Nuclear but not cytosolic phosphoinositide 3-kinase beta has an essential function in cell survival

Class I(A) phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival.     

Signal transduction pathways through cytoprotective, apoptotic and hypertrophic stimuli: a comparative study in adult cardiac myocytes

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen‐activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress‐ activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro‐apoptotic concentration of H₂O₂ activates strongly both MAPKs and PI3K/Akt pathways. Insulin‐like growth factor‐1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro‐survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway. Copyright © 2011 John Wiley & Sons, Ltd.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

On the vapour trail of an atmospheric imprint in insects

Terrestrial arthropods, at constant risk from desiccation, are highly sensitive to atmospheric temperature and humidity. A physiological marker of these abiotic conditions could highlight phenotypic adaptations, indicate niche partitioning, and predict responses to climate change for a group representing three-quarters of the Earth's animal species. We show that the (18)O composition of insect haemolymph is such a measure, providing a dynamic and quantitatively predictable signal for respiratory gas exchange and inputs from atmospheric humidity. Using American cockroaches (Periplaneta americana) under defined experimental conditions, we show that insects respiring at low humidity demonstrate the expected enrichment in the (18)O composition of haemolymph because of evaporation. At high humidity, however, diffusional influx of atmospheric water vapour into the animal forces haemolymph to become depleted in (18)O. Additionally, using cockroaches sampled from natural habitats, we show that the haemolymph (18)O signature is transferred to the organic material of the insect's exoskeleton. Insect cuticle, therefore, exhibits the mean atmospheric conditions surrounding the animals prior to moulting. This discovery will help to define the climatic tolerances of species and their habitat preferences, and offers a means of quantifying the balance between niche partitioning and 'neutral' processes in shaping complex tropical forest communities.     

Sharing the wealth: peroxisome inheritance in budding yeast

Eukaryotic cells have evolved molecular mechanisms to ensure the faithful partitioning of cellular components during cell division. The budding yeast Saccharomyces cerevisiae has to actively deliver about half of its organelles to the growing bud, while retaining the remaining organelles in the mother cell. Until lately, little was known about the inheritance of peroxisomes. Recent studies have identified the peroxisomal proteins Inp1p and Inp2p as two key regulators of peroxisome inheritance that perform antagonistic functions. Inp1p is required for the retention of peroxisomes in mother cells, whereas Inp2p promotes the bud-directed movement of these organelles. Inp1p anchors peroxisomes to the cell cortex by interacting with specific structures lining the cell periphery. On the other hand, Inp2p functions as the peroxisome-specific receptor for the class V myosin, Myo2p, thereby linking peroxisomes to the translocation machinery that propels peroxisome movement. Tight coordination between Inp1p and Inp2p ensures a fair and harmonious spatial segregation of peroxisomes upon cell division.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.     

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

Anaerobic Bioremediation of Groundwater Containing a Mixture of 1,1,2,2-Tetrachloroethane and Chloroethenes

This study investigated the biotransformation pathways of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) in the presence of chloroethenes (i.e. tetrachloroethene, PCE; trichloroethene, TCE) in anaerobic microcosms constructed with subsurface soil and groundwater from a contaminated site. When amended with yeast extract, lactate, butyrate, or H₂ and acetate, 1,1,2,2-TeCA was initially dechlorinated via both hydrogenolysis to 1,1,2-trichloroethane (1,1,2-TCA) (major pathway) and dichloroelimination to dichloroethenes (DCEs) (minor pathway), with both reactions occurring under sulfidogenic conditions. In the presence of only H₂, the hydrogenolysis of 1,1,2,2-TeCA to 1,1,2-TCA apparently required the presence of acetate to occur. Once formed, 1,1,2-TCA was degraded predominantly via dichloroelimination to vinyl chloride (VC). Ultimately, chloroethanes were converted to chloroethenes (mainly VC and DCEs) which persisted in the microcosms for very long periods along with PCE and TCE originally present in the groundwater. Hydrogenolysis of chloroethenes occurred only after highly reducing methanogenic conditions were established. However, substantial conversion to ethene (ETH) was observed only in microcosms amended with yeast extract (200 mg/l), suggesting that groundwater lacked some nutritional factors which were likely provided to dechlorinating microorganisms by this complex organic substrate. Bioaugmentation with an H₂-utilizing PCE-dechlorinating Dehalococcoides spp. -containing culture resulted in the conversion of 1,1,2,2-TeCA, PCE and TCE to ETH and VC. No chloroethanes accumulated during degradation suggesting that 1,1,2,2-TeCA was degraded through initial dichloroelimination into DCEs and then typical hydrogenolysis into ETH and VC.     

Effects of voltage‐gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage‐gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC α₁ subunit EGL‐19 and α₂/δ subunit UNC‐36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein‐based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction‐of‐function mutation in egl‐19 and significantly reduced by L‐type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L‐type channels. Transients did not depend directly on intracellular calcium stores, although a store‐independent 2‐APB and gadolinium‐sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc‐36, which encodes the main neuronal α₂/δ subunit in C. elegans. Interestingly, while egl‐19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc‐36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL‐19 and UNC‐36 in excitability and functional activity of the mechanosensory neurons. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006