Besides their commonly attributed role in the maintenance of low-copy number plasmids, toxin/antitoxin (TA) loci, also called 'addiction modules', have been found in chromosomes and associated to a number of biological functions such as: reduction of protein synthesis, gene regulation and retardation of cell growth under nutritional stress. The recent discovery of TA loci in obligatory intracellular species of the Rickettsia genus has prompted new research to establish whether they work as stress response elements or as addiction systems that might be toxic for the host cell. VapBC2 is a TA locus from R. felis, a pathogen responsible for flea-borne spotted fever in humans. The VapC2 toxin is a PIN-domain protein, whereas the antitoxin, VapB2, belongs to the family of swapped-hairpin β-barrel DNA-binding proteins. We have used a combination of biophysical and structural methods to characterize this new toxin/antitoxin pair. Our results show how VapB2 can block the VapC2 toxin. They provide a first structural description of the interaction between a swapped-hairpin β-barrel protein and DNA. Finally, these results suggest how the VapC2/VapB2 molar ratio can control the self-regulation of the TA locus transcription.

PubChem (http://pubchem.ncbi.nlm.nih.gov) is a public repository for biological activity data of small molecules and RNAi reagents. The mission of PubChem is to deliver free and easy access to all deposited data, and to provide intuitive data analysis tools. The PubChem BioAssay database currently contains 500,000 descriptions of assay protocols, covering 5000 protein targets, 30,000 gene targets and providing over 130 million bioactivity outcomes. PubChem's bioassay data are integrated into the NCBI Entrez information retrieval system, thus making PubChem data searchable and accessible by Entrez queries. Also, as a repository, PubChem constantly optimizes and develops its deposition system answering many demands of both high- and low-volume depositors. The PubChem information platform allows users to search, review and download bioassay description and data. The PubChem platform also enables researchers to collect, compare and analyze biological test results through web-based and programmatic tools. In this work, we provide an update for the PubChem BioAssay resource, including information content growth, data model extension and new developments of data submission, retrieval, analysis and download tools.

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.

The nuclear-encoded chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme controlling the malate valve, to allow the indirect export of reducing equivalents. Arabidopsis thaliana (L.) Heynh. T-DNA insertion mutants of NADP-MDH were used to assess the role of the light-activated NADP-MDH in a typical C(3) plant. Surprisingly, even when exposed to high-light conditions in short days, nadp-mdh knockout mutants were phenotypically indistinguishable from the wild type. The photosynthetic performance and typical antioxidative systems, such as the Beck-Halliwell-Asada pathway, were barely affected in the mutants in response to high-light treatment. The reactive oxygen species levels remained low, indicating the apparent absence of oxidative stress, in the mutants. Further analysis revealed a novel combination of compensatory mechanisms in order to maintain redox homeostasis in the nadp-mdh plants under high-light conditions, particularly an increase in the NTRC/2-Cys peroxiredoxin (Prx) system in chloroplasts. There were indications of adjustments in extra-chloroplastic components of photorespiration and proline levels, which all could dissipate excess reducing equivalents, sustain photosynthesis, and prevent photoinhibition in nadp-mdh knockout plants. Such metabolic flexibility suggests that the malate valve acts in concert with other NADPH-consuming reactions to maintain a balanced redox state during photosynthesis under high-light stress in wild-type plants.

