Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

Mycobacterium tuberculosis recall antigens suppress HIV-1 replication in anergic donor cells via CD8+ T cell expansion and increased IL-10 levels

Mycobacterium tuberculosis (MTb) is the leading cause of death in the setting of AIDS. MTb enhances the pathogenicity and accelerates the course of HIV disease and, furthermore, infection with HIV-1 increases the risk of reactivation or reinfection with MTb. In this study, we show that host-specific recall responses to one pathogen, MTb, has a direct effect upon the regulation of a second pathogen, HIV-1. Using cells from immunocompetent former tuberculosis (TB) patients who displayed either a persistently positive (responsive) or negative (anergic), delayed-type hypersensitivity (DTH) reaction to intradermal injection of purified protein derivative (PPD), we investigated the effect of recall Ags to MTb upon the replication of HIV-1 primary isolates in vitro. We show that HIV-1 replication of a T cell-tropic isolate was significantly impaired in MTb-stimulated PBMC from PPD-anergic donors. Furthermore, these donors displayed a significant increase in CD8(+) T cells and IL-10 levels and lower levels of IL-2 and TNF-alpha relative to PPD-responsive donors in response to PPD stimulation. Strikingly, CD8(+) T cell depletion and blocking of IL-10 significantly increased HIV-1 replication in these PPD-anergic donors, indicating that an immunosuppressive response to MTb recall Ags inhibits HIV-1 replication in PPD-anergic individuals. Therefore, immunotherapeutic approaches aimed at recapitulating Ag-specific MTb anergy in vivo could result in novel and effective approaches to inhibit HIV-1 disease progression in MTb/HIV-1 coinfection.     

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.