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Sample records for induces differential recruitment

  1. Differential recruitment of distinct amygdalar nuclei across appetitive associative learning

    PubMed Central

    Cole, Sindy; Powell, Daniel J.; Petrovich, Gorica D.

    2013-01-01

    The amygdala is important for reward-associated learning, but how distinct cell groups within this heterogeneous structure are recruited during appetitive learning is unclear. Here we used Fos induction to map the functional amygdalar circuitry recruited during early and late training sessions of Pavlovian appetitive conditioning. We found that a number of distinct amygdalar nuclei were differentially recruited by tone–food pairings during the early and late stages of training, suggesting evidence of learning-induced plasticity. Notably, these selectively activated nuclei belong to dissociable subsystems that are well placed to simultaneously inform cortical (cognitive) processing and behavioral control during tone–food learning. PMID:23676201

  2. Modulating rest-break length induces differential recruitment of automatic and controlled attentional processes upon task reengagement.

    PubMed

    Lim, Julian; Teng, James; Wong, Kian Foong; Chee, Michael W L

    2016-07-01

    Rest breaks are commonly administered as a countermeasure to reduce on-the-job fatigue, both physical and mental. However, this practice makes the assumption that recovery from fatigue, as measured by the reversal of performance declines, is the sole effect of taking a break on behavior. Here, through administering rest breaks of differing lengths in between blocks of a mentally demanding symbol decoding task, we show that this assumption may not be strictly true. First, we replicate previous work by showing that taking a longer break leads to two correlated effects: greater immediate rebound in performance, and greater subsequent time-on-task decline. Using fMRI, we reveal that time-on-task in this paradigm is associated with increasing recruitment of fronto-parietal areas associated with top-down control, and decreasing deactivation in the default-mode network. Finally, by analyzing individual differences, we reveal a potential neural basis for our behavioral observation: greater recovery following long breaks is associated with greater activity in the putamen, an area associated with the automatic generation of motor responses, followed by greater activity in left middle frontal gyrus by the end of those task periods. Taken together, this suggests a shift in the implicit engagement of automatic and controlled attentional processing following longer breaks. This shift may be undesirable or detrimental in real-world situations where maintaining a stable level of attention over time is necessary. PMID:27039697

  3. Differential recruitment of executive resources during mind wandering.

    PubMed

    Kam, Julia W Y; Handy, Todd C

    2014-05-01

    Recent research has shown that mind wandering recruits executive resources away from the external task towards inner thoughts. No studies however have determined whether executive functions are drawn away in a unitary manner during mind wandering episodes, or whether there is variation in specific functions impacted. Accordingly, we examined whether mind wandering differentially modulates three core executive functions-response inhibition, updating of working memory, and mental set shifting. In three experiments, participants performed one of these three executive function tasks and reported their attentional state as either on-task or mind wandering at random intervals. We found that mind wandering led to poorer performance in the response inhibition and working memory tasks, but not the set-shifting task. These findings suggest that mind wandering does not recruit executive functions in a monolithic manner. Rather, it appears to selectively engage certain executive functions, which may reflect the adaptive maintenance of ongoing task performance. PMID:24674757

  4. Differential adapter recruitment by TLR2 co-receptors.

    PubMed

    Piao, Wenji; Ru, Lisa W; Toshchakov, Vladimir Y

    2016-07-01

    TLR2 heterodimers with TLR1 or TLR6 recognize distinct pathogen-associated molecules such as tri- and di-acylated lipopeptides. The activated TLR2 heterodimers recruit Toll-IL-1R domain- (TIR-) containing adapter proteins, TIRAP and MyD88, through the receptor TIR domains. Molecular recognition mechanisms responsible for agonist-driven, TIR domain-mediated receptor-adapter interactions as well as the structure of resultant signaling complexes remain unknown. We previously reported that the cell-permeable peptide derived from helix D of TLR2 TIR (2R9) specifically binds TIRAP in vitro and in cells and thereby inhibits TIRAP-dependent TLR signaling. This study demonstrates that cell-permeable peptides from D helix of TLR1 or TLR6, peptides 1R9 and 6R9 respectively, inhibit signaling mediated by cognate TLR2 co-receptors. Interestingly, 1R9 and 6R9 bind different TLR2 adapters, as they selectively bind MyD88 and TIRAP TIR, respectively. Both peptides block the agonist-induced co-immunoprecipitation (co-IP) of TLR2 with TIRAP or MyD88, but not TLR2 co-IP with co-receptors. Our data suggest that D helices of TLR1 and TLR6 TIR domains are adapter recruitment sites in both co-receptors; yet the sites recruit different adapters. The D helix in TLR1 is the MyD88 docking site, whereas in TLR6 this site recruits TIRAP. PMID:27150837

  5. Differential Recruitment of Distinct Amygdalar Nuclei across Appetitive Associative Learning

    ERIC Educational Resources Information Center

    Cole, Sindy; Powell, Daniel J.; Petrovich, Gorica D.

