance increased by several-fold when fibroblasts attached and spread out over time. More notably, the sensors also detected the cellular response to the protein kinase C inhibitor, H-7. H-7 inhibits actomyosin contractility; thereafter, the loss of focal adhesion complexes occurs. The sensors, in turn, detected an impedance decrease after H-7 addition and an increase in impedance after H-7 removal.     

Intrinsic Migratory Properties of Cultured Schwann Cells Based on Single-Cell Migration Assay

The migration of Schwann cells is critical for development of peripheral nervous system and is essential for regeneration and remyelination after nerve injury. Although several factors have been identified to regulate Schwann cell migration, intrinsic migratory properties of Schwann cells remain elusive. In this study, based on time-lapse imaging of single isolated Schwann cells, we examined the intrinsic migratory properties of Schwann cells and the molecular cytoskeletal machinery of soma translocation during migration. We found that cultured Schwann cells displayed three motile phenotypes, which could transform into each other spontaneously during their migration. Local disruption of F-actin polymerization at leading front by a Cytochalasin D or Latrunculin A gradient induced collapse of leading front, and then inhibited soma translocation. Moreover, in migrating Schwann cells, myosin II activity displayed a polarized distribution, with the leading process exhibiting higher expression than the soma and trailing process. Decreasing this front-to-rear difference of myosin II activity by frontal application of a ML-7 or BDM (myosin II inhibitors) gradient induced the collapse of leading front and reversed soma translocation, whereas, increasing this front-to-rear difference of myosin II activity by rear application of a ML-7 or BDM gradient or frontal application of a Caly (myosin II activator) gradient accelerated soma translocation. Taken together, these results suggest that during migration, Schwann cells display malleable motile phenotypes and the extension of leading front dependent on F-actin polymerization pulls soma forward translocation mediated by myosin II activity.     

The Chimonanthus salicifolius genome provides insight into magnoliid evolution and flavonoid biosynthesis

Chimonanthus salicifolius, a member of the Calycanthaceae of magnoliids, is one of the most famous medicinal plants in Eastern China. Here, we report a chromosome‐level genome assembly of C. salicifolius, comprising 820.1 Mb of genomic sequence with a contig N50 of 2.3 Mb and containing 36 651 annotated protein‐coding genes. Phylogenetic analyses revealed that magnoliids were sister to the eudicots. Two rounds of ancient whole‐genome duplication were inferred in the C. salicifolious genome. One is shared by Calycanthaceae after its divergence with Lauraceae, and the other is in the ancestry of Magnoliales and Laurales. Notably, long genes with > 20 kb in length were much more prevalent in the magnoliid genomes compared with other angiosperms, which could be caused by the length expansion of introns inserted by transposon elements. Homologous genes within the flavonoid pathway for C. salicifolius were identified, and correlation of the gene expression and the contents of flavonoid metabolites revealed potential critical genes involved in flavonoids biosynthesis. This study not only provides an additional whole‐genome sequence from the magnoliids, but also opens the door to functional genomic research and molecular breeding of C. salicifolius.     

An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.