The impact of dietary fibers on dendritic cell responses in vitro is dependent on the differential effects of the fibers on intestinal epithelial cells
Bermudez-Brito, M. ; Sahasrabudhe, N.M. ; Rösch, C. ; Schols, H.A. ; Faas, M.M. ; Vos, P. de - \ 2015
Molecular Nutrition & Food Research 59 (2015)4. - ISSN 1613-4125 - p. 698 - 710.
immune function - receptor 2 - health - homeostasis - modulation - mortality - polysaccharides - activation - mechanisms - prebiotics
Scope In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied. Methods and results The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This was different when DCs and fibers were co-cultured together with supernatants from human epithelial cells (Caco spent medium). Caco spent medium enhanced the production of IL-12, IL-1Ra, IL-6, IL-8, TNF-a, MCP-1 (monocyte chemotactic protein), and MIP-1a but this was strongly attenuated by the dietary fibers. This attenuating effect on proinflammatory cytokines was dependent on the interaction of the fibers with Toll-like receptors as it was reduced by Pepinh-myd88. The interaction of galacto-oligosaccharides, chicory inulin, wheat arabinoxylan, barley ß-glucan with epithelial cells and DCs led to changes in the production of the Th1 cytokines in autologous T cells, while chicory inulin, and barley ß-glucan reduced the Th2 cytokine IL-6. The Treg-promoting cytokine IL-10 was induced by galacto-oligosaccharides whereas chicory inulin decreased the IL-10 production. Conclusions Our results suggest that dietary fibers can modulate the host immune system not only by the recognized mechanism of effects on microbiota but also by direct interaction with the consumer's mucosa. This modulation is dietary fiber type dependent.
Snooker Structure-Based Pharmacophore Model Explains Differences in Agonist and Blocker Binding to Bitter Receptor hTAS2R39
Roland, W.S.U. ; Sanders, M.P.A. ; Buren, L. van; Gouka, R.J. ; Gruppen, H. ; Vincken, J.P. ; Ritschel, T. - \ 2015
PLoS ONE 10 (2015)3. - ISSN 1932-6203
protein-coupled receptors - class-a gpcrs - taste receptors - activation - identification - requirements - peptides - family - assay - t2r1
The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4’-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.
Oral administration of Lactobacillus plantarum 299v modulates gene expression in the ileum of pigs: prediction of crosstalk between intestinal immune cells and sub-mucosal adipocytes
Hulst, M.M. ; Gross, G. ; Liu, Yapin ; Hoekman, A.J.W. ; Niewold, T. ; Meulen, J. van der; Smits, M.A. - \ 2015
Genes & Nutrition 10 (2015)3. - ISSN 1555-8932 - 13 p.
kappa-b - functional-analysis - in-vivo - inhibition - immunoglobulins - identification - adipogenesis - macrophages - metabolism - activation
To study host–probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 1012 CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer’s patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig’s body.
High fat challenges with different fatty acids affect distinct atherogenic gene expression pathways in immune cells from lean and obese subjects
Esser, D. ; Dijk, S.J. van; Oosterink, E. ; Lopez, S. ; Muller, M.R. ; Afman, L.A. - \ 2015
Molecular Nutrition & Food Research 59 (2015)8. - ISSN 1613-4125 - p. 1563 - 1572.
