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    ABSTRACT: Certain myrosinase-positive human gut bacteria can metabolize glucosinolates (GSLs) to produce isothiocyanates (ITC) as chemopreventive agents. We investigated glucoerucin, glucoiberin, and glucoraphanin (present in broccoli) metabolism by human gut strains. All tested bacteria metabolized glucoerucin to completion within 16 h to erucin and erucin nitrile (NIT). Lactobacillus agilis R16 metabolized only 10% of glucoiberin and glucoraphanin with no detectable products. Enterococcus casseliflavus CP1, however, metabolized 40-50% of glucoiberin and glucoraphanin producing relatively low concentrations of iberin and sulforaphane. Interestingly, Escherichia coli VL8 metabolized 80-90% of glucoiberin and glucoraphanin and also bioconverted glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively, producing erucin, erucin NIT, iberverin, and iberverin NIT from the two GSLs. The putative reductase enzyme in the cell-free extracts of this bacterium required both Mg(2+) and NAD(P)H as cofactors for bioconversion. The cell-free extract of E. coli VL8 containing the reductase enzyme was able to reduce both the GSL glucoraphanin and its hydrolysis product sulforaphane to glucoerucin and erucin/erucin NIT, respectively. The composition and metabolic activity of the human gut bacteria can indirectly impact on the potential chemopreventive effects of GSL-derived metabolites.
    Molecular Nutrition & Food Research 04/2014; 58(4). DOI:10.1002/mnfr.201300377
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    ABSTRACT: Recent research has indicated a new mode of intercellular communication facilitated by the movement of RNA between cells. There is evidence that RNA can transfer between cells in a multitude of ways, including in complex with proteins or lipids or in vesicles, including apoptotic bodies and exosomes. However, there remains little understanding of the function of nucleic acid transfer between human cells. In this article, we report that human macrophages transfer microRNAs (miRNAs) to hepato-carcinoma cells (HCCs) in a manner that required intercellular contact and involved gap junctions. Two specific miRNAs transferred efficiently between these cells-miR-142 and miR-223-and both were endogenously expressed in macrophages and not in HCCs. Transfer of these miRNAs influenced posttranscriptional regulation of proteins in HCCs, including decreased expression of reporter proteins and endogenously expressed stathmin-1 and insulin-like growth factor-1 receptor. Importantly, transfer of miRNAs from macrophages functionally inhibited proliferation of these cancerous cells. Thus, these data led us to propose that intercellular transfer of miRNA from immune cells could serve as a new defense against unwanted cell proliferation or tumor growth.
    The Journal of Immunology 11/2013; 191(12). DOI:10.4049/jimmunol.1301728
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    ABSTRACT: ABSTRACT Nitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency. IMPORTANCE Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling-sensed by the UTase (glnD) and PII (glnB), respectively-and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.
    mBio 10/2013; 4(6). DOI:10.1128/mBio.00881-13
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    ABSTRACT: The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission. Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingiameningoseptica. Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E. meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E. meningoseptica can be vertically transmitted from eggs to larvae and that E. meningoseptica-mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E. meningoseptica and Plasmodium on mosquito survival. The high pathogenicity and permanent association of E. meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.
    PLoS ONE 10/2013; 8(10):e77619. DOI:10.1371/journal.pone.0077619
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    ABSTRACT: The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection LtpD localized to the cytoplasmic face of the membrane of the Legionella containing vacuole (LCV). In A549 lung epithelial cells, ectopically-expressed LtpD localized to large vesicular structures which contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17 kDa fragment, LtpD471-626, which was essential for targeting ectopically-expressed LtpD to vesicular structures and for association of translocated LtpD with the LCV. LtpD471-626 bound directly to phosphatidylinositol-3-phosphate (PtdIns (3)P) in vitro and co-localized with the PtdIns (3)P markers FYVE and SetA in co-transfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector, which has a role in intracellular bacterial replication.