    2013-01-01

    The amygdala is important for reward-associated learning, but how distinct cell groups within this heterogeneous structure are recruited during appetitive learning is unclear. Here we used Fos induction to map the functional amygdalar circuitry recruited during early and late training sessions of Pavlovian appetitive conditioning. We found that a…

  6. Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

    PubMed Central

    Rampart, M.; Herman, A. G.

    1996-01-01

    The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB4 and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 × 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60–70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1. PMID:18475729

  7. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    SciTech Connect

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  8. 5 CFR 591.234 - Under what circumstances may people recruited locally receive a post differential?

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 5 Administrative Personnel 1 2014-01-01 2014-01-01 false Under what circumstances may people recruited locally receive a post differential? 591.234 Section 591.234 Administrative Personnel OFFICE OF... Post Differential-Nonforeign Areas Post Differentials § 591.234 Under what circumstances may...

  9. 5 CFR 591.234 - Under what circumstances may people recruited locally receive a post differential?

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Under what circumstances may people recruited locally receive a post differential? 591.234 Section 591.234 Administrative Personnel OFFICE OF... Post Differential-Nonforeign Areas Post Differentials § 591.234 Under what circumstances may...

  10. 5 CFR 591.234 - Under what circumstances may people recruited locally receive a post differential?

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 5 Administrative Personnel 1 2012-01-01 2012-01-01 false Under what circumstances may people recruited locally receive a post differential? 591.234 Section 591.234 Administrative Personnel OFFICE OF... Post Differential-Nonforeign Areas Post Differentials § 591.234 Under what circumstances may...

  11. 5 CFR 591.234 - Under what circumstances may people recruited locally receive a post differential?

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 1 2013-01-01 2013-01-01 false Under what circumstances may people recruited locally receive a post differential? 591.234 Section 591.234 Administrative Personnel OFFICE OF... Post Differential-Nonforeign Areas Post Differentials § 591.234 Under what circumstances may...

  12. 5 CFR 591.234 - Under what circumstances may people recruited locally receive a post differential?

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 5 Administrative Personnel 1 2011-01-01 2011-01-01 false Under what circumstances may people recruited locally receive a post differential? 591.234 Section 591.234 Administrative Personnel OFFICE OF... Post Differential-Nonforeign Areas Post Differentials § 591.234 Under what circumstances may...

  13. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS. PMID:25359783

  14. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation.

    PubMed

    Brady, Robert T; O'Brien, Fergal J; Hoey, David A

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24 hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. PMID:25721667

  15. Differential subcellular membrane recruitment of Src may specify its downstream signalling

    SciTech Connect

    Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah; D'Auria, Ludovic; Van Der Smissen, Patrick; Platek, Anna; Mettlen, Marcel; Caplanusi, Adrian; Hove, Marie-France van den; Tyteca, Donatienne; Courtoy, Pierre J.

    2008-04-15

    Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.

  16. Ubiquitination of RORγt at Lysine 446 Limits Th17 Differentiation by Controlling Coactivator Recruitment.

    PubMed

    He, Zhiheng; Wang, Fei; Ma, Jian; Sen, Subha; Zhang, Jing; Gwack, Yousang; Zhou, Yu; Sun, Zuoming

    2016-08-15

    The transcription factor retinoid acid-related orphan receptor γ t (RORγt) directs the differentiation of Th17 cells. Th17 cells mediate pathological immune responses responsible for autoimmune diseases, including psoriasis and multiple sclerosis. Previous studies focused on RORγt target genes and their function in Th17 differentiation. In this study, we assessed posttranscriptional regulation of RORγt and identified a functional ubiquitination site, K446. Mutation of K446 to arginine to prevent ubiquitination greatly enhanced recruitment of steroid receptor coactivator 1 (SRC1), a coactivator critical for RORγt activity. Correspondingly, the K446 to arginine mutation potentiated Th17 differentiation. We also showed that ubiquitin-specific protease (USP)15 interacted with RORγt, removed ubiquitin from K446, and stimulated RORγt activity by enhancing coactivator SRC1 recruitment. Knockdown of USP15 or expression of inactive USP15 impaired Th17 differentiation, suggesting a positive role for USP15-mediated deubiquitination of RORγt in Th17 differentiation. Therefore, ubiquitination of K446 limits RORγt-mediated Th17 differentiation by inhibiting the recruitment of coactivator SRC1. Our study will inform the development of treatments that target RORγt ubiquitination pathways to limit Th17-mediated autoimmunity. PMID:27430721

  17. Motivation and affective judgments differentially recruit neurons in the primate dorsolateral prefrontal and anterior cingulate cortex.

