The NADPH oxidase complex of neutrophils and macrophages is an important weapon used by these cells to kill microbial pathogens. The regulation of this enzyme complex is necessarily complicated by the diverse receptor types that are needed to trigger its activation and also the tight control that is required to deliver this activation at the appropriate time and place. As such, several signalling pathways have been established to regulate the NADPH oxidase downstream of cell surface receptors. Central amongst these are PI3K- (phosphoinositide 3-kinase)-dependent pathways, blockade of which severely limits activation of the oxidase to several soluble and particulate stimuli. The precise roles of the phosphoinositide products of PI3K activity in regulating NADPH oxidase assembly and activation are still unclear, but there is emerging evidence that they play a key role via regulation of guanine nucleotide exchange on Rac, a key component in the oxidase complex. There is also very strong evidence that the PI3K products PtdIns(3,4)P2 and PtdIns3P can bind directly to the PX (Phox homology) domains of the core oxidase components p47phox and p40phox respectively. However, the significance of these interactions in terms of membrane localization or allosteric consequences for the oxidase complex remains to be established.

InsP3 has two important functions in generating Ca2+ oscillations. It releases Ca2+ from the internal store and it can contribute to Ca2+ entry. A hypothesis has been developed to describe a mechanism for Ca2+ oscillations with particular emphasis on the way agonist concentration regulates oscillator frequency. The main idea is that the InsP3 receptors are sensitized to release Ca2+ periodically by cyclical fluctuations of Ca2+ within the lumen of the endoplasmic reticulum. Each time a pulse of Ca2+ is released, the luminal level of Ca2+ declines and has to be replenished before the InsP3 receptors are resensitized to deliver the next pulse of Ca2+. It is this loading of the internal store that explains why frequency is sensitive to external Ca2+ and may also account for how variations in agonist concentration are translated into changes in oscillation frequency. Variations in agonist-induced entry of external Ca2+, which can occur through different mechanisms, determine the variable rates of store loading responsible for adjusting the sensitivity of the InsP3 receptors to produce the periodic pulses of Ca2+. The Ca2+ oscillator is an effective analogue-to-digital converter in that variations in the concentration of the external stimulus are translated into a change in oscillator frequency.

The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for Galpha(o) and V2R, whereas V1R and Galpha(i) immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked Galpha(o). Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation.

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.

Breast radiotherapy as practised in the 1970s and 1980s resulted in significant myocardial exposure, and this was higher when the left breast was treated. It has been proposed that this difference might result in greater cardiovascular mortality following irradiation of the left breast when compared with the right.

The transcription factor hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tumour growth and progression, and HIF-1 is regulated through a number of signalling pathways. Here, we investigated the involvement of the mitogen-activated protein kinase (MAPK) signalling pathway in HIF-1 regulation. We found that overexpression of wild-type (WT) extracellular signal regulated protein kinase 1 (ERK1) greatly potentiated HIF-1 activation in hypoxia and HIF-1alpha induced in response to insulin growth-like factor 1 (IGF-1). Conversely, treatment of tumour cells with the MEK1/2 inhibitors PD98059 or U0216, or expression of a dominant-negative form of ERK1 blocked HIF-1 activation in hypoxia without affecting HIF-1alpha induction, localization or binding of HIF-1beta. Interestingly however, the highly selective MEK1/2 inhibitor PD184352 did not inhibit HIF-1 activity or vascular endothelial growth factor (VEGF) induced in response to hypoxia but blocked HIF-1alpha protein and HIF-1 activity induced by IGF-1 stimulation without affecting HIF-1alpha mRNA levels. Finally, we found that ERK5 phosphorylation status was not significantly affected by hypoxia in the presence or absence of PD184352. Taken together, our data suggest that although ERK1/2 signalling is important for HIF-1alpha induction and HIF-1 activity in response to IGF-1, it is dispensable for the induction of HIF-1alpha and activation of HIF-1 in response to hypoxia.