Defense against oxidative stress is executed by an antioxidant program that is tightly controlled by the transcription factor Nrf2. The stability of Nrf2 involves the interaction of two degradation domains, designated Neh2 and Neh6, with the E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, respectively. The regulation of Nrf2 through the Neh6 degron remains largely unexplored but requires GSK-3 to form a phosphodegron. In this study, the cancer-chemopreventive agent nordihydroguaiaretic acid (NDGA) increased the level of Nrf2 protein and expression of heme oxygenase-1 (HO-1) in kidney-derived LLC-PK1 and HEK293T cells and in wild-type mouse embryo fibroblasts (MEFs). However, NDGA did not induce HO-1 in Nrf2(-/-) MEFs, indicating that Nrf2 is required for induction. The relevance of the Nrf2/HO-1 axis to antioxidant protection was further demonstrated by the finding that the HO-1 inhibitor stannous-mesoporphyrin abolished protection against hydrogen peroxide conferred by NDGA. NDGA increased Nrf2 and HO-1 protein levels in Keap1(-/-) MEFs, implying that Keap1-independent mechanisms regulate Nrf2 stability. Mutants of the Neh2 or Nrh6 domain and chimeric proteins comprising cyan fluorescent protein fused to Neh2 and green fluorescent protein fused to Neh6 exhibited longer half-lives in the presence of NDGA, demonstrating that NDGA targets both the Neh2 and the Neh6 degrons. In common with other chemopreventive agents, NDGA activated the ERK1/2, p38, JNK, and PI3K pathways. By using selective kinase inhibitors we found that PI3K, JNK, and p38 were responsible for the stabilization of Nrf2 and induction of HO-1 by NDGA. To explain how NDGA might up-regulate Nrf2 in a Keap1-independent manner we explored the participation of GSK-3β because it controls the Neh6 phosphodegron. Importantly, NDGA caused inhibitory phosphorylation of GSK-3β at Ser9 and at Thr390, and this was associated with a substantial reduction in Neh6 phosphorylation. Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3β as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-like family. In the present study, we investigated the regulatory mechanism of STK38 and assessed its role in the cellular stress response. Among various environmental stresses, STK38 was specifically activated by H(2)O(2), and the phosphatidylinositol 3-kinase inhibitor wortmannin or AKT inhibitor IV suppressed this activation. STK38 was also activated by a constitutively active AKT1 or by GSK-3β inhibitor VII. The phosphorylation level of GSK-3β was correlated with the STK38 activity, in response to various stimuli and in different cell lines. Co-immunoprecipitation analysis revealed that GSK-3β physically interacted with STK38 in cells. GSK-3β overexpression inhibited the H(2)O(2)-stimulated STK38 activity. GSK-3β phosphorylated STK38 on residues S6 and T7 in vitro, depending largely on a PKA-mediated priming phosphorylation of STK38 on residues S10 and S11, respectively. STK38's H(2)O(2)-stimulated activity was enhanced by alanine substitution at its priming sites and/or at S6 and T7, and it was partially reduced by a phosphomimetic mutation at S6 or T7. STK38 knockdown enhanced the H(2)O(2)-induced JNK phosphorylation and cell death. Our results indicate that that GSK-3β inhibits STK38's full activation, and suggest that STK38 activation is required to prevent cell death in response to oxidative stress.

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ∼50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ∼80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ∼3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ∼50% decrease was seen in the expression of terminal osteogenic gene expression and a ∼50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ∼2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.

Mutations in Gjb2 and Gjb6 genes, coding for connexin26 (Cx26) and Cx30 proteins, respectively, are linked to about half of all cases of human autosomal non-syndromic prelingual deafness. Molecular mechanisms of the hearing impairments, however, are unclear. Most cochlear gap junctions (GJs) are co-assembled from Cx26 and Cx30 and deletion of either one of them causes deafness. Our previous studies have shown that normal hearing is possible in the absence of the Cx30 gene when Cx26 is over-expressed. To further test unique functional requirements for various types of connexins in the hearing, we investigated whether the hearing in the conditional Cx26 (cCx26) null mice could be rescued by genetically over-expressing Cx30. Multiple lines of control and experimental mouse models were used. Auditory brainstem response (ABR) measurements showed normal hearing in targeted gene deletion mice when the deleted Cx26 or Cx30 was transgenically expressed from integrated bacterial artificial chromosome (BAC), demonstrating the effectiveness of the BAC rescue approach. In contrast, severe hearing loss was found in cCx26 null mice in which Cx30 was over-expressed. Morphology observations were consistent with the ABR data. Cochleae of cCx26 null mice with and without the transgenic over-expression of Cx30 both showed the typical immature feature of postnatal cochlear development-the closed tunnel of Corti. Immunolabeling data and Western blot quantification indicated that the Cx26 protein expression preceded that of Cx30 during the early postnatal period in the cochlea. Null expression of Cx26 may therefore uniquely result in a transient period when a total elimination of GJs in functionally-important regions of the developing cochlea is possible. We conclude that Cx26 plays an essential role in the development of the auditory sensory epithelium and its unique developmental functions required for normal hearing is not replaceable by Cx30.

