It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2)], and phosphatidylinositol trisphosphate [PtdIns(3,4,5)P(3)], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [(32)P]inorganic phosphate or [(3)H]inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS(3). Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.

Phosphatidic acid and phosphatidylserine are negatively charged abundant phospholipids with well-recognized structural roles in cellular membranes. They are also signaling lipids since their regulated formation (or appearance) can constitute an important signal for downstream responses. The list of potential effectors for these lipids is expanding rapidly and includes proteins involved in virtually all aspects of cellular regulation. Because it is not always clear whether these effectors recognize the specific phospholipids or a general negatively-charged membrane environment, questions about specificity must be addressed on a case by case basis. In this review we present an up to date list of potential phosphatidic acid- and phosphatidylserine-binding proteins.

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.

The epigenetic phenomenon of genomic imprinting provides an additional level of gene regulation that is confined to a limited number of genes, frequently, but not exclusively, important for embryonic development. The evolution and maintenance of imprinting has been linked to the balance between the allocation of maternal resources to the developing fetus and the mother's well being. Genes that are imprinted in both the embryo and extraembryonic tissues show extensive conservation between a mouse and a human. Here we examine the human orthologues of mouse genes imprinted only in the placenta, assaying allele-specific expression and epigenetic modifications. The genes from the KCNQ1 domain and the isolated human orthologues of the imprinted genes Gatm and Dcn all are expressed biallelically in the human, from first-trimester trophoblast through to term. This lack of imprinting is independent of promoter CpG methylation and correlates with the absence of the allelic histone modifications dimethylation of lysine-9 residue of H3 (H3K9me2) and trimethylation of lysine-27 residue of H3 (H3K27me3). These specific histone modifications are thought to contribute toward regulation of imprinting in the mouse. Genes from the IGF2R domain show polymorphic concordant expression in the placenta, with imprinting demonstrated in only a minority of samples. Together these findings have important implications for understanding the evolution of mammalian genomic imprinting. Because most human pregnancies are singletons, this absence of competition might explain the comparatively relaxed need in the human for placental-specific imprinting.

In mammals, imprinted genes have an important role in feto-placental development. They affect the growth, morphology and nutrient transfer capacity of the placenta and, thereby, control the nutrient supply for fetal growth. In particular, the reciprocally imprinted Igf2-H19 gene complex has a central role in these processes and matches the placental nutrient supply to the fetal nutrient demands for growth. Comparison of Igf2P0 and complete Igf2 null mice has shown that interplay between placental and fetal Igf2 regulates both placental growth and nutrient transporter abundance. In turn, epigenetic modification of imprinted genes via changes in DNA methylation may provide a mechanism linking environmental cues to placental phenotype, with consequences for development both before and after birth. Changes in expression of imprinted genes, therefore, have major implications for developmental programming and may explain the poor prognosis of the infant born small for gestational age and the wide spectrum of adult-onset diseases that originate in utero.

Gnas is an enigmatic and rather complex imprinted gene locus. A single transcription unit encodes three, and possibly more, distinct proteins. These are determined by overlapping transcripts from alternative promoters with different patterns of imprinting. The canonical Gnas transcript codes for Gsalpha, a highly conserved signalling protein and an essential intermediate in growth, differentiation and homeostatic pathways. Monoallelic expression of Gnas is highly tissue-restricted. The alternative transcripts encode XLalphas, an unusual variant of Gsalpha, and the chromogranin-like protein Nesp55. These transcripts are expressed specifically from the paternal and maternal chromosomes, respectively. Their existence in the Gnas locus might imply functional connections amongst them or with Gsalpha. In this review, we consider how imprinting of Gnas was discovered, the phenotypic consequences of mutations in each of the gene products, both in the mouse and human, and provide some conjectures to explain why this elaborate imprinted locus has evolved in this manner in mammals.

Imprinted genes tend to be clustered in the genome. Most of these clusters have been found to be under the control of discrete DNA elements called imprinting centres (ICs) which are normally differentially methylated in the germline. ICs can regulate imprinted expression and epigenetic marks at many genes in the region, even those which lie several megabases away. Some of the molecular and cellular mechanisms by which ICs control other genes and regulatory regions in the cluster are becoming clear. One involves the insulation of genes on one side of the IC from enhancers on the other, mediated by the insulator protein CTCF and higher-order chromatin interactions. Another mechanism may involve non-coding RNAs that originate from the IC, targeting histone modifications to the surrounding genes. Given that several imprinting clusters contain CTCF dependent insulators and/or non-coding RNAs, it is likely that one or both of these two mechanisms regulate imprinting at many loci. Both mechanisms involve a variety of epigenetic marks including DNA methylation and histone modifications but the hierarchy of and interactions between these modifications are not yet understood. The challenge now is to establish a chain of developmental events beginning with differential methylation of an IC in the germline and ending with imprinting of many genes, often in a lineage dependent manner.

Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.

The mechanisms that regulate variable (V) gene selection during the development of the mouse IgH repertoire are not fully understood, due in part to the absence of the complete locus sequence. To better understand these processes, we have assembled the entire 2.5-Mb mouse IgH (Igh) V region sequence of the C57BL/6 strain from public sequences and present the first complete annotated map of the region, including V genes, pseudogenes, repeats, and nonrepetitive intergenic sequences. In so doing, we have discovered a new V gene family, VH16. We have identified clusters of conserved region-specific intergenic sequences and have verified our assembly by genic and intergenic Southern blotting. We have observed that V pseudogenes are not evenly spread throughout the V region, but rather cluster together. The largest J558 family, which spans more than half of the locus, has two strikingly different domains, which suggest points of evolutionary divergence or duplication. The 5' end contains widely spaced J558 genes interspersed with 3609 genes and is pseudogene poor. The 3' end contains closely spaced J558 genes, no 3609 genes, and is pseudogene rich. Each occupies a different branch of the phylogenetic tree. Detailed analysis of 500-bp upstream of all functional genes has revealed several conserved binding sites, general and B cell-specific, as well as key differences between families. This complete and definitive assembly of the mouse Igh V region will facilitate detailed study of promoter function and large-scale mechanisms associated with V(D)J recombination including locus contraction and antisense intergenic transcription.

We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1-2 mutation, pre-60S particles were rapidly degraded following transfer to 37 degrees C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1-2 strains at 37 degrees C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1-2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.

Genomic imprinting is limited to a subset of genes that play critical roles in fetal growth, development and behaviour. One of the most studied imprinted genes encodes insulin-like growth factor 2, and aberrant imprinting and DNA methylation of this gene is associated with the growth disorders Beckwith-Wiedemann and Silver-Russell syndromes and many human cancers. Specific isoforms of this gene have been shown to be essential for normal placental function, as mice carrying paternal null alleles for the Igf2-P0 transcript are growth restricted at birth. We report here the identification of three novel human transcripts from the IGF2 locus. One is equivalent to the mouse Igf2-P0 transcript, whereas the two others (INSIGF long and short) originate from the upstream INS gene that alternatively splices to downstream IGF2 exons. In order to elucidate the molecular mechanisms involved in the complex imprinting of these novel IGF2 transcripts, both the allele-specific expression and methylation for all the IGF2 promoters including P0 and the INSIGF transcripts were analysed in human tissues. Similar to the mouse, the human IGF2-P0 transcript is paternally expressed; however, its expression is not limited to placenta. This expression correlates with tissue-specific promoter methylation on the maternal allele. The two novel INSIGF transcripts reported here use the INS promoter and show highly restricted tissue expression profiles including the pancreas. As previously reported for INS in the yolk sac, we demonstrate complex, tissue-specific imprinting of these transcripts. The finding of additional transcripts within this locus will have important implications for IGF2 regulation in both cancer and metabolism.

Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways.

Intrauterine growth and development can impact upon the long-term health of an individual. The fetus is dependent upon the placenta for its supply of nutrients and oxygen from the mother. In turn, the functional capacity of the placenta to supply that demand is under the control of the fetal and maternal genomes. Recent evidence suggests that imprinted genes, a class of genes found in placental mammals whose expression depends on their parental origin, have multiple roles in the placenta. The imprinted genes regulate the growth and transport capacity of the placenta, thereby controlling the supply of nutrients. They may also regulate the growth rate of fetal tissues directly, thereby controlling nutrient demand by the fetus. Recent studies using mice with deletions or disruption of imprinted genes with an altered balance between placental and fetal growth and changes in placental efficiency are indicative of feto-placental signalling of fetal nutrient demand. We propose that signalling mechanisms involving growth demand signals and nutrient transporters are likely to occur and are important for fine tuning normal fetal growth.

