The Rac1 and Rac2 GTPases play important roles in many processes including cytoskeletal reorganization, proliferation, and survival, and are required for B-cell development. Previous studies had shown that deficiency in Rac2 did not affect T-cell development, whereas the function of Rac1 in this process has not been investigated. We now show that simultaneous absence of both GTPases resulted in a very strong developmental block at the pre-TCR checkpoint and in defective positive selection. Unexpectedly, deficiency of Rac1 and Rac2 also resulted in the aberrant survival of thymocytes lacking expression of TCR beta, showing hallmarks of hyperactive Notch signaling. Furthermore, we found a similar novel phenotype in the absence of Vav1, Vav2, and Vav3, which function as guanine nucleotide exchange factors for Rac1 and Rac2. These results show that a pathway containing Vav and Rac proteins may negatively regulate Notch signaling during early thymic development.

Interspecific hybridization in mammals causes hybrid dysgenesis effects, such as sterility and abnormal placentation. Here, we describe a novel obesity syndrome caused by interspecific hybridization in the genus Mus and show that this obesity, appearing sporadically in F1 littermates derived from inbred strains, has an epigenetic basis. Mus hybrids from various strains of M. musculus and M. spretus were generated and the sporadic obese phenotype was confirmed through assessment of physiological and biochemical parameters in littermates. To understand the underlying mechanisms, large-scale and candidate gene expression assays, global DNA methylation assays and allelic expression analysis were performed. Studies showed that obese hybrids are similar to other known models of obesity. While increased axial growth indicated a defect in POMC pathway, comparison of global gene expression patterns in brain of obese F1 and obese Pomc mutant mice showed little similarity. In F1 obese mice many genes involved in the maintenance of epigenetic states, as well as several imprinted genes, were differentially expressed. Global DNA methylation analysis in brain showed that increased methylation levels were associated with obesity. The imprinted gene Gnasxl, known to be important in lipid homeostasis, was found over expressed in the obese hybrids. Allelic expression and methylation analysis of Gnasxl showed that alterations of epigenetic marks underlying F1 obesity are probably many and multi-factorial.

Novel approaches to effectively reduce noise in data recorded from multi-trial physiology experiments have been investigated using two-dimensional filtering methods, adaptive Wiener filtering and reduced update Kalman filtering. Test data based on signal and noise model consisting of different conditions of signal components mixed with noise have been considered with filtering effects evaluated using analysis of frequency coherence and of time-dependent coherence. Various situations that may affect the filtering results have been explored and reveal that Wiener and Kalman filtering can considerably improve the coherence values between two channels of multi-trial data and suppress uncorrelated components. We have extended our approach to experimental data: multi-electrode array (MEA) local field potential (LFPs) recordings from the inferotemporal cortex of sheep and LFP vs. electromyogram (LFP-EMG) recording data during resting tremor in Parkinson's disease patients. Finally general procedures for implementation of these filtering techniques are described.

Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events affecting imprinted genes at 11p15.5. Most BWS cases are sporadic and result from imprinting errors (epimutations) involving either of the two 11p15.5 imprinting control regions (IC1 and IC2). Previously, we and other reported an association between sporadic BWS and assisted reproductive technologies (ARTs).

Chromosomal translocation requires formation of paired double-strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy-chain locus (IgH) and is found in Burkitt's lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation-induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here, we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.

Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies.

Physiologists have routinely used understanding of the immune system to generate antibodies against regulatory molecules, growth factors, plasma membrane receptors, and other mammalian molecules in the development of analytical tools and assays. In taking this notion further, antibodies have been used in vivo to modulate physiological systems and to improve our understanding of their molecular interactions. To develop antibodies with physiological activity (efficacy), physiologists have worked with immunologists in developing interdisciplinary insights, requiring basic knowledge of immune system function in designing strategies to generate antibodies that interact with endogenous molecules of physiological interest, in vivo. Antibodies in different physiological systems have been shown to enhance or inhibit endogenous molecular functions. Two approaches have been used: passive and active immunization. Antibodies in these contexts have provided tools to develop further insights into molecular physiological mechanisms. Perhaps surprisingly, enhancing antibodies have been developed against a diverse set of target molecules including several members of the growth hormone/insulin-like growth factor-I axes and those of the beta(2)-adrenoceptor axis. Antibodies that inhibit the actions of somatostatin have also been developed. A further novel approach has been the development of antibodies that interact with adipose cells in vivo. These have the potential to be used in therapeutic antiobesity approaches. Antibodies with efficacy in vivo have provided new insights into molecular physiological mechanisms, enhancing our understanding of these complex processes.

