Ammonia-oxidizing bacteria (AOB) remove intracellular nitrite to prevent its toxicity by a nitrifier denitrification pathway involving two denitrifying enzymes, nitrite reductase and nitric oxide reductase. Here, a Cu-containing nitrite reductase from Nitrosococcus oceani strain NS58, a gammaproteobacterial marine AOB, was expressed in Escherichia coli and purified to homogeneity. Sequence homology analysis indicated that the nitrite reductase from N. oceani was phylogenetically closer to its counterparts from denitrifying bacteria than that of the betaproteobacterium Nitrosomonas europaea. The recombinant enzyme was a homotrimer of a 32 kDa subunit molecule. The enzyme was green in the oxidized state with absorption peaks at 455 nm and 575 nm. EPR spectroscopy indicated the presence of type 2 Cu. Molecular activities and the affinity constant for the nitrite were determined to be 1.6×10(3) s(-1) and 52 μM, respectively.

Vancomycin-resistant Enterococcus faecalis, E. faecium and E. durans isolates with the genotype vanA were detected in 7 of 118 faecal samples (5.9%) of natural gilthead seabream recovered off the coast of Portugal, and one vancomycin-resistant isolate/sample was further characterized. The genes erm(B), tet(L), tet(M), aac(6')-aph(2"), aph(3')-IIIa and/or ant(6)-Ia were identified in most of the 7 vancomycin-resistant enterococci. Sequence types ST273, ST313 and ST76 were detected in three E. faecium isolates and ST6 in two E. faecalis isolates. VanA-containing enterococci are suggested to be disseminated in fish in marine ecosystems close to areas of human activity.

Nitric oxide (NO) plays a role in defence against hemibiotrophic pathogens mediated by salicylate (SA) and also necrotrophic pathogens influenced by jasmonate/ethylene (JA/Et). This study examined how NO-oxidizing haemoglobins (Hb) encoded by GLB1, GLB2, and GLB3 in Arabidopsis could influence both defence pathways. The impact of Hb on responses to the hemibiotrophic Pseudomonas syringae pathovar tomato (Pst) AvrRpm1 and the necrotrophic Botrytis cinerea were investigated using glb1, glb2, and glb3 mutant lines and also CaMV 35S GLB1 and GLB2 overexpression lines. In glb1, but not glb2 and glb3, increased resistance was observed to both pathogens but was compromised in the 35S-GLB1. A quantum cascade laser-based sensor measured elevated NO production in glb1 infected with Pst AvrRpm1 and B. cinerea, which was reduced in 35S-GLB1 compared to Col-0. SA accumulation was increased in glb1 and reduced in 35S-GLB1 compared to controls following attack by Pst AvrRpm1. Similarly, JA and Et levels were increased in glb1 but decreased in 35S-GLB1 in response to attack by B. cinerea. Quantitative PCR assays indicated reduced GLB1 expression during challenge with either pathogen, thus this may elevate NO concentration and promote a wide-ranging defence against pathogens.

NMDA receptors are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system. Recombinant GluN1-1a/GluN2D receptors are characterized by low channel open probability and prolonged deactivation time course following the removal of agonist. Here, we show that the deactivation time course, agonist potency, and single channel properties of GluN2D-containing NMDA receptors are modulated by alternative RNA splicing of GluN1. Our results demonstrate that GluN1 exon 5, which encodes a 21-amino-acid insert in the amino-terminal domain, is a key determinant of GluN1/GluN2D receptor function. GluN1-1b/GluN2D receptors, which contain the residues encoded by exon 5, deactivate with a dual exponential time course described by a τFAST of 410 ms and a τSLOW of 1100 ms. This time course is 3-fold more rapid than that for exon 5-lacking GluN1-1a/GluN2D, which deactivates with a τFAST of 1100 ms and a τSLOW of 3400 ms. Exon 5-containing NMDA receptors also have a two-fold higher open probability (0.037) than exon 5-lacking receptors (0.017). Furthermore, inclusion of exon 5-encoded residues within the GluN1-1b subunit decreases the potency for the endogenous agonist l-glutamate. Evaluation of receptor kinetics for NMDA receptors containing mutated GluN1-1b subunits and wild-type GluN2D identified residue Lys211 in GluN1-1b as a key determinant of exon 5 control of the deactivation time course and glutamate potency. Evaluation of a kinetic model of GluN1/GluN2D gating suggests that residues encoded by exon 5 influence several rate-limiting steps. These data demonstrate that the GluN1 subunit is a key determinant of the kinetic and pharmacological properties of GluN2D-containing NMDA receptors.