triglyceride-rich lipoproteins - blood mononuclear-cells - men - atherosclerosis - inflammation - activation - receptors - adherence - profiles - alpha
Scope - Early perturbations in vascular health can be detected by imposing subjects to a high fat (HF) challenge and measure response capacity. Subtle responses can be determined by assessment of whole-genome transcriptional changes. We aimed to magnify differences in health by comparing gene-expression changes in peripheral blood mononuclear cells toward a high MUFA or saturated fatty acids (SFA) challenge between subjects with different cardiovascular disease risk profiles and to identify fatty acid specific gene-expression pathways. Methods and results -In a cross-over study, 17 lean and 15 obese men (50–70 years) received two 95 g fat shakes, high in SFAs or MUFAs. Peripheral blood mononuclear cell gene-expression profiles were assessed fasted and 4-h postprandially. Comparisons were made between groups and shakes. During fasting, 294 genes were significantly differently expressed between lean and obese. The challenge increased differences to 607 genes after SFA and 2516 genes after MUFA. In both groups, SFA decreased expression of cholesterol biosynthesis and uptake genes and increased cholesterol efflux genes. MUFA increased inflammatory genes and PPAR-a targets involved in ß-oxidation. Conclusion - Based upon gene-expression changes, we conclude that an HF challenge magnifies differences in health, especially after MUFA. Our findings also demonstrate how SFAs and MUFAs exert distinct effects on lipid handling pathways in immune cells.
Systemic Regulation of RAS/MAPK Signaling by the Serotonin Metabolite 5-HIAA
Schmid, T. ; Snoek, L.B. ; Fröhli, E. ; Bent, M.L. van der; Kammenga, J.E. ; Hajnal, A. - \ 2015
Plos Genetics 11 (2015)5. - ISSN 1553-7404 - 16 p.
caenorhabditis-elegans - c-elegans - natural variation - vulvar induction - complex disease - receptor - protein - gene - kinase - activation
Human cancer is caused by the interplay of mutations in oncogenes and tumor suppressor genes and inherited variations in cancer susceptibility genes. While many of the tumor initiating mutations are well characterized, the effect of genetic background variation on disease onset and progression is less understood. We have used C. elegans genetics to identify genetic modifiers of the oncogenic RAS/MAPK signaling pathway. Quantitative trait locus analysis of two highly diverged C. elegans isolates combined with allele swapping experiments identified the polymorphic monoamine oxidase A (MAOA) gene amx-2 as a negative regulator of RAS/MAPK signaling. We further show that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA), which is a product of MAOA catalysis, systemically inhibits RAS/MAPK signaling in different organs of C. elegans. Thus, MAOA activity sets a global threshold for MAPK activation by controlling 5-HIAA levels. To our knowledge, 5-HIAA is the first endogenous small molecule that acts as a systemic inhibitor of RAS/MAPK signaling.
Covalent Attachment of 1-Alkenes to Oxidized Platinum Surfaces
Alonso Carnicero, J.M. ; Fabre, B. ; Trilling, A.K. ; Scheres, L.M.W. ; Franssen, M.C.R. ; Zuilhof, H. - \ 2015
Langmuir 31 (2015)9. - ISSN 0743-7463 - p. 2714 - 2721.
self-assembled monolayers - organic monolayers - gold - alkanethiols - functionalization - spectroscopy - activation - alkenes - layers - films
We report the formation of covalently bound alkyl layers onto oxidized Pt (PtOx) substrates by reaction with 1-alkenes as a novel way to bind organic molecules to metal surfaces. The organic layers were characterized by static contact angle, infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The grafted alkyl layers display a hydrolytic stability that is comparable to that of alkyl thiols on Au. PtOx-alkene attachment is compatible with terminal ester moieties enabling further anchoring of functional groups, such as redox-active ferrocene, and thus has great potential to extend monolayer chemistry on noble metals.
Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro
Schmeits, P.C.J. ; Shao, J. ; Krieken, D.A. van der; Volger, O.L. ; Loveren, H. van; Peijnenburg, A.A.C.M. ; Hendriksen, P.J.M. - \ 2015
Journal of Applied Toxicology 35 (2015)7. - ISSN 0260-437X - p. 831 - 841.
polycyclic aromatic-hydrocarbons - brominated flame retardants - tetrabromobisphenol-a - balb/c mice - vitamin-c - chlorpyrifos - activation - exposure - rats - kinase
Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6¿h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)–PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e.¿chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT–PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.