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily afflicting women. The reason for the gender bias is unclear, but genetic susceptibility, estrogen and environmental agents appear to play significant roles in SLE pathogenesis. Environmental agents can contribute to lupus susceptibility through epigenetic mechanisms. We used (C57BL/6xSJL)F1 mice transgenic for a dominant-negative MEK (dnMEK) that was previously shown to be inducibly and selectively expressed in T cells. In this model, induction of the dnMEK by doxycycline treatment suppresses T cell ERK signaling, decreasing DNA-methyltransferase expression and resulting in DNA demethylation, overexpression of immune genes Itgal (CD11a) and Tnfsf7 (CD70), and anti-dsDNA antibody. To examine the role of gender and estrogen in this model, male and female transgenic mice were neutered and implanted with time-release pellets delivering placebo or estrogen. Doxycycline induced IgG anti-dsDNA antibodies in intact and neutered, placebo-treated control female but not male transgenic mice. Glomerular IgG deposits were also found in the kidneys of female but not male transgenic mice, and not in the absence of doxycycline. Estrogen enhanced anti-dsDNA IgG antibodies only in transgenic, ERK-impaired female mice. Decreased ERK activation also resulted in overexpression and demethylation of the X-linked methylation-sensitive gene CD40lg in female but not male mice, consistent with demethylation of the second X chromosome in the females. The results show that both estrogen and female gender contribute to the female predisposition in lupus susceptibility through hormonal and epigenetic X-chromosome effects and through suppression of ERK signaling by environmental agents.

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.

We investigated whether group-level bias of a 24-h recall estimate of protein and potassium intake, as compared to biomarkers, varied across European centers and whether this was influenced by characteristics of individuals or centers.

Tuberculosis (TB) is a major infectious disease problem: 1.7 million people annually die due to TB. Emergence of drug-resistant Mycobacterium tuberculosis and the lack of new antibiotics have exacerbated the situation. There is an urgent need to develop or repurpose drugs against TB. We evaluated inhaled gentamicin as direct respiratory system-targeted therapy in a murine model of TB. Aerosolized-gentamicin-treated mice showed significantly reduced lung M. tuberculosis loads and fewer granulomas relative to untreated controls. These results suggest that direct delivery of antibiotics to the respiratory system may provide therapeutic benefit to conventional treatment regimes for treatment of pulmonary TB.

Mass spectrometry (MS)-based quantification of highly homologous proteins in complex samples has proven difficult due to subtle sequence variations and the wide dynamic range of protein isoforms present. Herein, we report the use of reductive dimethylation on intact proteins to quantitatively compare protein isoform expression in the nucleus and cytoplasm of mesenchymal stem cells (MSC) and normal stroma. By coupling fixed-charge MS/MS scanning, high-resolution UPLC FT-MS data-dependent acquisition and MASCOT-based data mining, hydrogen/deuterium-labeled dimethyl-lysine peptides were simultaneously captured allowing the accurate comparison of 123 protein isoforms in parallel LC MS/MS runs. Thirty-four isoforms were identified that had expression levels specific to MSC. Where possible, proteomic analyses were verified by Western blotting and were demonstrated to be divergent from the level of gene transcription detected for certain proteins. Our analysis provides a protein isoform signature specific to MSC and demonstrates the suitability of dimethyl-lysine labeling on intact proteins for quantifying highly homologous proteins on a proteome-wide scale.

Abundant evidence suggests that indirect inhibitory modulation of glutamatergic transmission, via metabotropic glutamatergic receptors (mGluR), may induce neuroprotection. The present study was designed to determine whether the selective antagonist of mGluR1 (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM), showed neuroprotection against the kainate (KA)-induced excitotoxicity in vitro and in vivo. In in vitro studies on mouse primary cortical and hippocampal neuronal cultures, incubation with KA (150 μM) induced strong degeneration [measured as lactate dehydrogenase (LDH) efflux] and apoptosis (measured as caspase-3 activity). EMQMCM (0.1-100 μM) added 30 min to 6 h after KA, significantly attenuated the KA-induced LDH release and prevented the increase in caspase-3 activity in the cultures. Those effects were dose- and time-dependent. In in vivo studies KA (2.5 nmol/1 μl) was unilaterally injected into the rat dorsal CA1 hippocampal region. Degeneration was calculated by counting surviving neurons in the CA pyramidal layer using stereological methods. It was found that EMQMCM (5-10 nmol/1 μl) injected into the dorsal hippocampus 30 min, 1 h, or 3 h (the higher dose only) after KA significantly prevented the KA-induced neuronal degeneration. In vivo microdialysis studies in rat hippocampus showed that EMQMCM (100 μM) significantly increased γ-aminobutyric acid (GABA) and decreased glutamate release. When perfused simultaneously with KA, EMQMCM substantially increased GABA release and prevented the KA-induced glutamate release. The obtained results indicate that the mGluR1 antagonist, EMQMCM, may exert neuroprotection against excitotoxicity after delayed treatment (30 min to 6 h). The role of enhanced GABAergic transmission in the neuroprotection is postulated.