Although there is evidence linking hematopoietic chimerism induction and solid organ transplant tolerance, the mechanistic requirements for chimerism-induced tolerance are not clearly elucidated. To address this, we used an MHC-defined primate model to determine the impact of impermanent, T cell-poor, mixed-chimerism on renal allograft survival. We compared two cohorts: one receiving a bone marrow and renal transplant ("BMT/renal") and one receiving only a renal transplant. Both cohorts received maintenance immunosuppression with CD28/CD40-directed costimulation blockade and sirolimus. As previously demonstrated, this transplant strategy consistently induced compartmentalized donor chimerism, (significant whole-blood chimerism, lacking T cell chimerism). This chimerism was not sufficient to prolong renal allograft acceptance: the BMT/renal mean survival time (MST, 76 days) was not significantly different than the renal transplant alone MST (85 days, p = 0.46), with histopathology documenting T cell mediated rejection. Flow cytometric analysis revealed significant enrichment for CD28-/CD95+ CD4+ and CD8+ Tem cells in the rejected kidney, suggesting a link between CD28-negative Tem and costimulation blockade-resistant rejection. These results suggest that in some settings, transient T cell-poor chimerism is not sufficient to induce tolerance to a concurrently placed renal allograft and that the presence of this chimerism per se is not an independent biomarker to identify tolerance.

Blockade of angiotensin II type 1 (AT1) receptors ameliorates brain inflammation, and reduces excessive brain interleukin-1 beta (IL-1β) production and release from cortical microglia. The aim of this study was to determine whether, in addition, AT1 receptor blockade directly attenuates IL-1β-induced inflammatory responses in neuronal cultures.

Polyunsaturated fatty acids (PUFA) have diverse biological effects, from promoting inflammation to preventing cancer and heart disease. Growing evidence suggests that individual PUFA may have independent effects in health and disease. The individual roles of the two essential PUFA, linoleic acid (LA) and α-linolenic acid (ALA), have been difficult to discern from the actions of their highly unsaturated fatty acid (HUFA) downstream metabolites. This issue has recently been addressed through the development of the Δ-6 desaturase knock out (D6KO) mouse, which lacks the rate limiting Δ-6 desaturase enzyme and therefore cannot metabolize LA or ALA. However, a potential confounder in this model is the production of novel Δ-5 desaturase (D5D) derived fatty acids when D6KO mice are fed diets containing LA and ALA, but void of arachidonic acid.

The suppressive role of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) complex-derived reactive oxygen species (ROS) in adaptive immunity-driven arthritis models is well established. In this study, we aimed to investigate the role of NOX2 complex-derived ROS in a model of innate immunity-driven arthritis and to identify the ROS-regulated innate receptors that control arthritis. We used collagen antibody-induced arthritis (CAIA), which is a T and B lymphocyte-independent model of the effector phase of arthritis and is induced by well-defined monoclonal arthritogenic antibodies and enhanced by injection of lipopolysaccharide (LPS). CAIA was induced in both wild-type and Ncf1 mutant mice that lack phagocyte oxidative burst, and stimulated with LPS and other agents to activate innate immune responses. We found that both LPS and lipomannan enhanced CAIA more potently in the presence of functional phagocyte ROS production than in its absence. The ROS-dependent enhancement of CAIA was regulated by TLR2, but not by TLR4 stimulation, and was driven by granulocytes, whereas macrophages did not contribute to the phenotype. In addition, we report that collagen-induced arthritis was not affected by the functionality of the TLR4. We report that TLR2 signaling as an important ROS-regulated proinflammatory pathway leads to severe neutrophil-dependent inflammation in murine CAIA and conclude that the TLR2 pathway is modulated by phagocyte ROS to stimulate the development of arthritis.

Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line.

Astrocyte elevated gene 1 (AEG-1), an important oncogene, has been shown to be overexpressed in several types of cancers. In colorectal cancer (CRC), the protein level of AEG-1 is up-regulated in tumour tissue compared to normal mucosa, showing prognostic significance. Since little is known about the transcriptional level of AEG-1 expression and its biological pathway in CRC the aim of the present study was to examine the relationship of AEG-1 mRNA expression, the protein level and clinicopathological variables as well as its biology pathway in CRC.