    PubMed

    Amemori, Ken-ichi; Amemori, Satoko; Graybiel, Ann M

    2015-02-01

    The judgment of whether to accept or to reject an offer is determined by positive and negative affect related to the offer, but affect also induces motivational responses. Rewarding and aversive cues influence the firing rates of many neurons in primate prefrontal and cingulate neocortical regions, but it still is unclear whether neurons in these regions are related to affective judgment or to motivation. To address this issue, we recorded simultaneously the neuronal spike activities of single units in the dorsolateral prefrontal cortex (dlPFC) and the anterior cingulate cortex (ACC) of macaque monkeys as they performed approach-avoidance (Ap-Av) and approach-approach (Ap-Ap) decision-making tasks that can behaviorally dissociate affective judgment and motivation. Notably, neurons having activity correlated with motivational condition could be distinguished from neurons having activity related to affective judgment, especially in the Ap-Av task. Although many neurons in both regions exhibited similar, selective patterns of task-related activity, we found a larger proportion of neurons activated in low motivational conditions in the dlPFC than in the ACC, and the onset of this activity was significantly earlier in the dlPFC than in the ACC. Furthermore, the temporal onsets of affective judgment represented by neuronal activities were significantly slower in the low motivational conditions than in the other conditions. These findings suggest that motivation and affective judgment both recruit dlPFC and ACC neurons but with differential degrees of involvement and timing. PMID:25653353

  18. Motivation and Affective Judgments Differentially Recruit Neurons in the Primate Dorsolateral Prefrontal and Anterior Cingulate Cortex

    PubMed Central

    Amemori, Ken-ichi; Amemori, Satoko

    2015-01-01

    The judgment of whether to accept or to reject an offer is determined by positive and negative affect related to the offer, but affect also induces motivational responses. Rewarding and aversive cues influence the firing rates of many neurons in primate prefrontal and cingulate neocortical regions, but it still is unclear whether neurons in these regions are related to affective judgment or to motivation. To address this issue, we recorded simultaneously the neuronal spike activities of single units in the dorsolateral prefrontal cortex (dlPFC) and the anterior cingulate cortex (ACC) of macaque monkeys as they performed approach–avoidance (Ap–Av) and approach–approach (Ap–Ap) decision-making tasks that can behaviorally dissociate affective judgment and motivation. Notably, neurons having activity correlated with motivational condition could be distinguished from neurons having activity related to affective judgment, especially in the Ap–Av task. Although many neurons in both regions exhibited similar, selective patterns of task-related activity, we found a larger proportion of neurons activated in low motivational conditions in the dlPFC than in the ACC, and the onset of this activity was significantly earlier in the dlPFC than in the ACC. Furthermore, the temporal onsets of affective judgment represented by neuronal activities were significantly slower in the low motivational conditions than in the other conditions. These findings suggest that motivation and affective judgment both recruit dlPFC and ACC neurons but with differential degrees of involvement and timing. PMID:25653353

  19. Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

    SciTech Connect

    Talhouk, Rabih S. Mroue, Rana; Mokalled, Mayssa; Abi-Mosleh, Lina; Nehme, Ralda; Ismail, Ayman; Khalil, Antoine; Zaatari, Mira; El-Sabban, Marwan E.

    2008-11-01

    Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.

  20. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling

    PubMed Central

    Gibert, Yann; Bernard, Laure; Debiais-Thibaud, Melanie; Bourrat, Franck; Joly, Jean-Stephane; Pottin, Karen; Meyer, Axel; Retaux, Sylvie; Stock, David W.; Jackman, William R.; Seritrakul, Pawat; Begemann, Gerrit; Laudet, Vincent

    2010-01-01

    One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity.—Gibert, Y., Bernard, L., Debiais-Thibaud, M., Bourrat, F., Joly, J.-S., Pottin, K., Meyer, A., Retaux, S., Stock, D. W., Jackman, W. R., Seritrakul, P., Begemann, G., Laudet, V. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling. PMID:20445074

  1. Bradykinin Induces TRPV1 Exocytotic Recruitment in Peptidergic Nociceptors

    PubMed Central

    Mathivanan, Sakthikumar; Devesa, Isabel; Changeux, Jean-Pierre; Ferrer-Montiel, Antonio

