A preformed complex of postsynaptic proteins is involved in excitatory synapse development

Nonsynaptic clusters of postsynaptic proteins have been documented; however, their role remains elusive. We monitored the trafficking of several candidate proteins implicated in synaptogenesis, when nonsynaptic clusters of scaffold proteins are most abundant. We find a protein complex consisting of two populations that differ in their content, mobility, and involvement in synapse formation. One subpopulation is mobile and relies on actin transport for delivery to nascent and existing synapses. These mobile clusters contain the scaffolding proteins PSD-95, GKAP, and Shank. A proportion of mobile clusters that exhibits slow movement and travels short distances contains neuroligin-1. The second group consists of stationary nonsynaptic scaffold complexes that mainly contain neuroligin-1, can recruit synaptophysin-containing axonal transport vesicles, and are readily transformed to functional presynaptic contacts that recycle the vital dye FM 4-64. These results postulate a mechanism whereby preformed scaffold protein complexes serve as predetermined postsynaptic hotspots for establishment of new functional excitatory synapses.     

Disruption of tubulin polymerization and cell proliferation by 1-naphthylarsonic acid

Arsenical compounds exhibit a differential toxicity to cancer cells. Microtubules are a primary target of a number of anticancer drugs, such as arsenical compounds. The interaction of 1-NAA (1-naphthylarsonic acid) has been investigated on microtubule polymerization under in vitro and cellular conditions. Microtubules were extracted from sheep brain. Transmission electron microscopy was used to show microtubule structure in the presence of 1-NAA. Computational docking method was applied for the discovery of ligand-binding sites on the microtubular proteins. Proliferation of HeLa cells and HF2 (human foreskin fibroblasts) was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay method following their incubation with 1-NAA. Fluorescence microscopic labelling was done with the help of α-tubulin monoclonal antibody and Tunel kit was used to investigate the apoptotic effects of 1-NAA on the HeLa cells. 1-NAA inhibits the tubulin polymerization by the formation of abnormal polymers having high affinity to the inner cell wall.     

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Mitochondrial and nuclear DNA damage induced by sulphur mustard in keratinocytes.

The extent and role of mitochondrial DNA damage in the mechanism of action of sulphur mustard (SM) is poorly understood. In this study, a combination of quantitative polymerase chain reaction and Southern hybridization was used to determine the levels of both total DNA adducts and DNA interstrand crosslinks in genomic and mitochondrial DNA isolated from normal human epidermal keratinocytes exposed to SM. The formation of both types of lesions occurred simultaneously in nuclear and mitochondrial DNA, however, SM produced significantly higher levels of both total adducts and crosslinks in genomic DNA than mitochondrial DNA. The total lesion frequency was 0.45 lesions/kb per 100 microM SM in the DHFR gene and 0.12 lesions/kb per 100 microM SM in the mitochondrial segment. Interstrand crosslinks occurred at a frequency of 0.28 crosslinks/10 kb per 100 microM SM in the DHFR gene and 0.05 crosslinks/10 kb per 100 microM SM in the mitochondrial segment. DNA interstrand crosslinks are thought to be the critical lesion produced by similar bi-functional alkylating agents. However, the levels of DNA cross-linking revealed in this study show that even at vesicating doses of SM mitochondrial DNA is still largely free of cross-links and the predominant form of DNA damage contributing to cell death occurs in the nucleus.     

Genetic differentiation and relationship of the dipodidsAllactaga andJaculus (Mammalia, Rodentia) in Egypt based on protein variation

Genetic differentiation among 14 populations representing all Egyptian dipodid (jerboa) species and subspecies was examined at 25 structural loci and interspecific relationships are discussed. Of the 25 loci, only 3 were monomorphic, with the same allele fixed in all taxa, 9 loci were monomorphic, but demonstrated intertaxon variation, and only 13 loci were polymorphic. The overall mean number of alleles per locus (A) was 1.23 ± 0.02 and the average percentage of polymorphic loci per taxon (P) was 23%. The overall mean of observed heterozygosity (H _o) was found significantly higher than that of expected heterozygosity (He); the overall means per taxon were 0.25 ± 0.017 and 0.085 ± 0.007. Mean levels of genetic identity (I) were 0.970 ± 0.003 among geographic populations, 0.718 ± 0.022 between subspecies, 0.590 ± 0.030 between congeneric species, and 0.368 ± 0.020 between genera. Phenetic analysis of genetic distance matrix produced a phenogram indicating a close association ofJaculus orientalis Erxleben, 1777 toJaculus jaculus (Linnaeus, 1758), particularly to its subspeciesJaculus jaculus butleri (Thomas, 1922), and indicating a distinct affinity between these latter two species andAllactaga tetradactyla (Lichtenstein, 1823). Estimates of genetic divergence demonstrated that J. orientalis appears to have shared a more recent common ancestor withJ. jaculus thanA. tetradactyla. Divergence of these species would have occurred by Miocene (ca 9.6 to 18.7 million years ago). The pattern of relationships of the dipodid species indicated in this study was closely consistent with the hypotheses derived from morphological and chromosomal data.     

Human β-Synuclein Rendered Fibrillogenic by Designed Mutations

Filamentous inclusions made of α-synuclein are found in nerve cells and glial cells in a number of human neurodegenerative diseases, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. The assembly and spreading of these inclusions are likely to play an important role in the etiology of common dementias and movement disorders. Both α-synuclein and the homologous β-synuclein are abundantly expressed in the central nervous system; however, β-synuclein is not present in the pathological inclusions. Previously, we observed a poor correlation between filament formation and the presence of residues 73–83 of α-synuclein, which are absent in β-synuclein. Instead, filament formation correlated with the mean β-sheet propensity, charge, and hydrophilicity of the protein (global physicochemical properties) and β-strand contiguity calculated by a simple algorithm of sliding averages (local physicochemical property). In the present study, we rendered β-synuclein fibrillogenic via one set of point mutations engineered to enhance global properties and a second set engineered to enhance predominantly β-strand contiguity. Our findings show that the intrinsic physicochemical properties of synucleins influence their fibrillogenic propensity via two distinct but overlapping modalities. The implications for filament formation and the pathogenesis of neurodegenerative diseases are discussed.