Constitutive overexpression of the pollen specific gene SKS13 in leaves reduces aphid performance on Arabidopsis thaliana
Chen, X. ; Zhang, Z. ; Visser, R.G.F. ; Vosman, B.J. ; Broekgaarden, C. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229
russian wheat aphid - green peach aphid - plant defense - transcriptome changes - resistance responses - peroxidase-activity - insect herbivores - myzus-persicae - watery saliva - activation
Background: Plants have developed a variety of mechanisms to counteract aphid attacks. They activate their defences by changing the expression of specific genes. Previously we identified an activation tag mutant of Arabidopsis thaliana on which Myzus persicae population development was reduced. Activation tag mutants are gain-of-function in which the expression of a gene is increased by the insertion of the Cauliflower mosaic virus 35S enhancer that acts on the natural promoter. By further characterizing this previously identified mutant we identified a gene that reduces performance of M. persicae and also provided clues about the mechanism involved. Results: We show that SKU5 SIMILAR 13 (SKS13), a gene whose expression in wild type plants is restricted to pollen and non-responsive to M. persicae attack, is overexpressed in the A. thaliana mutant showing reduced performance of M. persicae. Monitoring M. persicae feeding behaviour on SKS13 overexpressing plants indicated that M. persicae have difficulties feeding from the phloem. The constitutive expression of SKS13 results in accumulation of reactive oxygen species, which is possibly regulated through the jasmonic acid pathway. The enhanced resistance is not aphid species specific as also the population development of Brevicoryne brassicae was affected. Conclusions: We demonstrate that constitutive expression in leaves of the pollen-specific gene SKS13 can enhance plant defence, resulting in a reduction of M. persicae population development and also decreases the transmission of persistent viruses. Overexpression of SKS13 in A. thaliana also affects B. brassicae and possibly other phloem feeding insects as well. Identifying genes that can enhance plant defence against insects will be important to open up new avenues for the development of insect resistant crop plants.
TCR's genetically linked to CD28 and CD3e do not mispair with endogous TCR chains and mediate enhanced T cell persistance and anti-melanoma activity
Govers, C.C.F.M. ; Sebestyen, Z. ; Roszik, J. ; Brakel, M. van; Berrevoets, C. ; Szoor, A. ; Panoutsopoulou, K. ; Broertjes, M. ; Van, T. ; Vereb, G. ; Szollosi, J. ; Debets, R. - \ 2014
The Journal of Immunology 193 (2014)10. - ISSN 0022-1767 - p. 5315 - 5326.
chimeric-antigen-receptor - gene-transfer - metastatic melanoma - cancer regression - lymphocytes - therapy - alpha - activation - toxicity - survival
Adoptive transfer of T cells that are gene engineered to express a defined TCR represents a feasible and promising therapy for patients with tumors. However, TCR gene therapy is hindered by the transient presence and effectiveness of transferred T cells, which are anticipated to be improved by adequate T cell costimulation. In this article, we report the identification and characterization of a novel two-chain TCR linked to CD28 and CD3e (i.e., TCR:28e). This modified TCR demonstrates enhanced binding of peptide–MHC and mediates enhanced T cell function following stimulation with peptide compared with wild-type TCR. Surface expression of TCR:28e depends on the transmembrane domain of CD28, whereas T cell functions depend on the intracellular domains of both CD28 and CD3e, with IL-2 production showing dependency on CD28:LCK binding. TCR:28e, but not wild-type TCR, induces detectable immune synapses in primary human T cells, and such immune synapses show significantly enhanced accumulation of TCR transgenes and markers of early TCR signaling, such as phosphorylated LCK and ERK. Importantly, TCR:28e does not show signs of off-target recognition, as evidenced by lack of TCR mispairing, as well as preserved specificity. Notably, when testing TCR:28e in immune-competent mice, we observed a drastic increase in T cell survival, which was accompanied by regression of large melanomas with limited recurrence. Our data argue that TCR transgenes that contain CD28, and, thereby, may provide T cell costimulation in an immune-suppressive environment, represent candidate receptors to treat patients with tumors.