The activation of antigen receptors triggers two important signalling pathways originating from phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]. The first is phospholipase Cgamma (PLCgamma)-mediated hydrolysis of PtdIns(4,5)P(2), resulting in the activation of Ras, protein kinase C and Ca(2+) flux. This culminates in profound alterations in gene expression and effector-cell responses, including secretory granule exocytosis and cytokine production. By contrast, phosphoinositide 3-kinases (PI3Ks) phosphorylate PtdIns(4,5)P(2) to yield phosphatidylinositol(3,4,5)-trisphosphate, activating signalling pathways that overlap with PLCgamma or are PI3K-specific. Pathways that are PI3K-specific include Akt-mediated inactivation of Foxo transcription factors and transcription-independent regulation of glucose uptake and metabolism. The p110delta isoform of PI3K is the main source of PI3K activity following antigen recognition by B cells, T cells and mast cells. Here, we review the roles of p110delta in regulating antigen-dependent responses in these cell types.

Chemotaxis is the process by which organisms migrate toward nutrients and favorable environments and away from toxins and unfavorable environments. In many species of bacteria, this occurs when extracellular signals are detected by transmembrane receptors and relayed to flagellar motors, which control the cell's swimming behavior.

ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca(2+) signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca(2+) signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Nuclear envelope assembly is an essential event in each cell cycle but the proteins and lipids involved in its regulation remain mostly unknown. Assembly involves membrane fusions but neither specific SNAREs nor Rab GTPases have been identified in its control. We report that a precursor membrane population (MV1) required for NE assembly has a unique lipid composition consisting prominently of poly-phosphatidylinositides. The lipid composition was determined by adapting HPLC electrospray ionisation tandem mass spectrometry to phosphoinositide analysis, revealing the capacity of this technique to document dynamic lipid transitions of functional importance in natural membrane populations. MV1 is >100-fold enriched in endogenous PLCgamma and >25-fold enriched in the PLC substrate phosphatidylinositol bisphosphate (PtdInsP2) compared to the second membrane population, derived largely from endoplasmic reticulum (ER), that contributes most of the NE. During NE formation PLCgamma becomes transiently phosphorylated at the tyrosine 783 site indicative of its activation. In addition specific inhibition of PLCgamma blocks nuclear envelope formation. In vivo, PLCgamma is concentrated on vesicles of similar size to purified MV1. These associate with nuclei during the period of NE formation and are distinct from ER membranes. The unprecedented concentration of PLCgamma and its substrate PtdInsP2 in a subset of membranes that binds to only two regions of the nucleus, and activation of PLCgamma by GTP during initial stages of NE formation provide a mechanism for temporal control of NE assembly and offer an explanation for how such a process of membrane fusion can be spatially regulated.

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the gamma(A) and gamma(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control gamma(A) methylation during development. DNA methylation causes a 20-fold repression of gamma(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress gamma gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of gamma-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate gamma-globin expression in patients with beta-hemoglobinopathies.

Carboxypeptidase E (CPE) has important functions in processing of endocrine pro-peptides, such as pro-insulin, pro-opiomelanocortin, or pro-gonadotropin-releasing hormone, as evidenced by the hyper-pro-insulinemia, obesity, and sterility of Cpe mutant mice. Down-regulation of Cpe in enlarged placentas of interspecific hybrid (interspecies hybrid placental dysplasia (IHPD)) and cloned mice suggested that reduced CPE enzyme and receptor activity could underlie abnormal placental phenotypes. In this study, we have explored the role of Cpe in murine placentation by determining its expression at various stages of gestation, and by phenotypic analysis of Cpe mutant placentas. Our results show that Cpe and Carboxypeptidase D (Cpd), another carboxypeptidase with a very similar function, are strictly co-localized in the mouse placenta from late mid-gestation to term. We also show that absence of CPE causes a sporadic but striking placental phenotype characterized by an increase in giant and glycogen cell numbers and giant cell hypertrophy. Microarray-based transcriptional profiling of Cpe mutant placentas identified only a very small number of genes with altered expression, including Dtprp, which belongs to the prolactin gene family. Concordant deregulation of Cpe and Cpd in abnormal placentas of interspecies hybrids before the onset of IHPD phenotype and recapitulation of some phenotypes of IHPD hyperplastic placentas in Cpe mutant placentas suggests that these two genes are causally involved in IHPD and may function as speciation genes in the genus Mus.