    2016-01-01

    Transient receptor potential vanilloid I (TRPV1) sensitization in peripheral nociceptors is a prominent phenomenon that occurs in inflammatory pain conditions. Pro-algesic agents can potentiate TRPV1 activity in nociceptors through both stimulation of its channel gating and mobilization of channels to the neuronal surface in a context dependent manner. A recent study reported that ATP-induced TRPV1 sensitization in peptidergic nociceptors involves the exocytotic release of channels trafficked by large dense core vesicles (LDCVs) that cargo alpha-calcitonin gene related peptide alpha (αCGRP). We hypothesized that, similar to ATP, bradykinin may also use different mechanisms to sensitize TRPV1 channels in peptidergic and non-peptidergic nociceptors. We found that bradykinin notably enhances the excitability of peptidergic nociceptors, and sensitizes TRPV1, primarily through the bradykinin receptor 2 pathway. Notably, bradykinin sensitization of TRPV1 in peptidergic nociceptors was significantly blocked by inhibiting Ca2+-dependent neuronal exocytosis. In addition, silencing αCGRP gene expression, but not substance P, drastically reduced bradykinin-induced TRPV1 sensitization in peptidergic nociceptors. Taken together, these findings indicate that bradykinin-induced sensitization of TRPV1 in peptidergic nociceptors is partially mediated by the exocytotic mobilization of new channels trafficked by αCGRP-loaded LDCVs to the neuronal membrane. Our findings further imply a central role of αCGRP peptidergic nociceptors in peripheral algesic sensitization, and substantiate that inhibition of LDCVs exocytosis is a valuable therapeutic strategy to treat pain, as it concurrently reduces the release of pro-inflammatory peptides and the membrane recruitment of thermoTRP channels. PMID:27445816

  2. Differential recruitment of co-regulatory proteins to the human estrogen receptor 1 in response to xenoestrogens.

    PubMed

    Smith, L Cody; Clark, Jessica C; Bisesi, Joseph H; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-09-01

    The diverse biological effects of xenoestrogens may be explained by their ability to differentially recruit co-regulatory proteins to the estrogen receptor (ER). We employed high-throughput receptor affinity binding and co-regulatory protein recruitment screening assays based on fluorescence polarization and time resolved florescence resonance energy transfer (TR-FRET), respectively, to assess xenoestrogen-specific binding and co-regulatory protein recruitment to the ER. Then we used a functional proteomic assay based on co-immunoprecipitation of ER-bound proteins to isolate and identify intact co-regulatory proteins recruited to a ligand-activated ER. Through these approaches, we revealed differential binding affinity of bisphenol-A (BPA) and genistein (GEN) to the human ERα (ESR1) and ligand-dependent recruitment of SRC-1 and SRC-3 peptides. Recruitment profiles were variable for each ligand and in some cases were distinct compared to 17β-estradiol (E2). For example, E2 and GEN recruited both SRC-1 and -3 peptides whereas BPA recruited only SRC-1 peptides. Results of the functional proteomic assay showed differential recruitment between ligands where E2 recruited the greatest number of proteins followed by BPA then GEN. A number of proteins share previously identified relationships with ESR1 as determined by STRING analysis. Although there was limited overlap in proteins identified between treatments, all ligands recruited proteins involved in cell growth as determined by subnetwork enrichment analysis (p<0.05). A comparative, in silico analysis revealed that fewer interactions exist between zebrafish (Danio rerio) esr1 and zebrafish orthologs of proteins identified in our functional proteomic analysis. Taken together these results identify recruitment of known and previously unknown co-regulatory proteins to ESR1 and highlight new methods to assay recruitment of low abundant and intact, endogenous co-regulatory proteins to ESR1 or other nuclear receptors, in

  3. Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

    PubMed

    Hosoki, Koa; Aguilera-Aguirre, Leopoldo; Brasier, Allan R; Kurosky, Alexander; Boldogh, Istvan; Sur, Sanjiv

    2016-01-01

    Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation. PMID:26086549

  4. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes

    PubMed Central

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  5. Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.

    PubMed

    Sanders, M G; Parsons, M J; Howard, A G A; Liu, J; Fassio, S R; Martinez, J A; Bouchier-Hayes, L