Deficiency of macrophage stimulating protein results in spontaneous inflammation and increased susceptibility towards epithelial damage in zebrafish
Witte, M. ; Huitema, L.F. ; Nieuwenhuis, E.E.S. ; Brugman, S. - \ 2014
Zebrafish 11 (2014)6. - ISSN 1545-8547 - p. 542 - 550.
receptor tyrosine kinase - transgenic zebrafish - crohns-disease - ron - activation - cell - mouse - msp - pathogenesis - induction
Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP–RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP–RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (mspt34230) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP–RON signaling in the context of intestinal inflammation.
Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis
Zhao, G. ; Hatting, M. ; Nevzorova, Y.A. ; Peng, J. ; Hu, W.Y. ; Boekschoten, M.V. ; Müller, M.R. - \ 2014
Gut 63 (2014)7. - ISSN 0017-5749 - p. 1159 - 1172.
signal-transduction pathway - tnf-alpha - pathogenic role - fibrosis - activation - mice - proliferation - induction - injury - phosphorylation
Objective The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. Design Jnk1f/f wildtype (WT), Jnk1-/- and Jnk1¿hepa (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. Results Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1-/- compared with WT and Jnk1¿hepa mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and a-smooth muscle actin expression were downregulated in Jnk1-/- compared with WT and Jnk1¿hepa livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow–derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1-/- livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. Conclusions Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.
DON shares a similar mode of action as the ribotoxic stress inducer anisomycin while TBTO shares ER stress patterns with the ER stress inducer Thapsigargin based on comparative gene expression profiling in Jurkat T cells
Schmeits, P.C.J. ; Katika, M.R. ; Peijnenburg, A.A.C.M. ; Loveren, H. van; Hendriksen, P.J.M. - \ 2014
Toxicology Letters 224 (2014)3. - ISSN 0378-4274 - p. 395 - 406.
tri-n-butyltin - unfolded protein response - tributyltin-oxide tbto - mouse thymoma cells - deoxynivalenol don - induced apoptosis - plasma-membrane - ribosomal-rna - in-vivo - activation
Previously, we studied the effects of deoxynivalenol (DON) and tributyltin oxide (TBTO) on whole genome mRNA expression profiles of human T lymphocyte Jurkat cells. These studies indicated that DON induces ribotoxic stress and both DON and TBTO induced ER stress which resulted into T-cell activation and apoptosis. The first goal of the present study was to provide final proof for these mode of actions by comparing the effects of 6 h exposure to DON and TBTO on mRNA expression to those of positive controls of ribotoxic stress (anisomycin), ER stress (thapsigargin) and T cell activation (ionomycin). Genes affected by anisomycin and the majority of genes affected by thapsigargin were affected in the same direction by DON and TBTO, respectively, confirming the expected modes of action. Pathway analysis further sustained that DON induces ribotoxic stress and both DON and TBTO induce unfolded protein response (UPR), ER stress, T cell activation and apoptosis. The second goal was to assess whether DON and/or TBTO affect other pathways above those detected before. TBTO induced groups of genes that are involved in DNA packaging and heat shock response that were not affected by thapsigargin. DON did not affect other genes than anisomycin indicating the effect of DON to be restricted to ribotoxic stress. This study also demonstrates that comparative gene expression analysis is a very promising tool for the identification of modes of action of immunotoxic compounds.
The tomato phosphatidylinositol-phospholipase C2 (SlPLC2) is required for defense gene induction by the fungal elicitor xylanase
Gonorazky, G. ; Ramirez, L. ; Abd-El-Haliem, A. ; Vossen, J.H. ; Lamattina, L. ; Have, A. ten; Joosten, M.H.A.J. ; Laxalt, A.M. - \ 2014
Journal of Plant Physiology 171 (2014)11. - ISSN 0176-1617 - p. 959 - 965.
phosphatidic-acid accumulation - cultured rice cells - nitric-oxide - disease resistance - c/diacylglycerol kinase - arabidopsis-thaliana - signaling pathways - activation - responses - plants
The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J.