Extracellular single unit activity was recorded from medial prefrontal cortex (mPFC) of postpartum dams over the course of 3 days while they engaged in spontaneous pup-directed behaviors and non-specific exploratory behavior. Out of 109 units identified over the course of the experiment, 15 units were observed to be pup-responsive and 15 increased their discharge rates non-specifically while not attending to pups. An association between neuronal activity and typical maternal behaviors (e.g., retrieval, pup-grooming, nursing) was not observed. Instead, brief bouts of snout contact with pups were accompanied by phasic increases and decreases in spike rates. The observed pup contact responsive cells might play a role in processing of sensory feedback from pups or the transmission of modulatory output to other subcortical maternal brain areas.

Peroxisomes undergo rapid, selective autophagic degradation (pexophagy) when the metabolic pathways they contain are no longer required for cellular metabolism. Pex3 is central to the formation of peroxisomes and their segregation because it recruits factors specific for these functions. Here, we describe a novel Saccharomyces cerevisiae protein that interacts with Pex3 at the peroxisomal membrane. We name this protein Atg36 as its absence blocks pexophagy, and its overexpression induces pexophagy. We have isolated pex3 alleles blocked specifically in pexophagy that cannot recruit Atg36 to peroxisomes. Atg36 is recruited to mitochondria if Pex3 is redirected there, where it restores mitophagy in cells lacking the mitophagy receptor Atg32. Furthermore, Atg36 binds Atg8 and the adaptor Atg11 that links receptors for selective types of autophagy to the core autophagy machinery. Atg36 delivers peroxisomes to the preautophagosomal structure before being internalised into the vacuole with peroxisomes. We conclude that Pex3 recruits the pexophagy receptor Atg36. This reinforces the pivotal role played by Pex3 in coordinating the size of the peroxisome pool, and establishes its role in pexophagy in S. cerevisiae.

Glioblastoma (GBM), a highly malignant brain tumor of adults and children, diffusely invades within the non-neoplastic brain. Despite aggressive current therapeutic interventions, improved therapeutic strategies are greatly needed. Interactions between the tumor and constituents of its microenvironment are known to regulate malignancy, and heparan sulfate proteoglycans (HSPGs) are important as they bind diverse extracellular proteins, including growth factors and cell adhesion molecules, regulating the activity of several ligand-mediated signaling pathways. Recent work from our group described a mechanism by which GBM regulates PDGFR-alpha signaling via enzymatic alteration of heparan sulfate proteoglycans (HSPGs) in the extracellular microenvironment. Blocking tumor-induced alterations of HSPGs, which can be achieved by pharmacological strategies, would potentially inhibit multiple oncogenic signaling pathways in tumor cells and disrupt critical tumormicroenvironment interactions. Here we examine HSPGs and the enzymes that modify them in GBM. We compare their expression across tumor subtypes, their potential roles in oncogenesis, and their potential as novel therapeutic targets in GBM.

Human immunodeficiency virus (HIV) and Simian immunodeficiency virus (SIV) disease progression is associated with multifocal damage to the gastrointestinal tract epithelial barrier that correlates with microbial translocation and persistent pathological immune activation, but the underlying mechanisms remain unclear. Investigating alterations in mucosal immunity during SIV infection, we found that damage to the colonic epithelial barrier was associated with loss of multiple lineages of interleukin (IL)-17-producing lymphocytes, cells that microarray analysis showed expressed genes important for enterocyte homeostasis, including IL-22. IL-22-producing lymphocytes were also lost after SIV infection. Potentially explaining coordinate loss of these distinct populations, we also observed loss of CD103+ dendritic cells (DCs) after SIV infection, which associated with the loss of IL-17- and IL-22-producing lymphocytes. CD103+ DCs expressed genes associated with promotion of IL-17/IL-22+ cells, and coculture of CD103+ DCs and naïve T cells led to increased IL17A and RORc expression in differentiating T cells. These results reveal complex interactions between mucosal immune cell subsets providing potential mechanistic insights into mechanisms of mucosal immune dysregulation during HIV/SIV infection, and offer hints for development of novel therapeutic strategies to address this aspect of AIDS virus pathogenesis.

