Rap1a deficiency modifies cytokine responses and MAPK-signaling in vitro and impairs the in vivo inflammatory response

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo.     

Design and dosimetric analysis of a 385 MHz TETRA head exposure system for use in human provocation studies

A new head exposure system for double‐blind provocation studies investigating possible effects of terrestrial trunked radio (TETRA)‐like exposure (385 MHz) on central nervous processes was developed and dosimetrically analyzed. The exposure system allows localized exposure in the temporal brain, similar to the case of operating a TETRA handset at the ear. The system and antenna concept enables exposure during wake and sleep states while an electroencephalogram (EEG) is recorded. The dosimetric assessment and uncertainty analysis yield high efficiency of 14 W/kg per Watt of accepted antenna input power due to an optimized antenna directly worn on the subject's head. Beside sham exposure, high and low exposure at 6 and 1.5 W/kg (in terms of maxSAR10g in the head) were implemented. Double‐blind control and monitoring of exposure is enabled by easy‐to‐use control software. Exposure uncertainty was rigorously evaluated using finite‐difference time‐domain (FDTD)‐based computations, taking into account anatomical differences of the head, the physiological range of the dielectric tissue properties including effects of sweating on the antenna, possible influences of the EEG electrodes and cables, variations in antenna input reflection coefficients, and effects on the specific absorption rate (SAR) distribution due to unavoidable small variations in the antenna position. This analysis yielded a reasonable uncertainty of <±45% (max to min ratio of 4.2 dB) in terms of maxSAR10g in the head and a variability of <±60% (max to min ratio of 6 dB) in terms of mass‐averaged SAR in different brain regions, as demonstrated by a brain region‐specific absorption analysis. Bioelectromagnetics 33:594–603, 2012. © 2012 Wiley Periodicals, Inc.     

Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β₂-adrenergic receptor (β₂AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β₂AR signaling network revealed the differential contribution of G_s-, G_i- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²⁺ in the impedance response and led to the discovery of a novel β₂AR-promoted Ca²⁺ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.     

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H(2)O(2) accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response-causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H(2)O(2) during both gene-for-gene and nonhost resistance responses.     

Inhibition of STAT3 promotes the efficacy of adoptive transfer therapy using type-1 CTLs by modulation of the immunological microenvironment in a murine intracranial glioma

A variety of cancers, including malignant gliomas, show aberrant activation of STAT3, which plays a pivotal role in negative regulation of antitumor immunity. We hypothesized that inhibition of STAT3 signals would improve the efficacy of T cell adoptive transfer therapy by reversal of STAT3-induced immunosuppression in a murine GL261 intracranial glioma model. In vitro treatment of GL261 cells with JSI-124, a STAT3 inhibitor, reversed highly phosphorylated status of STAT3. Systemic i.p. administration of JSI-124 in glioma-bearing immunocompetent mice, but not athymic mice, resulted in prolonged survival, suggesting a role of adaptive immunity in the antitumor effect. Furthermore, JSI-124 promoted maturation of tumor-infiltrating CD11c(+) dendritic cells and activation of tumor-conditioned cytotoxic T cells, enhanced dendritic cells and GL261 production of CXCL-10, a critical chemokine for attraction of Tc1 cells. When i.p. JSI-124 administration was combined with i.v. transfer of Pmel-I mouse-derived type-1 CTLs (Tc1), glioma-bearing mice exhibited prolonged survival compared with i.p. JSI-124 or i.v. Tc1 therapy alone. Flow cytometric analyses of brain infiltrating lymphocytes revealed that JSI-124-treatment enhanced the tumor-homing of i.v. transferred Tc1 cells in a CXCL-10-dependent fashion. Systemic JSI-124 administration also up-regulated serum IL-15 levels, and promoted the persistence of transferred Tc1 in the host. These data suggest that systemic inhibition of STAT3 signaling can reverse the suppressive immunological environment of intracranial tumor bearing mice both systemically and locally, thereby promoting the efficacy of adoptive transfer therapy with Tc1.     