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage-induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp(52)/iso-Asp(66) species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage-induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

PI3K signalling pathways link cell surface receptors to the control of several intracellular functions including cell growth, survival and movement. Filamins are important regulators of cortical actin structure and function. LL5beta is a filamin binding protein that is an effector of the PI3K signalling pathway. We define an N-terminal region of LL5beta that is responsible for binding to the C-terminus of filamins. Under conditions of very low PI3K activity, we show that this region, together with an additional domain of the protein, is responsible for localising the complex to punctate structures that are also decorated by L-FILIP (a protein previously characterised to bind filamin and accelerate its destruction). Under conditions of significant PI3K activity, PtdIns(3,4,5)P(3) binding to the C-terminal PH domain in LL5beta prevents localisation to these structures. These observations start to define the basis for PI3K regulation of filamin through LL5beta.

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

The loss of all, or part of an X chromosome, in Turner syndrome (TS, 45,XO) results in deficits in attentional functioning.

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.

A correlation multi-variate analysis of variance (MANOVA) test to statistically analyze changing patterns of multi-electrode array (MEA) electrophysiology data is developed. The approach enables us not only to detect significant mean changes, but also significant correlation changes in response to external stimuli. Furthermore, a method to single out hot-spot variables in the MEA data both for the mean and correlation is provided. Our methods have been validated using both simulated spike data and recordings from sheep inferotemporal cortex.

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma.

Productive T-cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. The costimulatory molecule CD28 plays an essential role in the initiation of T-cell-mediated immunity. We investigated the possibility that CD28 may also regulate migration of primed T cells to target tissue. In vitro, CD28-mediated signals enhanced T-cell transendothelial migration, integrin clustering, and integrin-mediated migration. In vivo, T cells bearing a mutation in the CD28 cytoplasmic domain, which abrogates PI3K activation, displayed normal clonal expansion but defective localization to antigenic sites following antigenic rechallenge. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T-cell migration to extra-lymphoid tissue, which occurred independently of T-cell receptor (TCR)-derived signals and homing-receptor expression. Finally, we provide evidence that CD28- and CTLA-4-mediated signals exert opposite effects on T-cell trafficking in vivo. These findings highlight a novel physiologic function of CD28 that has crucial implications for the therapeutic manipulation of this and other costimulatory molecules.

The field of Computational Systems Neurobiology is maturing quickly. If one wants it to fulfil its central role in the new Integrative Neurobiology, the reuse of quantitative models needs to be facilitated. The community has to develop standards and guidelines in order to maximise the diffusion of its scientific production, but also to render it more trustworthy. In the recent years, various projects tackled the problems of the syntax and semantics of quantitative models. More recently the international initiative BioModels.net launched three projects: (1) MIRIAM is a standard to curate and annotate models, in order to facilitate their reuse. (2) The Systems Biology Ontology is a set of controlled vocabularies aimed to be used in conjunction with models, in order to characterise their components. (3) BioModels Database is a resource that allows biologists to store, search and retrieve published mathematical models of biological interests. We expect that those resources, together with the use of formal languages such as SBML, will support the fruitful exchange and reuse of quantitative models.

Alzheimer's disease (AD) is characterized by Abeta peptide-containing plaques and tau-containing neurofibrillary tangles (NFTs). Both pathologies have been combined by crossing Abeta plaque-forming APP mutant mice with NFT-forming P301L tau mutant mice or by stereotaxically injecting beta-amyloid peptide 1-42 (Abeta42) into brains of P301L tau mutant mice. In cell culture, Abeta42 induces filamentous tau aggregates. To understand which processes are disrupted by Abeta42 in the presence of tau aggregates, we applied comparative proteomics to Abeta42-treated P301L tau-expressing neuroblastoma cells and the amygdala of P301L tau transgenic mice stereotaxically injected with Abeta42. Remarkably, a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. We also identified model-specific effects of Abeta42 treatment such as differences in cell signaling proteins in the cellular model and of cytoskeletal and synapse associated proteins in the amygdala. By Western blotting (WB) and immunohistochemistry (IHC), we were able to show that 72% of the tested candidates were altered in human AD brain with a major emphasis on stress-related unfolded protein responsive candidates. These data highlight these processes as potentially important initiators in the Abeta42-mediated pathogenic cascade in AD and further support the role of unfolded proteins in the course of AD.