As the amount of biomedical information available in the literature continues to increase, databases that aggregate this information continue to grow in importance and scope. The population of databases can occur either through fully automated text mining approaches or through manual curation by human subject experts. We here report our experiences in populating the National Institute of Allergy and Infectious Diseases sponsored Immune Epitope Database and Analysis Resource (IEDB, http://iedb.org), which was created in 2003, and as of 2012 captures the epitope information from approximately 99% of all papers published to date that describe immune epitopes (with the exception of cancer and HIV data). This was achieved using a hybrid model based on automated document categorization and extensive human expert involvement. This task required automated scanning of over 22 million PubMed abstracts followed by classification and curation of over 13 000 references, including over 7000 infectious disease-related manuscripts, over 1000 allergy-related manuscripts, roughly 4000 related to autoimmunity, and 1000 transplant/alloantigen-related manuscripts. The IEDB curation involves an unprecedented level of detail, capturing for each paper the actual experiments performed for each different epitope structure. Key to enabling this process was the extensive use of ontologies to ensure rigorous and consistent data representation as well as interoperability with other bioinformatics resources, including the Protein Data Bank, Chemical Entities of Biological Interest, and the NIAID Bioinformatics Resource Centers. A growing fraction of the IEDB data derives from direct submissions by research groups engaged in epitope discovery, and is being facilitated by the implementation of novel data submission tools. The present explosion of information contained in biological databases demands effective query and display capabilities to optimize the user experience. Accordingly, the development of original ways to query the database, on the basis of ontologically driven hierarchical trees, and display of epitope data in aggregate in a biologically intuitive yet rigorous fashion is now at the forefront of the IEDB efforts. We also highlight advances made in the realm of epitope analysis and predictive tools available in the IEDB.

A number of recent studies have examined the ability of stem cells derived from different sources to differentiate into dopamine-producing cells and ameliorate behavioural deficits in Parkinsonian models. Recently, using the approach of cell reprogramming by small cell-permeable biological active compounds that involved in the regulation of chromatin structure and function, and interfere with specific cell signalling pathways that promote neural differentiation we have been able to generate neural-like cells from human bone marrow (BM)-derived MSCs (hMSCs). Neurally induced hMSCs (NI-hMSCs) exhibited several neural properties and exerted beneficial therapeutic effect on tissue preservation and locomotor recovery in spinal cord injured rats. In this study, we aimed to determine whether hMSCs neuralized by this approach can generate dopaminergic (DA) neurons. Immunocytochemisty studies showed that approximately 50-60% of NI-hMSCs expressed early and late dopaminergic marker such as Nurr-1 and TH that was confirmed by Western blot. ELISA studies showed that NI-hMSCs also secreted neurotrophins and dopamine. Hypoxia preconditioning prior to neural induction increased hMSCs proliferation, viability, expression TH and the secretion level of dopamine induced by ATP. Taken together, these studies demonstrated that hMSCs neurally modified by this original approach can be differentiated towards DA-like neurons.

NMDA receptor (NMDAR) channels allow Ca(2+) influx only during correlated activation of both pre- and postsynaptic cells; a Mg(2+) block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg(2+) block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg(2+) block, we found that Mg(2+) block mutants are defective for LTM formation but not associative learning. We demonstrate that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg(2+) block defective NMDARs. Furthermore, we show that genetic and pharmacological reduction of Mg(2+) block significantly increases expression of a CREB repressor isoform. Our results suggest that Mg(2+) block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction.

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results.

Pain contains both sensory and affective dimensions. Using a rodent visceral pain assay that combines the colorectal distension (CRD) model with the conditioned place avoidance (CPA) paradigms, we measured a learned behavior that directly reflects the affective component of visceral pain, and showed that perigenual anterior cingulate cortex (pACC) activation is critical for memory processing involved in long-term visceral affective state and prediction of aversive stimuli by contextual cue. Progress has been made and suggested that activation of vagal afferents plays a role in the behavioral control nociception and memory storage processes.In human patients, electrical vagus nerve stimulation enhanced retention of verbal learning performance. Cholecystokinin-octapeptide (CCK), which is a gastrointestinal hormone released during feeding, has been shown to enhance memory retention. Mice access to food immediately after training session enhanced memory retention. It has been well demonstrated that CCK acting on vagal afferent fibers mediates various physiological functions. We hypothesize that CCK activation of vagal afferent enhances visceral pain-related affective memory.

The purpose of this paper is to examine the acceptability, feasibility, reliability and validity of a new decision quality instrument that assesses the extent to which patients are informed and receive treatments that match their goals.