We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110delta leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110delta. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110delta mutant mice, indicating a critical role for the p110delta isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110delta mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110delta double mutant B cells. These results demonstrate an epistatic relationship between p110delta and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.

Nascent transcription occurs at nuclear foci of concentrated, hyperphosphorylated RNA polymerase II (RNAPII). We investigate RNAPII localization, distal gene co-association, and Hbb locus conformation during inhibition of transcription. Our results show distal active genes remain associated with RNAPII foci and each other in the absence of elongation. When initiation is inhibited, active genes dissociate from RNAPII foci and each other, suggesting initiation is necessary to tether distal active genes to shared foci. In the absence of transcription RNAPII foci remain, indicating they are not simple accumulations of RNAPII on transcribed genes but exist as independent nuclear subcompartments.

Stem cells that can be derived from fetal membranes represent an exciting field of research that bears tremendous potential for developmental biology and regenerative medicine. In this report we summarize contributions to a workshop in which newest insights into the characteristics, subtypes and molecular determinants of stem cells from trophoblast and endometrial tissues were presented.

Null mutations that cripple T cell receptor (TCR) signaling explain rare primary immunodeficiencies, but it is not understood why more common polymorphisms that lead to subtle TCR signaling defects are paradoxically associated with autoimmunity. Here we analyzed how a series of Zap70 variants with step-wise decreases in TCR signaling impacted upon opposing TCR functions of immunity and tolerance. One Zap70 variant, murdock, moderately decreased TCR signaling and thymic selection without compromising immunological tolerance, whereas a more severe Zap70 defect, mrtless, abolished thymic-positive selection and led to immunodeficiency. Signaling capacities between these two thresholds disproportionately compromised negative selection and Foxp3(+) regulatory T cell formation, creating a cellular imbalance between immunogenic and tolerogenic functions that resulted in the excessive production of autoantibodies and immunoglobulin E (IgE). The pleiotropic functions of ZAP-70 and their differential response to graded variation provide a paradigm for understanding the complex outcomes of human genetic variation.

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

Extraembryonic development in rodents depends on the differentiation and function of trophoblast giant cells. Morphologically striking, giant cells exhibit many extraordinary characteristics adapted to ensure the success of pregnancy. This review summarizes some of the intriguing aspects of giant cell morphology and function. Giant cells are highly polyploid as a result of a switch from a mitotic to an endoreduplicative cell cycle. They further partition their genome content into various fragments which may represent a mechanism to maximize protein synthesis. Similar to metastatic tumour cells, they breach basement membranes and invade deeply into a foreign tissue, the maternal decidualized uterine stroma. Their angiogenic and vasodilatory properties, combined with the ability to remodel arterial walls, enable them to redirect maternal blood flow towards the implantation site. Recent advances have recognized that the giant cell population is more diverse than previously recognized and future studies will have to show how these subtypes differ functionally and how their differentiation is controlled.

The Minimal Information Requested In the Annotation of biochemical Models (MIRIAM) is a set of guidelines for the annotation and curation processes of computational models, in order to facilitate their exchange and reuse. An important part of the standard consists in the controlled annotation of model components, based on Uniform Resource Identifiers. In order to enable interoperability of this annotation, the community has to agree on a set of standard URIs, corresponding to recognised data types. MIRIAM Resources are being developed to support the use of those URIs.

microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.

The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles in preparation for neurotransmission. Samples of cerebellum, cortex, hippocampus, substantia nigra and striatum from VMAT2-deficient mice were compared to age-matched control mice. Multivariate statistical analyses of (1)H NMR spectral profiles separated VMAT2-deficient mice from controls for all five brain regions. Although the data show that metabolic alterations are region- and age-specific, in general, analyses indicated decreases in the concentrations of taurine and creatine/phosphocreatine and increases in glutamate and N-acetyl aspartate in VMAT2-deficient mouse brain tissues. This study demonstrates the efficacy of metabolomics as a functional genomics phenotyping tool for mouse models of neurological disorders, and indicates that mild reductions in the expression of VMAT2 affect normal brain metabolism.

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.

While the cytokine IL-17 has been cloned and described more than 10 years ago [Yao Z, Fanslow WC, Seldin MF, Rousseau AM, Painter SL, Comeau MR, et al. Herpesvirus Saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor. Immunity 1995;3(6):811-21; Kennedy J, Rossi DL, Zurawski SM, Vega Jr F, Kastelein RA, Wagner JL, et al. Mouse IL-17: a cytokine preferentially expressed by alpha beta TCR+CD4-CD8-T cells. J Interferon Cytokine Res 1996;16(8):611-7], it was only 2 years ago that IL-17 producing T cells have been classified as a new distinct CD4 T cell subset [Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 2005;6(11):1123-32] and only in 2006 the molecular mechanisms underlying their differentiation were identified [Veldhoen M, Hocking RJ, Atkins CJ, Locksley RM, Stockinger B. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 2006;24(2):179-89; Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441(7090):235-8; Mangan PR, Harrington LE, O'Quinn DB, Helms WS, Bullard DC, Elson CO, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 2006;441(7090):231-4]. Since then the literature on IL-17 producing cells has grown steadily and many reviews of the field are already outdated by the time they are published, a fate that no doubt will affect this review as well. In order to avoid too many repetitions we focus this review mainly on publications in 2006 and 2007 and refer to a number of reviews, which cover earlier aspects of Th17/IL-17 biology.

Trf4 is the poly(A) polymerase component of TRAMP4, which stimulates nuclear RNA degradation by the exosome. We report that in Saccharomyces cerevisiae strains lacking Trf4, cryptic transcripts are detected from regions of repressed chromatin at telomeres and the rDNA intergenic spacer region (IGS1-R), and at CEN3. Degradation of the IGS1-R transcript was reduced in strains lacking TRAMP components, the core exosome protein Mtr3 or the nuclear-specific exosome component Rrp6. IGS1-R has potential binding sites for the RNA-binding proteins Nrd1/Nab3, and was stabilized by mutation of Nrd1. IGS1-R passes through the replication fork barrier, a region required for rDNA copy number control. Strains lacking Trf4 showed sporadic changes in rDNA copy number, whereas loss of both Trf4 and either the histone deacetylase Sir2 or the topoisomerase Top1 caused dramatic loss of rDNA repeats. Chromatin immunoprecipitation analyses showed that Trf4 is co-transcriptionally recruited to IGS1-R, consistent with a direct role in rDNA stability. Co-transcriptional RNA binding by Trf4 may link RNA and DNA metabolism and direct immediate IGS1-R degradation by the exosome following transcription termination.

Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigen-presenting cells (APCs). The relevant PI3K catalytic isoform and relative contribution of the TcR and CD28 to PI3K activity at the immune synapse have not been determined unequivocally. Using a quantitative imaging-based assay, we show that the PI3K activity at the T cell-APC contact area is dependent on the p110delta, but not the p110gamma, isoform of PI3K. CD28 enhanced PIP3 production at the T-cell synapse independently of its YMNM PI3K-recruitment motif that instead was required for efficient PKC recruitment. CD28 could partially compensate for the lack of p110delta activity during T-cell activation, which indicates that CD28 and p110delta act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110delta double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110delta activity and CD28 costimulation has potent therapeutic potential.

Protein homeostasis maintains proper intracellular balance by promoting protein folding and clearance mechanisms while minimizing the stress caused by the accumulation of misfolded and damaged proteins. Chronic expression of aggregation-prone proteins is deleterious to the cell and has been linked to a wide range of conformational disorders. The molecular response to misfolded proteins is highly conserved and generally studied as a cell-autonomous process. Here, we provide evidence that neuronal signaling is an important modulator of protein homeostasis in post-synaptic muscle cells. In a forward genetic screen in Caenorhabditis elegans for enhancers of polyglutamine aggregation in muscle cells, we identified unc-30, a neuron-specific transcription factor that regulates the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We used additional sensors of protein conformational states to show that defective GABA signaling or increased acetylcholine (ACh) signaling causes a general imbalance in protein homeostasis in post-synaptic muscle cells. Moreover, exposure to GABA antagonists or ACh agonists has a similar effect, which reveals that toxins that act at the neuromuscular junction are potent modifiers of protein conformational disorders. These results demonstrate the importance of intercellular communication in intracellular homeostasis.

The nucleus is a highly heterogeneous structure, containing various 'landmarks' such as the nuclear envelope and regions of euchromatin or dense heterochromatin. At a morphological level, regions of the genome that are permissive or repressive to gene expression have been associated with these architectural features. However, gene position within the nucleus can be both a cause and a consequence of transcriptional regulation. New results indicate that the spatial distribution of genes within the nucleus contributes to transcriptional control. In some cases, position seems to ensure maximal expression of a gene. In others, it ensures a heritable state of repression or correlates with a developmentally determined program of tissue-specific gene expression. In this review, we highlight mechanistic links between gene position, repression and transcription. Recent findings suggest that architectural features have multiple functions that depend upon organization into dedicated subcompartments enriched for distinct enzymatic machinery.

Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).

Biobanks are well-organized resources comprising biological samples and associated information that are accessible to scientific investigation. Across Europe, millions of samples with related data are held in different types of collections. While individual collections can be well organized and accessible, the resources are subject to fragmentation, insecurity of funding and incompleteness. To address these issues, a Biobanking and BioMolecular Resources Infrastructure (BBMRI) is to be developed across Europe, thereby implementing a European 'roadmap' for research infrastructures that was developed by a forum of EU member states and that has been received by the European Commission. In this review, we describe the work involved in preparing for the construction of BBMRI in a European and global context.

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Ca(2+) increases in the heart control both contraction and transcription. To accommodate a short-term increased cardiovascular demand, neurohormonal modulators acting on the cardiac pacemaker and individual myocytes induce an increase in frequency and magnitude of myocyte contraction respectively. Prolonged, enhanced function results in hypertrophic growth of the heart, which is initially also associated with greater Ca(2+) signals and cardiac contraction. As a result of disease, however, hypertrophy progresses to a decompensated state and Ca(2+) signalling capacity and cardiac output are reduced. Here, the role that Ca(2+) plays in the induction of hypertrophy as well as the impact that cardiac hypertrophy and failure has on Ca(2+) fluxes will be discussed.

This chapter outlines methods that can be applied to determine the levels of lipids in cells and tissues. In particular, the methods focus upon the extraction and analysis of those lipids critical for monitoring signal transduction pathways. The methods address the analysis of the phosphoinositides, the lipid agonists lysophosphatidic acid and sphingosine 1-phosphate, and the neutral lipid messengers diacylglycerol and ceramide. Additionally, because of the increasing need to determine the dynamics of signaling, the analysis of phospholipids synthesis using stable isotope methods is described. The use of these methods as described or adaptation to permit both approaches should allow investigators to determine changes in signaling lipids and to better understand such processes in most cell types. The increasing appreciation of the central roles played by lipid signaling pathways has dispelled the misconception that lipids are inert structural components that are involved solely in keeping a cell intact. Advances in our understanding of cell-signaling pathways have identified particular lipids that act to regulate the functions of a number of proteins either by controlling enzyme activity directly, or by localizing proteins to particular intracellular compartments where they perform a specialized role. These lipid-binding domains (e.g., PH, PX, FYVE) have been found in many proteins, and considerable detail is recorded of the structural basis of lipid protein interaction. Additional lipid-binding domains exist, which remain less well characterized (e.g., those that bind phosphatidic acid [PA] or ceramide); however, the important regulatory roles that these lipids play and the pathways involving these messengers are increasingly appreciated. While the downstream targets are thus being defined, the actual changes in lipid concentration in a stimulated cell or membrane are less characterized. The primary reason for this lack has been a deficiency in methodology. Much of the reported studies of lipid messengers in stimulated cells have depended upon monitoring changes in radio-labeled cells. Many well-documented problems are associated with this type of methodology, including lack of isotopic equilibrium, distinct pools with different turnover rates, and inadequate separation of radio-labeled metabolites; however, much important information has been generated. The second approach has been to make use of the lipid-binding properties of the target protein domains and to generate a tagged fusion protein, generally GFP, which permits identification of a region rich in a signaling lipid (Guillou et al., 2007). This has proved useful in monitoring PI-3-kinase activation in stimulated cells; however, considerable caveats must be raised, not least the problems associated with lipid specificity and the fact that many of these domains have associated protein-binding regions that can compromise the findings. A further problem associated with these two methodologies is that they tend to group lipids together and take no account of the multiple acyl chain structures that occur in all lipids. These concerns point to the need to determine actual changes in lipid compositions. Until relatively recently, such an analysis was unachievable; however, advances in both chromatographic separation and mass spectrometry (MS) have permitted the development of lipidomic analysis. This chapter outlines a number of methods that allow determination of changes in signaling lipids. Adaptation of the methods here for the analysis of other molecules should be relatively straightforward in the future. Much of the lipidomic research in the United Kingdom is focused upon signaling lipidomics, with particular foci upon phosphoinositide-related signaling in Birmingham and Cambridge (Wakelam) and London (Larijani), upon eicosanoids in Cardiff (O'Donnell), and steroids in London (Griffiths). Meanwhile, the use of stable isotopes has been particularly developed in Southampton (Postle).