Nucleosome-remodelling factors are key facilitators of chromatin dynamics. At the level of single nucleosomes, they are involved in nucleosome-repositioning, altering histone-DNA interactions, disassembly of nucleosomes, and the exchange of histones with variants of different properties. The fundamental nature of chromatin dictates that nucleosome-remodelling affects all aspects of eukaryotic DNA metabolism, but much less is known about the functional interactions of nucleosome-remodelling factors with folded chromatin fibres. Because remodelling machines are abundant constituents of eukaryotic nuclei and, therefore, have ample potential to interact with chromatin, they might also affect higher-order chromatin architecture. Recent observations support roles for nucleosome-remodelling factors at the supra-nucleosomal level.

Phosphoinositide 3-kinase (PI3K) gamma has been implicated in a vast array of physiological settings including the activation of different leukocyte species and the regulation of myocardial contractility. Activation of PI3Kgamma is primarily mediated by Gbetagamma subunits of heterotrimeric G proteins, which are recognized by a p101 regulatory subunit. Here, we describe the identification and characterization of a novel regulatory subunit of PI3Kgamma, which we termed p87(PIKAP) (PI3Kgamma adapter protein of 87 kDa). It is homologous to p101 in areas that we have recently shown that they mediate binding to the catalytic p110gamma subunit and to Gbetagamma. Like p101, p87(PIKAP) binds to both p110gamma and Gbetagamma and mediates activation of p110gamma downstream of G protein-coupled receptors. In contrast to p101, p87(PIKAP) is highly expressed in heart and may therefore be crucial to PI3Kgamma cardiac function. Moreover, p87(PIKAP) and p101 are both expressed in dendritic cells, macrophages, and neutrophils, raising the possibility of regulatory subunit-dependent differences in PI3Kgamma signaling within the same cell type. We further provide evidence that p87(PIKAP) physically interacts with phosphodiesterase (PDE) 3B, suggesting that p87(PIKAP) is also involved in the recently described noncatalytic scaffolding interaction of p110gamma with PDE3B. However, coexpression of PDE3B and PI3Kgamma subunits was not sufficient to reconstitute the regulatory effect of PI3Kgamma on PDE3B activity observed in heart, implying further molecules to be present in the complex regulating PDE3B in heart.

An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate PI(4,5)P2 by phospholipase Cbeta (PLCbeta) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates its levels, mediating both amplification and response termination. In this study, we demonstrate that lazaro (laza) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. We demonstrate that the synergistic activity of laza and rdgA regulates response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in phosphatidic acid (PA) levels and an associated reduction in phosphatidylinositol (PI) levels. Together our results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo.

CellML and SBML are XML-based languages for storage and exchange of molecular biological and physiological reaction models. They use very similar subsets of MathML to specify the mathematical aspects of the models. CellML2SBML is implemented as a suite of XSLT stylesheets that, when applied consecutively, convert models expressed in CellML into SBML without significant loss of information. The converter is based on the most recent stable versions of the languages (CellML version 1.1; SBML Level 2 Version 1), and the XSLT used in the stylesheets adheres to the XSLT version 1.0 specification. Of all 306 models in the CellML repository in April 2005, CellML2SBML converted 91% automatically into SBML. Minor manual changes to the unit definitions in the originals raised the percentage of successful conversions to 96%.

We describe de novo generation of IL-17-producing T cells from naive CD4 T cells, induced in cocultures of naive CD4 T cells and naturally occurring CD4+ CD25+ T cells (Treg) in the presence of TLR3, TLR4, or TLR9 stimuli. Treg can be substituted by TGFbeta1, which, together with the proinflammatory cytokine IL-6, supports the differentiation of IL-17-producing T cells, a process that is amplified by IL-1beta and TNFalpha. We could not detect a role for IL-23 in the differentiation of IL-17-producing T cells but confirmed its importance for their survival and expansion. Transcription factors GATA-3 and T-bet, as well as its target Hlx, are absent in IL-17-producing T cells, and they do not express the negative regulator for TGFbeta signaling, Smad7. Our data indicate that, in the presence of IL-6, TGFbeta1 subverts Th1 and Th2 differentiation for the generation of IL-17-producing T cells.