    2015-01-01

    The human inflammatory caspases, including caspase-1, -4, -5 and -12, are considered as key regulators of innate immunity protecting from sepsis and numerous inflammatory diseases. Caspase-1 is activated by proximity-induced dimerization following recruitment to inflammasomes but the roles of the remaining inflammatory caspases in inflammasome assembly are unclear. Here, we use caspase bimolecular fluorescence complementation to visualize the assembly of inflammasomes and dimerization of inflammatory caspases in single cells. We observed caspase-1 dimerization induced by the coexpression of a range of inflammasome proteins and by lipospolysaccharide (LPS) treatment in primary macrophages. Caspase-4 and -5 were only dimerized by select inflammasome proteins, whereas caspase-12 dimerization was not detected by any investigated treatment. Strikingly, we determined that certain inflammasome proteins could induce heterodimerization of caspase-1 with caspase-4 or -5. Caspase-5 homodimerization and caspase-1/-5 heterodimerization was also detected in LPS-primed primary macrophages in response to cholera toxin subunit B. The subcellular localization and organization of the inflammasome complexes varied markedly depending on the upstream trigger and on which caspase or combination of caspases were recruited. Three-dimensional imaging of the ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)/caspase-1 complexes revealed a large spherical complex of ASC with caspase-1 dimerized on the outer surface. In contrast, NALP1 (NACHT leucine-rich repeat protein 1)/caspase-1 complexes formed large filamentous structures. These results argue that caspase-1, -4 or -5 can be recruited to inflammasomes under specific circumstances, often leading to distinctly organized and localized complexes that may impact the functions of these proteases. PMID:26158519

  6. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis.

    PubMed

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing; Persson, Staffan; Van Damme, Daniël; Li, Chuanyou; Bednarek, Sebastian Y; Pan, Jianwei

    2016-05-01

    In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2μ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2μ, was required for SA- and tyrphostin A23-dependent inhibition of CME Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells. PMID:26945051

  7. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis1[OPEN

    PubMed Central

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing

    2016-01-01

    In plants, clathrin-mediated endocytosis (CME) is dependent on the function of clathrin and its accessory heterooligomeric adaptor protein complexes, ADAPTOR PROTEIN2 (AP-2) and the TPLATE complex (TPC), and is negatively regulated by the hormones auxin and salicylic acid (SA). The details for how clathrin and its adaptor complexes are recruited to the plasma membrane (PM) to regulate CME, however, are poorly understood. We found that SA and the pharmacological CME inhibitor tyrphostin A23 reduce the membrane association of clathrin and AP-2, but not that of the TPC, whereas auxin solely affected clathrin membrane association, in Arabidopsis (Arabidopsis thaliana). Genetic and pharmacological experiments revealed that loss of AP2μ or AP2σ partially affected the membrane association of other AP-2 subunits and that the AP-2 subunit AP2σ, but not AP2μ, was required for SA- and tyrphostin A23-dependent inhibition of CME. Furthermore, we show that although AP-2 and the TPC are both required for the PM recruitment of clathrin in wild-type cells, the TPC is necessary for clathrin PM association in AP-2-deficient cells. These results indicate that developmental signals may differentially modulate the membrane recruitment of clathrin and its core accessory complexes to regulate the process of CME in plant cells. PMID:26945051

  8. PROGRESSIVE RECRUITMENT OF RUNX2 TO GENOMIC TARGETS DESPITE DECREASING EXPRESSION DURING OSTEOBLAST DIFFERENTIATION

    PubMed Central

    Pregizer, Steven; Baniwal, Sanjeev K.; Yan, Xiting; Borok, Zea; Frenkel, Baruch

    2008-01-01

    The mRNAs encoding Runx2, a master osteoblast transcription factor, and its target gene Osteocalcin (OC), are commonly used as markers of osteoblast differentiation. We found that while OC mRNA levels do indeed increase during development of the osteoblast phenotype in MC3T3-E1 cultures, Runx2 mRNA levels surprisingly decrease. Neither translational control of Runx2 (based on Western analysis) nor regulation of its DNA-binding ability (assessed by electrophoretic mobility shift assay) could explain the unexpected opposite patterns of Runx2 and OC expression. Instead, a series of ChIP assays during osteoblast differentiation revealed that early on, when Runx2 protein amount and DNA-binding activity are maximal, it is practically absent from the OC promoter. At later stages, Runx2 is recruited to the OC promoter while Runx2 mRNA, protein, and in vitro DNA binding progressively decrease. We also followed Runx2 occupancy at a novel genomic target discovered by ChIP-Chip analysis of cells in which the OC promoter is maximally occupied. The results revealed that Runx2 is recruited to this locus and to the OC promoter with a remarkably similar temporal pattern . These observations highlight a mechanism that restrains Runx2-mediated transcriptional control by confining its access to genomic targets to a narrow window of time. The need for such stringent control is consistent with the severe consequences of Runx2 over-expression in vivo. PMID:18821584