GenBank® is a comprehensive database that contains publicly available nucleotide sequences for more than 250,00 formally described species. These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole-genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through the NCBI Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI home page: www.ncbi.nlm.nih.gov.

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ES(I) (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ES(I) causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.

Endosulfan is an organochlorine insecticide and has been implicated in neurotoxicity, hepatotoxicity, immunosuppression and teratogenicity. However, the molecular mechanism of endosulfan toxicity is not yet clear. Recent studies demonstrated that oxidative stress induced by endosulfan is involved in its toxicity and accumulating evidence suggests that endosulfan can modulate the activities of stress-responsive signal transduction pathways including extracellular signal regulated kinases (ERK) 1/2. However, none of the previous studies investigated the ability of endosulfan to modulate activating protein-1 (AP-1) binding and antioxidant response element (ARE)-mediated transcription as an underlying mechanism of endosulfan toxicity. In this report, we show that treatment of HepG2 cells with endosulfan significantly increased oxidative stress-responsive transcription via AP-1 activation. In addition, endosulfan-induced transcription was enhanced in cells depleted of glutathione by buthionine sulfoximine (BSO) treatment. Exposure to endosulfan resulted in a significant increase in the activities of MAPKs, ERK1/2 and p38. Endosulfan-induced increases in enzymatic activities of these MAPKs were consistent with MAPK phosphorylation. Endosulfan exposure also caused an increase in c-Jun phosphorylation. These results suggest a model for endosulfan toxicity in which endosulfan increases ERK1/2 and p38 activities and these activated MAPKs then increase c-Jun phosphorylation. Phosphorylated c-Jun, in turn, increases AP-1 activity, which results in activation of ARE-mediated transcription.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7-81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23-57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Dendritic cells (DC) take up pathogens through phagocytosis and process them into protein and lipid fragments for presentation to T cells. So far, the proteome of the human DC phagosome, a detrimental compartment for antigen processing and presentation as well as for DC activation, remains largely uncharacterized. Here we have analyzed the protein composition of phagosomes from human monocyte-derived DC. For LC-MS/MS analysis we purified phagosomes from DC using latex beads targeted to DC-SIGN, and quantified proteins using a label-free method. We used organellar enrichment ranking (OER) to select proteins with a high potential to be relevant for phagosome function. The method compares phagosome protein abundance with protein abundance in whole DC. Phagosome enrichment indicates specific recruitment to the phagosome rather than co-purification or passive incorporation. Using OER we extracted the most enriched proteins that we further complemented with functionally associated proteins to define a set of 90 phagosomal proteins that included many proteins with established relevance on DC phagosomes as well as high potential novel candidates. We already experimentally confirmed phagosomal recruitment of Galectin-9, which has not been previously associated with phagocytosis, to both bead and pathogen containing phagosomes, suggesting a role for Galectin-9 in DC phagocytosis.

Research on the neural bases of learning has mainly focused on reinforcement learning where the central role of the dopaminergic system is well established. However, in everyday life many decisions are not followed by feedback, in which case humans have been shown to code the most probable outcome into memory. We used functional magnetic resonance imaging (fMRI) to examine the neural basis of internally generated signals on correctness and decision confidence in the complete absence of feedback in a categorization task. During test trials after observational training activation in dopaminergic target regions was modulated by the correctness of the answer similarly as during feedback-based training. Moreover, activation in the nucleus accumbens and putamen was correlated with the prediction error on confidence as estimated by a reinforcement learning model. In this model subjective confidence ratings acquired after each trial served as outcome measure. Activation in the striatum therefore follows a similar pattern in response to prediction errors on confidence as it does during reinforcement learning in response to reward prediction errors, but with respect to internally generated signals based on knowledge of the structure of the environment. Furthermore, ventral striatal activation decreased with stimulus novelty, which might support the allocation of attention to unfamiliar stimuli. These results provide a parsimonious account for the neural bases of learning, indicating overlapping neural substrates of reinforcement learning and learning when outcome information has to be internally constructed.