Post-transcriptional control of gene expression is an important mechanism for maintaining cellular homoeostasis and regulating the immune response to infection. It allows control of mRNA abundance, translation and localization. Mechanisms for post-transcriptional control involve RNA-binding proteins and miRNAs (microRNAs). The TTP(tristetraprolin) family of proteins recognize and bind AU-rich elements. Deletion of TTP led to a systemic autoimmune syndrome with excess circulating TNFalpha (tumour necrosis factor alpha) and GM-CSF (granulocyte/macrophage colony-stimulating factor) due to aberrantly stabilized mRNA. The family may also have a role in control of lymphocyte development and function. miRNAs regulate gene expression by promoting decay or inhibiting translation of transcripts with base pair complementarity. The importance of miRNAs in lymphocytes is highlighted by the T-cell-specific deletion of Dicer, an enzyme required for miRNA-mediated processing and from the phenotype of bic (B-cell integration cluster)/miR-155 (miRNA 155)-deficient mice.

B-cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF-signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF-mediated responses of B cells deficient for the PI3K catalytic subunit P110delta. We find that the loss of P110delta impairs the BAFF-mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110delta was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110delta in regulating B-cell responses to BAFF.

Recent studies indicate that pharmaceutical blockade of phosphoinositide 3-kinase (PI3K) signaling enzymes might be effective in reducing allergic airway inflammation. Signals generated by the p110delta PI3K isoform play critical roles in signaling through antigen and cytokine receptors and were shown to be required for induction of type 2, but not type 1, cytokine responses.

Studies on control of fluid secretion by an insect salivary gland led to the discovery of inositol trisphosphate (IP3) and its role in calcium signalling. Many cell stimuli act on receptors that are coupled to phospholipase C that hydrolyses phosphatidylinosol 4,5-bisphosphate (PIP2) to release IP3 to the cytosol. IP3 receptors located on the endoplasmic reticulum respond to this elevation of IP3 by releasing Ca2+, which is often organized into characteristic spatial (elementary events and waves) and temporal (Ca2+ oscillations) patterns. This IP3/Ca2+ pathway is a remarkably versatile signalling system that has been adapted to control processes as diverse as fertilization, proliferation, contraction, cell metabolism, vesicle and fluid secretion and information processing in neuronal cells.

A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted--monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.

Inflammatory activation of monocytes is an essential part of both innate immune responses and the pathogenesis of conditions such as atherosclerosis. However, the mechanisms which modulate the response of monocytes to inflammatory stimuli are still poorly understood. Here, we report that tribbles-2 (trb-2) is a novel regulator of inflammatory activation of monocytes. Down-regulation of trb-2 levels potentiates LPS-induced IL-8 production via enhanced activation of the extracellular signal-regulated kinase and jun kinase mitogen-activated protein kinase (MAPK) pathways. In keeping with this, the endogenous level of trb-2 expression in human primary monocytes is inversely correlated to the cell's ability to produce IL-8. We show that trb-2 is a binding partner and a negative regulator of selected MAPKs. The potential in vivo relevance of these findings is highlighted by the observation that modified low-density lipoprotein profoundly down-regulates trb-2 expression, which may, in turn, significantly contribute to the inflammatory processes in the development of vascular disease. Taken together, our results define trb-2 as a potent novel regulator of monocyte biology, controlling the activation of these cells.