  9. How to assess positive end-expiratory pressure-induced alveolar recruitment?

    PubMed

    Lu, Q

    2013-01-01

    Randomized trials fail to demonstrate a decrease in mortality when high Positive End-Expiratory Pressure (PEEP) is applied to patients with acute respiratory distress syndrome. Use of PEEP in all patients without taking into consideration specific lung morphology, potential for recruitment and risk of lung hyperinflation could be one of explanations. Assessment of alveolar recruitment in each individual patient appears to reach a good compromise between optimization of mechanical ventilation and reduction of lung injury due to systematic application of high PEEP. The purpose of the review was to discuss different methods to measure alveolar recruitment aimed at selecting optimal PEEP. The revision of the literature includes relevant human and animal studies published in the past ten years describing validated and promising methods. Computed tomography remains the reference method to assess regional PEEP-induced alveolar recruitment and hyperinflation. Lung ultrasound and pressure-volume (P-V) curve method are simple and repeatable at the bedside, but they can't provide information on lung hyperinflation. Electrical impedance tomography allows bedside assessment of tidal recruitment in dependent and nondependent regions. By measuring functional residual capacity, alveolar recruitment and strain can be estimated. Decremental PEEP titration preceded by recruitment maneuver has been suggested to define optimal PEEP that sustains oxygenation benefit of recruitment maneuver. Different methods are available to assess PEEP-induced alveolar recruitment. Lung ultrasound and P-V curve method can be easily used at bedside to assess lung recruitability and test optimal PEEP. Further development is required for bedside assessment combing alveolar recruitment with hyperinflation. PMID:23135694

  10. Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements▿§

    PubMed Central

    De, Supriyo; Wurster, Andrea L.; Precht, Patricia; Wood, William H.; Becker, Kevin G.; Pazin, Michael J.

    2011-01-01

    T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes. PMID:21262765

  11. UV Radiation Induces the Epidermal Recruitment of Dendritic Cells that Compensate for the Depletion of Langerhans Cells in Human Skin.

    PubMed

    Achachi, Amine; Vocanson, Marc; Bastien, Philippe; Péguet-Navarro, Josette; Grande, Sophie; Goujon, Catherine; Breton, Lionel; Castiel-Higounenc, Isabelle; Nicolas, Jean-François; Gueniche, Audrey

    2015-08-01

    UVR causes skin injury and inflammation, resulting in impaired immune function and increased skin cancer risk. Langerhans cells (LCs), the immune sentinels of the epidermis, are depleted for several days following a single UVR exposure and can be reconstituted from circulating monocytes. However, the differentiation pathways leading to the recovery of a normal pool of LCs is still unclear. To study the dynamic changes in human skin with UV injury, we exposed a cohort of 29 healthy human volunteers to a clinically relevant dose of UVR and analyzed sequential epidermal biopsies for changes in leukocyte and dendritic cell (DC) subsets. UV-induced depletion of CD1a(high) LC was compensated by sequential appearance of various epidermal leukocytes. CD14(+) monocytes were recruited as early as D1 post exposure, followed by recruitment of two inflammatory DC subsets that may represent precursors of LCs. These CD1a(low) CD207(-) and the heretofore unknown CD1a(low) CD207(+) DCs appeared at day 1 and day 4 post UVR, respectively, and were endowed with T-cell-activating properties similar to those of LCs. We conclude that recruitment of monocytes and inflammatory DCs appear as a physiological response of the epidermis in order to repair UVR-induced LC depletion associated with immune suppression. PMID:25806853

  12. Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium

    PubMed Central

    McGettrick, Helen M; Buckley, Christopher D; Filer, Andrew; Ed Rainger, G; Nash, Gerard B

    2010-01-01

    Stromal fibroblasts modify the initial recruitment of leucocytes by endothelial cells (EC), but their effects on subsequent transendothelial migration remain unclear. Here, EC and dermal or synovial fibroblasts were cultured on opposite surfaces of 3-μm pore filters and incorporated in static or flow-based migration assays. Fibroblasts had little effect on tumour necrosis factor-α-induced transendothelial migration of neutrophils, but tended to increase the efficiency of migration away from the endothelium. Surprisingly, similar close contact between EC and fibroblasts strongly reduced lymphocyte migration in static assays, and nearly abolished stable lymphocyte adhesion from flow. Fibroblasts did not alter endothelial surface expression of adhesion molecules or messenger RNA for chemokines. Inhibition of attachment did not occur when EC-fibroblast contact was restricted by using 0·4-μm pore filters, but under these conditions pre-treatment with heparinase partially inhibited adhesion. In the 3-μm pore co-cultures, inhibition of metalloproteinase activity partially recovered lymphocyte adhesion, but addition of CXCL12 (SDF-1α) to the endothelial surface did not. Hence, the ability of EC to present activating chemokines for lymphocytes may have been enzymatically inhibited by direct contact with fibroblasts. To avoid contact, we cultured EC and fibroblasts on separate 3-μm pore filters one above the other. Here, fibroblasts promoted the transendothelial migration of lymphocytes. Fibroblasts generate CXCL12, but blockade of CXCL12 receptor had no effect on lymphocyte migration. While stromal cells can provide signal(s) promoting leucocyte migration away from the sub-endothelial space, direct cell contact (which might occur in damaged tissue) may cause disruption of chemokine signalling, specifically inhibiting lymphocyte rather than neutrophil recruitment. PMID:20518822