Dynamic hydrolase activities precede hypersensitive tissue collapse in tomato seedlings
Sueldo, D. ; Ali, A. ; Misas-Villamil, J. ; Colby, T. ; Tameling, W.I.L. ; Joosten, M.H.A.J. ; Hoorn, R. van der - \ 2014
New Phytologist 203 (2014)3. - ISSN 0028-646X - p. 913 - 925.
programmed cell-death - vacuolar processing enzyme - pathogenesis-related proteins - disease resistance - cysteine proteases - defense responses - plant-pathogen - gene-expression - arabidopsis - activation
Hydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins. We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity. ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs. In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse.
Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity
Hof, W.F.P.M. ; Summeren, A. van; Lommen, A. ; Coonen, M.L.J. ; Brauers, K. ; Herwijnen, M. van; Wodzig, W.K.W.H. ; Kleinjans, J.C.S. - \ 2014
Toxicology 324 (2014). - ISSN 0300-483X - p. 18 - 26.
drug-induced hepatotoxicity - gene-expression - micrornas - repression - normalization - accumulation - metabonomics - translation - activation - biomarkers
The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites.
Exploring the molecular link between swim-training and caudal fin development in zebrafish (Danio rerio) larvae
Fiaz, A.W. ; Leon, K.M. ; Leeuwen, J.L. van; Kranenbarg, S. - \ 2014
Journal of Applied Ichthyology 30 (2014)4. - ISSN 0175-8659 - p. 753 - 761.
transcription factor - mechanical control - gene-expression - bone - activation - forces - tissue - transhydrogenase - osteoblasts - contributes
In vitro and in vivo studies have shown that mechanical forces play an important role during development. The molecular mechanisms via which mechanical forces regulate development have been extensively investigated by in vitro studies. However, knowledge about the molecular pathways that mediate the effect of mechanical forces during development in vivo is limited. Previously, we showed that swim-training increased maximum normalized curvatures in the caudal fin (suggesting that the caudal fin experienced increased mechanical loads) and prioritized the development of skeletal structures in the caudal fin. Therefore, we used the zebrafish caudal fin to explore the molecular link between an increased swimming activity and development in vivo. Whole genome microarray analysis of caudal fins of zebrafish subjected to swim-training and control fish identified 46 genes which were up-regulated with a fold change of 1.5 or larger at 10 dpf. Fourteen genes were expressed specifically in the following tissues in the caudal fin: the neural tube, the tissue surrounding the hypurals, the finfold, or muscle fibers. Subsequently, we identified two muscle specific genes, aste1 (asteroid homolog 1) and zgc:65811, which showed an increased expression specifically in the caudal fin in response to swim-training. This makes these genes interesting candidate genes for further research on the molecular link between mechanical forces and caudal fin development. Our study is the first to investigate the molecular link between swim-training and caudal fin development and offers a system that can provide a deeper understanding of the link between mechanical and molecular signals during development in vivo.
Control of Competence for DNA Transformation in Streptococcus suis by Genetically Transferable Pherotypes
Zaccaria, E. ; Baarlen, P. van; Greeff, A. de; Morrison, D.A. ; Smith, H. ; Wells, J.M. - \ 2014
PLoS ONE 9 (2014)6. - ISSN 1932-6203 - 8 p.
horizontal gene-transfer - bacterial transformation - haemophilus-influenzae - peptide pheromone - pneumoniae - thermophilus - activation - expression - regulator - system
Here we show that S. suis, a major bacterial pathogen of pigs and emerging pathogen in humans responds to a peptide pheromone by developing competence for DNA transformation. This species does not fall within any of the phylogenetic clusters of streptococci previously shown to regulate competence via peptide pheromones suggesting that more species of streptococci may be naturally competent. Induction of competence was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon, extracellular addition of a comX-inducing peptide (XIP), and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. Different comS alleles are present among strains of S. suis. These comS alleles are not functionally equivalent and appear to operate in conjuction with a cognate ComR to regulate comX through a conserved comR-box promoter. We demonstrate that these ‘pherotypes’ can be genetically transferred between strains, suggesting that similar approaches might be used to control competence induction in other lactic acid bacteria that lack ComR/ComS homologues but possess comX and the late competence regulon. The approaches described in this paper to identify and optimize peptide-induced competence may also assist other researchers wishing to identify natural competence in other bacteria. Harnessing natural competence is expected to accelerate genetic research on this and other important streptococcal pathogens and to allow high-throughput mutation approaches to be implemented, opening up new avenues for research.
