Store-operated Ca 2+ entry is mediated by Ca 2+ release-activated Ca 2+ (CRAC) channels following Ca 2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca 2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.Receptor-mediated signaling in nonexcitable cells, immune cells in particular, involves an initial rise in intracellular Ca 2+ due to release from the intracellular stores. The resulting depletion of the intracellular stores induces Ca 2+ entry through the plasma membrane through CRAC channels (1-4). This phenomenon is central to many physiological processes such as T cell proliferation, gene transcription, and cytokine release (3, 5-7). Biophysically, CRAC currents have been well characterized (2,8,9), but the identity of the CRAC channel itself and the pathway resulting in its activation are still unknown. Recently, STIM1 (for stromal interaction molecule in Drosophila) was identified as an essential component of store-operated calcium entry (10,11). This protein is located in intracellular compartments that likely represent parts of the endoplasmic reticulum (ER). It has a single transmembranespanning domain with a C-terminal Ca 2+ -binding motif that appears to be crucial for its hypothesized function as the ER sensor for luminal Ca 2+ concentration. When stores become depleted, STIM1 redistributes into distinct structures (punctae) that move toward Fig. 1, B and C, from cells treated with dsRNA against Rho1 (mock) and stim1, as well as two genes we later identified as CRAC modulators 1 and 2 (CRACM1 and CRACM2). On the basis of inhibitory efficacy relative to positive and negative controls, we identified ~1500 genes that reduced Ca 2+ influx to varying degrees (table S1). After eliminating numerous genes based on artifactual fluorescence signals or because they represent known housekeeping genes, cell cycle regulators, and so on, we eventually arrived at 27 candidate genes (table S2) that were subsequently evaluated in a secondary screen using single-cell patch-clamp assays.From the secondary patch-clamp screen, we identified two novel genes that are essential for CRAC channel function, CRACM1 (encoded by olf186-F in Drosophila and FLJ14466 in human) and CRACM2 (encoded by dpr3 in Drosophila, with no human ortholog). We measured CRAC currents in Drosophila Kc cells after inositol 1,4,5-trisphosphate (IP 3 )-mediated depletion of Ca 2+ from intracellular stores. Both untreated control wild-type cells and cells treated with an irrelevant dsRNA against Rho1 (mock) responded by ra...

The Fc epsilonRI complex forms a high-affinity cell-surface receptor for the Fc region of antigen-specific immunoglobulin E (IgE) molecules. Fc epsilonRI is multimeric and is a member of a family of related antigen/Fc receptors which have conserved structural features and similar roles in initiating intracellular signalling cascades. In humans, Fc epsilonRI controls the activation of mast cells and basophils, and participates in IgE-mediated antigen presentation. Multivalent antigens bind and crosslink IgE molecules held at the cell surface by Fc epsilonRI. Receptor aggregation induces multiple signalling pathways that control diverse effector responses. These include the secretion of allergic mediators and induction of cytokine gene transcription, resulting in secretion of molecules such as interleukin-4, interleukin-6, tumour-necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor. Fc epsilonRI is therefore central to the induction and maintenance of an allergic response and may confer physiological protection in parasitic infections.

No abstract

Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)–dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation.

In response to the recognized need for high throughput biodosimetry methods for use after large scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. We describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or γ-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine, to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in-situ analysis in multi-well plates, implementation of robotic plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to highthroughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here we describe the development of each of the individual modules of the RABIT system, and show preliminary data from key modules. Ongoing is system integration, followed by calibration and validation.

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstickderived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparincoated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

After a large-scale nuclear accident or an attack with an improvised nuclear device, rapid biodosimetry would be needed for triage. As a possible means to address this need, we previously defined a gene expression signature in human peripheral white blood cells irradiated ex vivo that predicts the level of radiation exposure with high accuracy. We now demonstrate this principle in vivo using blood from patients receiving total-body irradiation (TBI). Whole genome microarray analysis has identified genes responding significantly to in vivo radiation exposure in peripheral blood. A 3-nearest neighbor classifier built from the TBI patient data correctly predicted samples as exposed to 0, 1.25 or 3.75 Gy with 94% accuracy (P < 0.001) even when samples from healthy donor controls were included. The same samples were classified with 98% accuracy using a signature previously defined from ex vivo irradiation data. The samples could also be classified as exposed or not exposed with 100% accuracy. The demonstration that ex vivo irradiation is an appropriate model that can provide meaningful prediction of in vivo exposure levels, and that the signatures are robust across diverse disease states and independent sample sets, is an important advance in the application of gene expression for biodosimetry.

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via FcεRI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.