  13. A splice variant of RILP induces lysosomal clustering independent of dynein recruitment

    SciTech Connect

    Marsman, Marije; Jordens, Ingrid; Rocha, Nuno; Kuijl, Coenraad; Janssen, Lennert; Neefjes, Jacques . E-mail: j.neefjes@nki.nl

    2006-06-09

    The small GTPase Rab7 controls fusion and transport of late endocytic compartments. A critical mediator is the Rab7 effector RILP that recruits the minus-end dynein-dynactin motor complex to these compartments. We identified a natural occurring splice variant of RILP (RILPsv) lacking only 27 amino acids encoded by exon VII. Both variants bind Rab7, prolong its GTP-bound state, and induce clustering of late endocytic compartments. However, RILPsv does not recruit the dynein-dynactin complex, implicating exon VII in motor recruitment. Clustering might still occur via dimerization, since both RILP and RILPsv are able to form hetero- and homo-dimers. Moreover, both effectors compete for Rab7 binding but with different outcome for dynein-dynactin recruitment and transport. Hence, RILPsv provides an extra dimension to the control of vesicle fusion and transport by the small GTPase Rab7.

  14. Nicorandil inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in mice.

    PubMed

    Matsui, Tamires C; Coura, Giovanna M E; Melo, Ivo S F; Batista, Carla R A; Augusto, Paulo Sérgio A; Godin, Adriana M; Araújo, Débora P; César, Isabela C; Ribeiro, Lucas S; Souza, Danielle G; Klein, André; de Fátima, Ângelo; Machado, Renes R; Coelho, Márcio M

    2015-12-15

    Nicorandil is a drug characterized by the coupling of a nitric oxide (NO) donor to nicotinamide. We have previously demonstrated that nicotinamide exhibits activity in different models of pain and inflammation. Now, we investigated the effects induced by per os (p.o.) administration of nicorandil (25, 50 or 100mg/Kg) on neutrophil recruitment in a carrageenan-induced model of pleurisy in mice. Effects induced by nicorandil (100mg/kg) were compared with those induced by equimolar doses of nicotinamide (58mg/kg) and N-(2-hydroxyethyl)-nicotinamide (NHN; 79mg/kg). We also investigated whether effects on the production of inflammatory mediators play a role in the activity of nicorandil. P.o. nicorandil, 0.5h before and 1h after the i.pl. injection of carrageenan, reduced neutrophil recruitment. However, equimolar doses of nicotinamide or NHN failed to induce such effect. Single treatment (previous or late) with nicorandil (100mg/Kg, p.o.) also reduced neutrophils recruitment, although to a lesser extent when compared to the double treatment. Nicorandil reduced the concentrations of interleukin-1β, CXCL-1 and prostaglandin E2 in the pleural exudate. Concluding, we demonstrated the activity of nicorandil in a model of pleurisy induced by carrageenan. This activity was characterized by reduction of the neutrophil accumulation and inhibition of production of inflammatory mediators. The effects induced by nicorandil on the leukocytes recruitment and production of inflammatory mediators contribute to a better understanding of its clinical benefits and indicate that these benefits may be due to its vasodilating and anti-inflammatory activities. PMID:26607465

  15. NK cells promote neutrophil recruitment in the brain during sepsis-induced neuroinflammation

    PubMed Central

    He, Hao; Geng, Tingting; Chen, Piyun; Wang, Meixiang; Hu, Jingxia; Kang, Li; Song, Wengang; Tang, Hua

    2016-01-01

    Sepsis could affect the central nervous system and thus induces neuroinflammation, which subsequently leads to brain damage or dysfunction. However, the mechanisms of generation of neuroinflammation during sepsis remain poorly understood. By administration of lipopolysaccharides (LPS) in mice to mimic sepsis, we found that shortly after opening the blood–brain barrier, conventional CD11b+CD27+ NK subset migrated into the brain followed by subsequent neutrophil infiltration. Interestingly, depletion of NK cells prior to LPS treatment severely impaired neutrophil recruitment in the inflamed brain. By in vivo recruitment assay, we found that brain-infiltrated NK cells displayed chemotactic activity to neutrophils, which depended on the higher expression of chemokines such as CXCL2. Moreover, microglia were also responsible for neutrophil recruitment, and their chemotactic activity was significantly impaired by ablation of NK cells. Furthermore, depletion of NK cells could significantly ameliorate depression-like behavior in LPS-treated mice. These data indicated a NK cell-regulated neutrophil recruitment in the blamed brain, which also could be seen on another sepsis model, cecal ligation and puncture. So, our findings revealed an important scenario in the generation of sepsis-induced neuroinflammation. PMID:27270556