    Infection and immunity 09/2013; DOI:10.1128/IAI.01054-13
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    ABSTRACT: Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development.
    Science Signaling 07/2013; 6(285):ra62. DOI:10.1126/scisignal.2003947
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    ABSTRACT: Ovarian cancer is associated with limited overall survival, due to problems in early detection and therapy. Membrane ion channels have been proposed to play a significant, concerted role in the cancer process, from initial proliferation to metastasis, and promise to be early, functional biomarkers. We review the evidence for ion channel and aquaporin expression and functioning in human ovarian cancer cells and tissues. In vitro, K(+) channels, mainly voltage-gated, including Ca(2+)-activated channels, have been found to control the cell cycle, as in other cancers. Voltage-gated, volume-regulated and intracellular Cl(-) channels have been detected in vitro and in vivo and shown to be involved in proliferation, adhesion and invasion. Evidence for 'transient receptor potential', voltage-gated sodium and calcium channels, which have been shown to contribute to pathogenesis of other carcinomas, is also emerging in ovarian cancer. Aquaporins may be involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. It is concluded that functional expression of ion channels and their regulation by steroid hormones and growth factors are an integral part of ovarian cancer development and progression. Furthermore, ion channels may be involved in multidrug resistance, commonly associated with treatment of ovarian cancer. We propose that ion channel studies can facilitate our understanding of the pathobiology of ovarian cancer and, ultimately, can serve as viable novel targets for its clinical management.
    European journal of cancer (Oxford, England: 1990) 05/2013; 49(10). DOI:10.1016/j.ejca.2013.03.016
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    ABSTRACT: Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacteria. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in haemocytes showed signs of instability and leakage. Furthermore, in contrast to wild type L. pneumophila, a ΔsdhA mutant caused a transient depletion of haemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.
    Infection and immunity 05/2013; DOI:10.1128/IAI.00296-13
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    ABSTRACT: Rituximab, which binds CD20 on B cells, is one of the best characterised antibodies used in the treatment of B cell malignancies and autoimmune diseases. Rituximab triggers Natural Killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC - nor what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B cell surface, independent of antibody cross-linking or intercellular contact. Unexpectedly, other proteins, including ICAM-1 and moesin, were selectively recruited to the cap of CD20 and the MTOC became polarised towards the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarised compared to being un-polarised. Polarised B cells were lysed more frequently still, when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarisation of B cells and this augments its therapeutic function in triggering NK cell-mediated ADCC.
    Blood 04/2013; DOI:10.1182/blood-2013-02-482570
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    ABSTRACT: We report the solution structures of the VPg proteins from feline calicivirus (FCV) and murine norovirus (MNV), which have been determined by nuclear magnetic resonance spectroscopy. In both cases the core of the protein adopts a compact helical structure flanked by flexible N and C-termini. Remarkably, while the core of FCV VPg contains a well-defined three-helix bundle, the MNV VPg core has just the first two of these secondary structure elements. In both cases the VPg cores are stabilised by networks of hydrophobic and salt-bridge interactions. The Tyr residue in VPg that is nucleotidylated by the viral NS7 polymerase (Y24 in FCV, Y26 in MNV) occurs in a conserved position within the first helix of the core. Intriguingly, given its structure, VPg would appear to be unable to bind to the viral polymerase so as to place this Tyr in the active site without a major conformation change to VPg or the polymerase. However, mutations that destabilised the VPg core either had no effect on or reduced both the ability of the protein to be nucleotidylated and virus infectivity and did not reveal a clear structure-activity relationship. The precise role of the calicivirus VPg core in virus replication remains to be determined but knowledge of its structure will facilitate future investigations.
    Journal of Virology 03/2013; DOI:10.1128/JVI.03151-12
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Philosophical Transactions of The Royal Society B Biological Sciences 02/2015; 370(20130551). DOI:10.1098/rstb.2013.0551
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