Human mutation in the anti-apoptotic heat shock protein 20 abrogates its cardioprotective effects

The small heat shock protein Hsp20 protects cardiomyocytes against apoptosis, and phosphorylation at its Ser16 site enhances its cardioprotection. To determine whether genetic variants exist in human Hsp20, which may modify these beneficial effects, we sequenced the coding region of the Hsp20 gene in 1347 patients suffering from dilated cardiomyopathy and 744 subjects with no heart disease. We identified a C59T substitution in the human Hsp20 gene in one patient and three individuals without heart disease. All subjects were heterozygous for this mutation, which changes a fully conserved proline residue into leucine at position 20 (P20L), resulting in secondary structural alterations. To examine the potential functional significance of the P20L-Hsp20 human variant, adult rat cardiomyocytes were infected with Ad.GFP (where Ad is adenovirus and GFP is green fluorescent protein), Ad.WT-Hsp20 (where WT is wild-type), and Ad.P20L-Hsp20 and subjected to simulated ischemia/reperfusion injury. Expression of WT-Hsp20 resulted in significant attenuation of apoptosis compared with the GFP control. However, the P20L-Hsp20 mutant showed no protection against apoptosis, assessed by Hoechst staining and DNA fragmentation. The loss of cardioprotection by the mutant Hsp20 was associated with its diminished phosphorylation at Ser16 compared with WT-Hsp20. Furthermore, maximal stimulation of cardiomyocytes with isoproterenol or protein kinase A-mediated phosphorylation in vitro confirmed the impaired ability of the mutant Hsp20 to become phosphorylated at Ser16. In conclusion, we have identified a P20L substitution in human Hsp20, which is associated with diminished phosphorylation at Ser16 and complete abrogation of the Hsp20 cardioprotective effects which may adversely affect the ability of human carriers to cope with cellular stress.     

Patterns of host-plant choice in bees of the genus Chelostoma: the constraint hypothesis of host-range evolution in bees

To trace the evolution of host-plant choice in bees of the genus Chelostoma (Megachilidae), we assessed the host plants of 35 Palearctic, North American and Indomalayan species by microscopically analyzing the pollen loads of 634 females and reconstructed their phylogenetic history based on four genes and a morphological dataset, applying both parsimony and Bayesian methods. All species except two were found to be strict pollen specialists at the level of plant family or genus. These oligolectic species together exploit the flowers of eight different plant orders that are distributed among all major angiosperm lineages. Based on ancestral state reconstruction, we found that oligolecty is the ancestral state in Chelostoma and that the two pollen generalists evolved from oligolectic ancestors. The distinct pattern of host broadening in these two polylectic species, the highly conserved floral specializations within the different clades, the exploitation of unrelated hosts with a striking floral similarity as well as a recent report on larval performance on nonhost pollen in two Chelostoma species clearly suggest that floral host choice is physiologically or neurologically constrained in bees of the genus Chelostoma. Based on this finding, we propose a new hypothesis on the evolution of host range in bees.     

Programmed cell death of primordial germ cells in Drosophila is regulated by p53 and the Outsiders monocarboxylate transporter

Primordial germ cell development uses programmed cell death to remove abnormal, misplaced or excess cells. Precise control of this process is essential to maintain the continuity and integrity of the germline, and to prevent germ cells from colonizing locations other than the gonads. Through careful analyses of primordial germ cell distribution in developing Drosophila melanogaster embryos, we show that normal germ cell development involves extensive programmed cell death during stages 10-12 of embryogenesis. This germ cell death is mediated by Drosophila p53 (p53). Mutations in p53 result in excess primordial germ cells that are ectopic to the gonads. Initial movements of the germ cells appear normal, and wild-type numbers of germ cells populate the gonads, indicating that p53 is required for germ cell death, but not migration. To our knowledge, this is the first report of a loss-of-function phenotype for Drosophila p53 in a non-sensitized background. The p53 phenotype is remarkably similar to that of outsiders (out) mutants. Here, we show that the out gene encodes a putative monocarboxylate transporter. Mutations in p53 and out show nonallelic noncomplementation. Interestingly, overexpression of p53 in primordial germ cells of out mutant embryos partially suppresses the out germ cell death phenotype, suggesting that p53 functions in germ cells either downstream of out or in a closely linked pathway. These findings inform models in which signaling between p53 and cellular metabolism are integrated to regulate programmed cell death decisions.