No abstract

Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through a poorly defined mechanism. Here we determined that dysregulated redox stress in ALS mice caused by NADPH oxidases Nox1 and Nox2 significantly influenced the progression of motor neuron disease caused by mutant SOD1 G93A expression. Deletion of either Nox gene significantly slowed disease progression and improved survival. However, 50% survival rates were enhanced significantly more by Nox2 deletion than by Nox1 deletion. Interestingly, female ALS mice containing only 1 active X-linked Nox1 or Nox2 gene also had significantly delayed disease onset, but showed normal disease progression rates. Nox activity in spinal cords from Nox2 heterozygous female ALS mice was approximately 50% that of WT female ALS mice, suggesting that random X-inactivation was not influenced by Nox2 gene deletion. Hence, chimerism with respect to Noxexpressing cells in the spinal cord significantly delayed onset of motor neuron disease in ALS. These studies define what we believe to be new modifier gene targets for treatment of ALS.

Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.

Nature © Macmillan Publishers Ltd 19988 letters to nature NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com 921 cipitated beads were resuspended in 23 l lysis buffer and aliquoted as indicated. Histone deacetylase assays. Assays were done as described 4 ; where indicated, ATP was added to 2.5 mM in the presence of 5 mM MgCl 2 , and trapoxin was added to 200 nM. 8-azido-adenosine-g-32 P-ATP crosslinking. NRD antibody-immobilized bead complexes were incubated with 2 Ci 8-azido-adenosine-5Ј-g-32 Ptriphosphate (ICN) for 3 min at room temperature in 50 l buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 5 mM MgCl 2 , 1 m ZnSO 4 , 0.1% Tween-20. The reaction mixture was irradiated for 5 min with 254-nm UV light (1,120 W cm −2 from 3 cm away). Beads were washed three times with 1 ml 20 mM HEPES, pH 7.6, 100 mM KCl, 0.1% Tween-20. Bound proteins were resolved by SDS-PAGE and imaged using a phosphorimager. Nucleosome-disruption assays. the 155-bp pTPT MluI/EcoRI fragment was prepared and footprinted as described 13 . 0.2, 1.0 and 5.0 l HDAC/CHD immunoprecipitates, 1.0 and 5.0 l peptide-blocked HDAC/CHD immunoprecipitates, and 0.04, 0.16, 0.6 and 2.4 l of hSWI/SNF were tested for disruptive activity. 2 mM ATP/MgCl 2 was added where indicated; after 45 min, reactions were treated with 0.12 U DNase I (0.012 U for bare DNA). Plasmid chromatin reconstitution. HeLa cells were radiolabelled as described previously 4 and hyperacetylated histones were purified by hydroxyapatite chromatography as described 14 . 16g of a 9-kb plasmid was linearized, purified, and mixed with an equal mass of radiolabelled, hyperacetylated core histones in 50 l of 15 mM HEPES, pH 7.5, 1 M NaCl, 0.2 mM EDTA and 0.2 mM PMSF. Stepwise dilution of salt was done by addition of the same buffer without NaCl over 3 h at 10-min intervals until the final concentration of NaCl was 100 mM. Non-nucleosomal histones were then separated by repeat Centricon-500 (Amicon) filtration. Nucleosomal structure was confirmed by micrococcal nuclease digestion of the product.

During embryogenesis, inductive interactions underlie the development of much of the body plan. In Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone; members of both the transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) ligand families seem to have critical roles in this process. Here we report the identification and characterization of laloo, a novel participant in the signal transduction cascade linking extracellular, mesoderm-inducing signals to the nucleus, where alteration of cell fate is driven by changes in gene expression. Overexpression of laloo, a member of the Src-related gene family, in Xenopus embryos gives rise to ectopic posterior structures that frequently contain axial tissue. Laloo induces mesoderm in Xenopus ectodermal explants; this induction is blocked by reagents that disrupt the FGF signalling pathway. Conversely, expression of a dominant-inhibitory Laloo mutant blocks mesoderm induction by FGF and causes severe posterior truncations in vivo. This work provides the first evidence that a Src-related kinase is involved in vertebrate mesoderm induction.