To like or not to like: Neural substrates of subjective flavor preferences
Bosch, I. van den; Dalenberg, J.R. ; Renken, R. ; Langeveld, A.W.B. van; Smeets, P.A.M. ; Griffioen-Roose, S. ; Horst, G.J. ter; Graaf, C. de; Boesveldt, S. - \ 2014
Behavioural Brain Research 269 (2014). - ISSN 0166-4328 - p. 128 - 137.
human orbitofrontal cortex - human brain - different representations - selective attention - prefrontal cortex - taste stimuli - humans - reward - pleasantness - activation
Flavor preferences vary; what one enjoys may be disgusting to another. Previous research has indicated several brain regions associated with flavor preferences. However, by using different stimuli or different internal states to obtain differences in liking, results of these studies may be confounded. Therefore, we used one target stimulus (grapefruit juice) and fMRI to compare brain activation patterns between participants that either liked (n = 16) or disliked (n = 18) this stimulus. Our first aim was to investigate whether differential neural activation exists that accounts for the difference in subjective flavor preference for the target stimulus. Secondly, multivariate analysis was used to investigate whether differences in subjective liking for the target revealed similar activation patterns as differences in general liking for a sweet and bitter solution. A direct comparison of likers and dislikers of the target stimulus revealed only small differences in activations in orbitofrontal cortex (OFC) and dorsal anterior cingulate cortex (dACC). However, when using multivariate analysis, a broader activation pattern (including OFC, dACC, pregenual anterior cingulate, anterior insula and ventral striatum) was identified that discriminated likers from dislikers with an 88% success rate. Interestingly though, little overlap was found between this pattern and the pattern that discriminates liking for the sweet and bitter solutions and lesser voxels contributed to the former compared with the latter. These differences between patterns discerning innate versus learned preferences may suggest that different mechanisms are at work and highlight the importance of elucidating the neural processes of how subjective preferences are learned and acquired.
Potato and Mushroom Polyphenol Oxidase Activities Are Differently Modulated by Natural Plant Extracts
Kuijpers, T.F.M. ; Herk, T. van; Vincken, J.P. ; Janssen, R.H. ; Narh, D.L. ; Berkel, W.J.H. van; Gruppen, H. - \ 2014
Journal of Agricultural and Food Chemistry 62 (2014)1. - ISSN 0021-8561 - p. 214 - 221.
tyrosinase inhibitors - chlorogenic acid - ilex-paraguariensis - constituents - activation - licorice - identification - mechanism - agents - sds
Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom (Agaricus bisporus, AbPPO) and PPO from potato (Solanum tuberosum, StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate (Ilex paraguariensis) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones.
The leucine-rich repeat receptor kinase BIR2 is a negative regulator of BAK1 in plant immunity
Halter, T. ; Imkampe, J. ; Mazzotta, S. ; Wierzba, M. ; Postel, S. ; Bücherl, C.A. ; Kiefer, C. ; Stahl, M. ; Chinchilla, D. ; Wang, X. ; Nürnberger, T. ; Zipfel, C. ; Clouse, S. ; Borst, J.W. ; Boeren, S. ; Vries, S.C. de; Tax, F. ; Kemmerling, B. - \ 2014
Current Biology 24 (2014)2. - ISSN 0960-9822 - p. 134 - 143.
innate immunity - cell-death - necrotrophic pathogens - molecular-patterns - protein-kinase - arabidopsis - complex - bri1 - perception - activation
Background Transmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively. Results Identification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex. Conclusions Our results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands.