Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell-autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b)-ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1)-synoviolin and ataxin 3, and a related sequence in M(r) 78,000 glycoprotein-Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP-Cdc48p. However, the differing molecular architecture of VCP-Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms.

Several epidemiological studies have shown an association between the season in which certain cancers are diagnosed and survival, with diagnosis in summer and autumn being associated with better survival. In this study, we have added resolution to the analysis of seasonality in cancer survival by considering mortality within several nonoverlapping time periods following diagnosis, thereby quantifying the separate contributions of mechanisms operating in the short term and in the longer term. We found evidence of seasonality acting on mortality within 2 distinct periods following diagnosis. Diagnosis in the summer was associated with substantially decreased mortality within the first month of diagnosis compared with winter in men with prostate cancer, those of both sexes with colorectal or lung cancer, and most strikingly, amongst women with breast cancer (hazard ratio 0.81 [95% confidence interval 0.75-0.86]). Adjusting for monthly variations in general mortality greatly attenuated the seasonal effects on short-term mortality. At long-term follow-up (>5 years), there was a consistent shift in the seasonality pattern, with autumn diagnosis alone being associated with decreased mortality, both in female breast cancer cases and in lung cancer cases of both sexes. We conclude that the higher survival observed amongst patients diagnosed in summer and autumn is predominantly a short-term phenomenon that is largely attributable to generally higher mortality in winter. However, the distinct mortality reduction observed in the long term amongst those diagnosed in the autumn, especially amongst breast cancer patients, may indicate the presence of a seasonally variable protective mechanism.

Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld(S)) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld(S) acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld(S) significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld(S) mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld(S) for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld(S) redistribution. Cytoplasmic Wld(S) fractionated predominantly with mitochondria and microsomes. We conclude that Wld(S) can act in one or more non-nuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.

Autophosphorylation of the activation loop in cis is an underappreciated and poorly understood mode of activation of protein kinases. Here, I describe four examples of protein kinases that are activated in this way, concentrating on their biochemical properties and how their autophosphorylation in cis is achieved.

There is increasing evidence for specialised processing of social cues in the brain. This review considers how the main olfactory system of mammals is designed to process social odours and the effects of learning in a social context. It focuses mainly on extensive research carried out on offspring, mate or conspecific learning carried out in sheep and rodents. Detailing the roles of the olfactory bulb and its projections, classical neurotransmitters, nitric oxide, oestrogen and neuropeptides such as oxytocin and vasopressin in mediating plasticity changes in the olfactory system arising from these different social learning contexts. The relative simplicity of the organisation of the olfactory system, the speed and robustness of these forms of social learning together with the similarity in brain regions and neurochemical contributions across the different learning paradigms make them important and useful models for investigating general principles of learning and memory in the brain.

Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.

Active DNA demethylation underlies key facets of reproduction in flowering plants and mammals and serves a general genome housekeeping function in plants. A family of 5-methylcytosine DNA glycosylases catalyzes plant demethylation via the well-known DNA base-excision-repair process. Although the existence of active demethylation has been known for a longer time in mammals, the means of achieving it remain murky and mammals lack counterparts to the plant demethylases. Several intriguing experiments have indicated, but not conclusively proven, that DNA repair is also a plausible mechanism for animal demethylation. Here, we examine what is known from flowering plants about the pathways and function of enzymatic demethylation and discuss possible mechanisms whereby DNA repair might also underlie global demethylation in mammals.

Genomic imprinting requires the differential marking by DNA methylation of genes in male and female gametes. In the female germline, acquisition of methylation imprint marks depends upon the de novo methyltransferase Dnmt3a and its cofactor Dnmt3L, but the reasons why specific sequences are targets for Dnmt3a and Dnmt3L are still poorly understood. Here, we investigate the role of transcription in establishing maternal germline methylation marks. We show that at the Gnas locus, truncating transcripts from the furthest upstream Nesp promoter disrupts oocyte-derived methylation of the differentially methylated regions (DMRs). Transcription through DMRs in oocytes is not restricted to this locus but occurs across the prospective DMRs at many other maternally marked imprinted domains, suggesting a common requirement for transcription events. The transcripts implicated here in gametic methylation are protein-coding, in contrast to the noncoding antisense transcripts involved in the monoallelic silencing of imprinted genes in somatic tissues, although they often initiate from alternative promoters in oocytes. We propose that transcription is a third essential component of the de novo methylation system, which includes optimal CpG spacing and histone modifications, and may be required to create or maintain open chromatin domains to allow the methylation complex access to its preferred targets.

The POU family transcription factor Brn-3a is required for the differentiation and survival of sensory neurones, and is phosphorylated in neuroblastoma cells following treatment with all-trans retinoic acid (RA). Mutation of serines-121 and -122 of Brn-3a to alanine blocks its phosphorylation and impairs RA-mediated neurite outgrowth. Here we show that this deficit in differentiation is mimicked by a single mutation at serine-122, and demonstrate a similar requirement for a second residue, threonine-39. Like Brn-3a, the neuropeptide Galanin has been implicated in the development of sensory neurones. We show that Brn-3a over-expression acts synergistically with RA treatment to up-regulate Galanin promoter activity; that the activity of the N-terminal transcriptional activation domain of Brn-3a is increased following RA treatment; and that both these effects require threonine-39 and serine-122. In addition, we demonstrate that the RA-mediated activation of Galanin promoter activity and Brn-3a N-terminal transcriptional activity are both blocked by pan-MEK inhibitors, and show that the expression of a constitutively-active mutant of MEK1, but not MEK5, is sufficient to increase Brn-3a activity. These results reveal an important role for the ERK1/2 pathway in Brn-3a regulation during RA-mediated neuronal differentiation and define the neuropeptide Galanin as a novel target of this transcription factor.

The molecular association between APP and its mammalian homologs has hardly been explored. In systematically addressing this issue, we show by live cell imaging that APLP1 mainly localizes to the cell surface, whereas APP and APLP2 are mostly found in intracellular compartments. Homo- and heterotypic cis interactions of APP family members could be detected by FRET and co-immunoprecipitation analysis and occur in a modular mode. Only APLP1 formed trans interactions, supporting the argument for a putative specific role of APLP1 in cell adhesion. Deletion mutants of APP family members revealed two highly conserved regions as important for the protein crosstalk. In particular, the N-terminal half of the ectodomain was crucial for APP and APLP2 interactions. By contrast, multimerization of APLP1 was only partially dependent on this domain but strongly on the C-terminal half of the ectodomain. We further observed that coexpression of APP with APLP1 or APLP2 leads to diminished generation of Abeta42. The current data suggest that this is due to the formation of heteromeric complexes, opening the way for novel therapeutic strategies targeting these complexes.

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.

Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

The myeloproliferative disorders are clonal disorders with frequent somatic gain-of-function alterations affecting tyrosine kinases. In these diseases, there is an increase in DNA damage and a risk of progression to acute leukemia. The molecular mechanisms in myeloproliferative disorders that prevent apoptosis induced by damaged DNA are obscure.

In 2005, the International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a "Comprehensive Classification System for Lipids" based on well-defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable. This classification system, which is compatible with contemporary databasing and informatics needs, has now been accepted internationally and widely adopted. In response to considerable attention and requests from lipid researchers from around the globe and in a variety of fields, the comprehensive classification system has undergone significant revisions over the last few years to more fully represent lipid structures from a wider variety of sources and to provide additional levels of detail as necessary. The details of this classification system are reviewed and updated and are presented here, along with revisions to its suggested nomenclature and structure-drawing recommendations for lipids.

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.