Genomic imprinting results in allele-specific silencing according to parental origin. Silencing is brought about by imprinting control regions (ICRs) that are differentially marked in gametogenesis. The group of imprinted transcripts in the mouse Gnas cluster (Nesp, Nespas, Gnasxl, Exon 1A and Gnas) provides a model for analyzing the mechanisms of imprint regulation. We previously identified an ICR that specifically regulates the tissue-specific imprinted expression of the Gnas gene. Here we identify a second ICR at the Gnas cluster. We show that a paternally derived targeted deletion of the germline differentially methylated region (DMR) associated with the antisense Nespas transcript unexpectedly affects both the expression of all transcripts in the cluster and methylation of two DMRs. Our results establish that the Nespas DMR is the principal ICR at the Gnas cluster and functions bidirectionally as a switch for modulating expression of the antagonistically acting genes Gnasxl and Gnas. Uniquely, the Nespas DMR acts on the downstream ICR at exon 1A to regulate tissue-specific imprinting of the Gnas gene.

The generation of T cell receptor (TCR) sequence diversity is the strength of adaptive immunity, yet is also the Achilles' heel. To purge highly self-reactive T cells from the immune system, generation of diversity has coevolved with a mechanism of negative selection. Recent studies have revealed new insights addressing the why and how of negative selection by examining situations in which negative selection has failed in human and animals models of autoimmunity. Both thymocyte extrinsic and intrinsic mechanisms are required to restrict the TCR repertoire to a non-autoreactive set. Negative selection also ensures that T cells emerge with receptors that are focussed on the peptide moiety of MHC-peptide complexes.

The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Emu) only modestly reduce D(H)-->J(H) rearrangements when assessed in peripheral B cells. However, deletion of Emu severely impairs recombination of V(H) gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Emu deletions on primary D(H)-->J(H) rearrangement: 1) Accessibility at the D(H)J(H) cluster is controlled by a redundant cis-element in the absence of Emu. One candidate for this element lies 5' to D(Q52) (PD(Q52)) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, D(H)-->J(H) recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PD(Q52) and Emu in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PD(Q52) promoter is dispensable for germline transcription and recombination of the D(H)J(H) cluster. In contrast, we demonstrate that Emu directly regulates accessibility of the D(H)J(H) region. These findings reveal a significant role for Emu in the control mechanisms that activate IgH gene assembly and suggest that impaired V(H)-->D(H)J(H) rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in D(H)-->J(H) recombination.

The typically lysosomal family of cysteine cathepsin proteases has been implicated in the development of the placenta in particular, from studies in the mouse. Here, we analysed overall expression, regulation and presence of transcript isoforms of cysteine cathepsins during human extra-embryonic development. All 11 family members are expressed in human placental tissues, and many are differentially regulated during gestation. Several cysteine cathepsins exhibit deregulated expression levels in placentas from pregnancies complicated by pre-eclampsia. The localization of cathepsin B predominantly in placental and decidual macrophages suggests a role in the physiological functions of these cells in mediating villous angiogenesis and decidual apoptosis. Cathepsin L levels are highest in a subpopulation of invasive cytotrophoblasts. Reflecting the expression pattern of two murine cathepsins, these data give insights into the evolutionary conservation of cathepsin function that is not necessarily exhibited by gene pairs defined by highest sequence similarity. Furthermore, cathepsin L protein localization in uterine epithelial cells demonstrates the in vivo occurrence of intranuclear cathepsin L isoforms. The zonally restricted expression of cathepsin in the syncytiotrophoblast may be important for the metabolic breakdown of maternal nutrients. Overall, the distribution and abnormal expression levels in pre-eclamptic placentas indicate that cysteine cathepsins may play important roles during normal placentation and in the etiology of pre-eclampsia.

Rho and Arf family small GTPases control dynamic actin rearrangements and vesicular trafficking events. ARAP3 is a dual GAP for RhoA and Arf6 that is regulated by phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], a product of the phosphoinositide 3-kinase (PI3K) signalling pathway. To investigate the physiological function of ARAP3, we used an RNAi-based approach in an endothelial cell model. ARAP3-deficient cells showed increased activities of RhoA and Arf6. Phenotypically, they were more rounded than control counterparts and displayed very fine stress fibres. ARAP3-deficient cells were not capable of producing lamellipodia upon growth factor stimulation, a process known to depend on PI3K and Rac activities. Rac was transiently activated in stimulated ARAP3 RNAi cells although its cellular localisation was altered, a likely consequence of increased Arf6 activity. We conclude that ARAP3 recruitment to sites of elevated PtdIns(3,4,5)P(3) is crucial to allow localised inactivation of RhoA and cycling of Arf6, both of which are necessary to allow growth factor-stimulated formation of lamellipodia.