Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal

We identified a new calmodulin kinase I (CaMKI) substrate, cytidyltransferase (CCTα), a crucial enzyme required for maintenance of cell membranes. CCTα becomes activated with translocation from the cytoplasm to the nuclear membrane, resulting in increased membrane phospholipids. Calcium-activated CCTα nuclear import is mediated by binding of its C-terminus to 14-3-3 ζ, a regulator of nuclear trafficking. Here CaMK1 phosphorylates residues within this C-terminus that signals association of CCTα with 14-3-3 ζ to initiate calcium-induced nuclear entry. CaMKI docks within the CCTα membrane-binding domain (residues 290-299), a sequence that displays similarities to a canonical nuclear export signal (NES) that also binds CRM1/exportin 1. Expression of a CFP-CCTα mutant lacking residues 290-299 in cells results in cytosolically retained enzyme. CRM1/exportin 1 was required for CCTα nuclear export, and its overexpression in cells was partially sufficient to trigger CCTα nuclear export despite calcium stimulation. An isolated CFP-290-299 peptide remained in the nucleus in the presence of leptomycin B but was able to target to the cytoplasm with farnesol. Thus CaMKI vies with CRM1/exportin 1 for access to a NES, and assembly of a CaMKI-14-3-3 ζ-CCTα complex is a key effector mechanism that drives nuclear CCTα translocation.     

Comparing Proteomics and RISC Immunoprecipitations to Identify Targets of Epstein-Barr Viral miRNAs

Epstein-Barr virus is a gamma-herpes virus that is causally associated with several lymphomas and carcinomas. This virus encodes at least 25 pre-miRNAs, which are expressed in infected cells to yield more than 50 detected mature miRNAs. miRNAs are small, non-coding RNAs that inhibit gene expression by promoting the inhibition of translation or of degradation of mRNAs. Currently, the function of these viral miRNAs and the contribution they provide to EBV’s life-cycle remain largely unknown, due to difficulties in identifying cellular and viral genes regulated by these miRNAs. We have compared and contrasted two methods to identify targets of viral miRNAs in order to identify the advantages and limitations of each method to aid in uncovering the functions of EBV’s miRNAs.     

Desert ants learn vibration and magnetic landmarks

The desert ants Cataglyphis navigate not only by path integration but also by using visual and olfactory landmarks to pinpoint the nest entrance. Here we show that Cataglyphis noda can additionally use magnetic and vibrational landmarks as nest-defining cues. The magnetic field may typically provide directional rather than positional information, and vibrational signals so far have been shown to be involved in social behavior. Thus it remains questionable if magnetic and vibration landmarks are usually provided by the ants' habitat as nest-defining cues. However, our results point to the flexibility of the ants' navigational system, which even makes use of cues that are probably most often sensed in a different context.     

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.     

Sensing the Underground - Ultrastructure and Function of Sensory Organs in Root-Feeding Melolontha melolontha (Coleoptera: Scarabaeinae) Larvae

Introduction

Below ground orientation in insects relies mainly on olfaction and taste. The economic impact of plant root feeding scarab beetle larvae gave rise to numerous phylogenetic and ecological studies. Detailed knowledge of the sensory capacities of these larvae is nevertheless lacking. Here, we present an atlas of the sensory organs on larval head appendages of Melolontha melolontha. Our ultrastructural and electrophysiological investigations allow annotation of functions to various sensory structures.

Results

Three out of 17 ascertained sensillum types have olfactory, and 7 gustatory function. These sensillum types are unevenly distributed between antennae and palps. The most prominent chemosensory organs are antennal pore plates that in total are innervated by approximately one thousand olfactory sensory neurons grouped into functional units of three-to-four. In contrast, only two olfactory sensory neurons innervate one sensillum basiconicum on each of the palps. Gustatory sensilla chaetica dominate the apices of all head appendages, while only the palps bear thermo-/hygroreceptors. Electrophysiological responses to CO₂, an attractant for many root feeders, are exclusively observed in the antennae. Out of 54 relevant volatile compounds, various alcohols, acids, amines, esters, aldehydes, ketones and monoterpenes elicit responses in antennae and palps. All head appendages are characterized by distinct olfactory response profiles that are even enantiomer specific for some compounds.

Conclusions

Chemosensory capacities in M. melolontha larvae are as highly developed as in many adult insects. We interpret the functional sensory units underneath the antennal pore plates as cryptic sensilla placodea and suggest that these perceive a broad range of secondary plant metabolites together with CO₂. Responses to olfactory stimulation of the labial and maxillary palps indicate that typical contact chemo-sensilla have a dual gustatory and olfactory function.     

Role of IL-10 in the resolution of airway inflammation

IL-10 can be considered an important agent in the resolution of inflammation. Originally named "cytokine synthesis inhibitory factor" for its ability to inhibit IFN-gamma and IL-2 production in Th2 cells, it is secreted by monocytes, macrophages, mast cells, T and B lymphocytes, and dendritic cells (DCs). IL-10 production and release by monocytic cells in response to allergic challenge is upregulated by TNF-alpha, and by negative feedback regulation of itself. However, it is also secreted by T regulatory cells (Tregs), under the control of IL-2. Importantly in the context of asthma, IL-10 inhibits eosinophilia, by suppression of IL-5 and GM-CSF, by direct effects on eosinophil apoptosis, and effects on cell proliferation through down-regulation of IL-1. A number of its cytokine suppressive characteristics are now thought to occur through its upregulation of suppressor of cytokine signaling (SOCS)-3. IL-10 is also a suppressor of nitric oxide (NO) production, which may have ramifications for its role in airway inflammatory diseases. Initial clinical trials have demonstrated relative safety and few clinically adverse events at doses of recombinant human IL-10 below 50 microg/kg, with mixed success in treatment of patients with inflammatory bowel disease and psoriasis. However, both steroid therapy and allergen specific immunotherapy are known to elevate endogenous IL-10 levels, which may account for their efficacy, suggesting that further study of IL-10 as a target for treatment of airway inflammatory diseases such as asthma and COPD is warranted.     

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.     

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen.

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.