Recent investigations have implicated long antisense noncoding RNAs in the epigenetic regulation of chromosomal domains. Here we show that Kcnq1ot1 is an RNA polymerase II-encoded, 91 kb-long, moderately stable nuclear transcript and that its stability is important for bidirectional silencing of genes in the Kcnq1 domain. Kcnq1ot1 interacts with chromatin and with the H3K9- and H3K27-specific histone methyltransferases G9a and the PRC2 complex in a lineage-specific manner. This interaction correlates with the presence of extended regions of chromatin enriched with H3K9me3 and H3K27me3 in the Kcnq1 domain in placenta, whereas fetal liver lacks both chromatin interactions and heterochromatin structures. In addition, the Kcnq1 domain is more often found in contact with the nucleolar compartment in placenta than in liver. Taken together, our data describe a mechanism whereby Kcnq1ot1 establishes lineage-specific transcriptional silencing patterns through recruitment of chromatin remodeling complexes and maintenance of these patterns through subsequent cell divisions occurs via targeting the associated regions to the perinucleolar compartment.

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Uterine NK (uNK) cells are a prominent feature of the uterine mucosa and regulate placentation. NK cell activity is regulated by a balance of activating and inhibitory receptors, however the receptor repertoire of mouse uNK cells is unknown. We describe herein two distinct subsets of CD3(-)CD122(+) NK cells in the mouse uterus (comprising decidua and mesometrial lymphoid aggregate of pregnancy) at mid-gestation: a small subset indistinguishable from peripheral NK cells, and a larger subset that expresses NKp46 and Ly49 receptors, but not NK1.1 or DX5. This larger subset reacts with Dolichus biflores agglutinin, a marker of uNK cells in the mouse, and is adjacent to the invading trophoblast. By multiparametric analysis we show that the phenotype of uNK cells is unique and unprecedented in terms of adhesion, activation, and MHC binding potential. Thus, the Ly49 repertoire and the expression of other differentiation markers strikingly distinguish uNK cells from peripheral NK cells, suggesting that a selection process shapes the receptor repertoire of mouse uNK cells.

Since the discovery of T helper type 1 and type 2 effector T cell subsets 20 years ago, inducible regulatory T cells and interleukin 17 (IL-17)-producing T helper cells have been added to the 'portfolio' of helper T cells. It is unclear how many more effector T cell subsets there may be and to what degree their characteristics are fixed or flexible. Here we show that transforming growth factor-beta, a cytokine at the center of the differentiation of IL-17-producing T helper cells and inducible regulatory T cells, 'reprograms' T helper type 2 cells to lose their characteristic profile and switch to IL-9 secretion or, in combination with IL-4, drives the differentiation of 'T(H)-9' cells directly. Thus, transforming growth factor-beta constitutes a regulatory 'switch' that in combination with other cytokines can 'reprogram' effector T cell differentiation along different pathways.

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, long-range chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of alphabeta T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of alphabeta T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.

Several advances in recent years have focused increasing attention on the role of the RAF-MEK-ERK1/2 pathway in promoting cell survival. The demonstration that BRAF is a human oncogene mutated at high frequency in melanoma, thyroid and colon cancer has provided a pathophysiological context, whilst the description of potent and highly selective inhibitors of BRAF or MEK has allowed a more informed and rational intervention in both normal and tumour cells. In addition, separate studies have uncovered new mechanisms by which the ERK1/2 pathway can control the activity or abundance of members of the BCL-2 protein family to promote cell survival. It is now apparent that various oncogenes co-opt ERK1/2 signalling to de-regulate these BCL-2 proteins and this contributes to, and even underpins, survival signalling in some tumours. New oncogene-targeted therapies allow direct or indirect inhibition of ERK1/2 signalling and can cause quite striking tumour cell death. In other cases, inhibition of the ERK1/2 pathway may be more effective in combination with other conventional and novel therapeutics. Here, we review recent advances in our understanding of how the ERK1/2 pathway regulates BCL-2 proteins to promote survival, how this is de-regulated in tumour cells and the opportunities this might afford with the use of new targeted therapies.

Genomic data allow the large-scale manual or semi-automated assembly of metabolic network reconstructions, which provide highly curated organism-specific knowledge bases. Although several genome-scale network reconstructions describe Saccharomyces cerevisiae metabolism, they differ in scope and content, and use different terminologies to describe the same chemical entities. This makes comparisons between them difficult and underscores the desirability of a consolidated metabolic network that collects and formalizes the 'community knowledge' of yeast metabolism. We describe how we have produced a consensus metabolic network reconstruction for S. cerevisiae. In drafting it, we placed special emphasis on referencing molecules to persistent databases or using database-independent forms, such as SMILES or InChI strings, as this permits their chemical structure to be represented unambiguously and in a manner that permits automated reasoning. The reconstruction is readily available via a publicly accessible database and in the Systems Biology Markup Language (http://www.comp-sys-bio.org/yeastnet). It can be maintained as a resource that serves as a common denominator for studying the systems biology of yeast. Similar strategies should benefit communities studying genome-scale metabolic networks of other organisms.

In the cerebellar cortex, Bergmann glia enclose the synapses of both parallel and climbing fiber inputs to the Purkinje neuron. The glia express Ca(2+)-permeable AMPA receptors, and the GLAST and GLT-1 classes of glutamate transporter, which are activated by glutamate released during synaptic transmission. We have previously reported that parallel fiber to Bergmann glial transmission in rat cerebellar slices exhibits a form of frequency-dependent plasticity, namely long-term depression, following repetitive stimulation at 0.1-1 Hz. Here, we report that this form of plasticity is also present at the climbing fiber input, that climbing and parallel fibers can be depressed independently, that discrete parallel fiber inputs can also be depressed independently, and that depression is maintained when a distributed array of parallel fibers are stimulated (in contrast to several forms of synaptic plasticity at the Purkinje neuron). Depression of glutamate transporter currents does not correlate with a decrease in the stringency with which Purkinje neuron synapses are isolated. Rather, postsynaptic currents in Purkinje neurons decay more rapidly and perisynaptic metabotropic glutamate receptors are activated less effectively after stimulation at 0.2 and 1 Hz, suggesting that depression arises from a decrease in extrasynaptic glutamate concentration and not from impairment of glutamate clearance in and around the synapse. These results indicate that neuron-glial plasticity is activity dependent, input specific and does not require spillover between adjacent synapses to manifest. They also argue against a withdrawal of the glial sheath from synaptic regions as the putative mechanism of plasticity.

Mouse ES cells can differentiate into all three germ layers of the embryo but are generally excluded from the trophoblast lineage. Here we show that ES cells deficient in DNA methylation can differentiate efficiently into trophoblast derivatives. In a genome-wide screen we identified the transcription factor Elf5 as methylated and repressed in ES cells, and hypomethylated and expressed in TS and methylation-deficient ES cells. Elf5 creates a positive-feedback loop with the TS cell determinants Cdx2 and Eomes that is restricted to the trophoblast lineage by epigenetic regulation of Elf5. Importantly, the late-acting function of Elf5 allows initial plasticity and regulation in the early blastocyst. Thus, Elf5 functions as a gatekeeper, downstream of initial lineage determination, to reinforce commitment to the trophoblast lineage or to abort this pathway in epiblast cells. This epigenetic restriction of cell lineage fate provides a molecular mechanism for Waddington's concept of canalization of developmental pathways.

It has been shown previously that certain bacteria rapidly (3 h) up-regulated in vivo microfold cell (M cell)-mediated transport of Ag across the follicle-associated epithelium of intestinal Peyer's patch. Our aim was to determine whether soluble mediators secreted following host-bacteria interaction were involved in this event. A combination of proteomics and immunohistochemical analyses was used to identify molecules produced in the gut in response to bacterial challenge in vivo; their effects were then tested on human intestinal epithelial cells in vitro. Macrophage migration inhibitory factor (MIF) was the only cytokine produced rapidly after in vivo bacterial challenge by CD11c(+) cells located beneath the M cell-rich area of the follicle-associated epithelium of the Peyer's patch. Subsequently, in vitro experiments conducted using human Caco-2 cells showed that, within hours, MIF induced the appearance of cells that showed temperature-dependent transport of microparticles and M cell-specific bacterium Vibrio cholerae, and acquired biochemical features of M cells. Furthermore, using an established in vitro human M cell model, we showed that anti-MIF Ab blocked Raji B cell-mediated conversion of Caco-2 cells into Ag-sampling cells. Finally, we report that MIF(-/-) mice, in contrast to wild-type mice, failed to show increased M cell-mediated transport following in vivo bacterial challenge. These data show that MIF plays a role in M cell-mediated transport, and cross-talk between bacteria, gut epithelium, and immune system is instrumental in regulating key functions of the gut, including M cell-mediated Ag sampling.