  16. NK cells promote neutrophil recruitment in the brain during sepsis-induced neuroinflammation.

    PubMed

    He, Hao; Geng, Tingting; Chen, Piyun; Wang, Meixiang; Hu, Jingxia; Kang, Li; Song, Wengang; Tang, Hua

    2016-01-01

    Sepsis could affect the central nervous system and thus induces neuroinflammation, which subsequently leads to brain damage or dysfunction. However, the mechanisms of generation of neuroinflammation during sepsis remain poorly understood. By administration of lipopolysaccharides (LPS) in mice to mimic sepsis, we found that shortly after opening the blood-brain barrier, conventional CD11b(+)CD27(+) NK subset migrated into the brain followed by subsequent neutrophil infiltration. Interestingly, depletion of NK cells prior to LPS treatment severely impaired neutrophil recruitment in the inflamed brain. By in vivo recruitment assay, we found that brain-infiltrated NK cells displayed chemotactic activity to neutrophils, which depended on the higher expression of chemokines such as CXCL2. Moreover, microglia were also responsible for neutrophil recruitment, and their chemotactic activity was significantly impaired by ablation of NK cells. Furthermore, depletion of NK cells could significantly ameliorate depression-like behavior in LPS-treated mice. These data indicated a NK cell-regulated neutrophil recruitment in the blamed brain, which also could be seen on another sepsis model, cecal ligation and puncture. So, our findings revealed an important scenario in the generation of sepsis-induced neuroinflammation. PMID:27270556

  17. RORγt modulates macrophage recruitment during a hydrocarbon oil-induced inflammation.

    PubMed

    Wu, Qi; Sun, Xin; Chi, Ruo; Xu, Long; Li, Xue; Feng, Jing; Chen, Huaiyong

    2013-01-01

    Hydrocarbon oils are often utilized as adjuvants in vaccines. In response to naturally occurring hydrocarbon oils, inflammation is initiated and persists with the continuous recruitment of immune cells such as macrophages and neutrophils. However, the mechanism underlying the chronic inflammation in response to hydrocarbon oils is not fully defined. In this study, we revealed an essential role of retinoid-related orphan receptor gamma t (RORγt) in sustaining the recruitment of macrophages following pristane treatment. RORγt absence resulted in the incompetent formation of mesenteric oil granulomas which may associate to a reduction in the migration of macrophages into the mesentery during pristane-induced inflammation. This is at least partially dependent on the expression of the monocyte chemoattractant protein-1 (MCP-1) in the mesentery and the decrease in the macrophage reservoir in the spleen. However, the absence of RORγt had no impact on the recruitment of neutrophils to the mesentery after pristane treatment. Our data uncovered an important role of RORγt in the recruitment of macrophages during hydrocarbon oil-induced chronic inflammation. PMID:24260235

  18. RORγt Modulates Macrophage Recruitment during a Hydrocarbon Oil-Induced Inflammation

    PubMed Central

    Chi, Ruo; Xu, Long; Li, Xue; Feng, Jing; Chen, Huaiyong

    2013-01-01

    Hydrocarbon oils are often utilized as adjuvants in vaccines. In response to naturally occurring hydrocarbon oils, inflammation is initiated and persists with the continuous recruitment of immune cells such as macrophages and neutrophils. However, the mechanism underlying the chronic inflammation in response to hydrocarbon oils is not fully defined. In this study, we revealed an essential role of retinoid-related orphan receptor gamma t (RORγt) in sustaining the recruitment of macrophages following pristane treatment. RORγt absence resulted in the incompetent formation of mesenteric oil granulomas which may associate to a reduction in the migration of macrophages into the mesentery during pristane-induced inflammation. This is at least partially dependent on the expression of the monocyte chemoattractant protein-1 (MCP-1) in the mesentery and the decrease in the macrophage reservoir in the spleen. However, the absence of RORγt had no impact on the recruitment of neutrophils to the mesentery after pristane treatment. Our data uncovered an important role of RORγt in the recruitment of macrophages during hydrocarbon oil-induced chronic inflammation. PMID:24260235

  19. Particulate matter phagocytosis induces tissue factor in differentiating macrophages.

    PubMed

    Milano, M; Dongiovanni, P; Artoni, A; Gatti, S; Rosso, L; Colombo, F; Bollati, V; Maggioni, M; Mannucci, P M; Bertazzi, P A; Fargion, S; Valenti, L

    2016-01-01

    Airborne exposure to particulate matter with diameter < 10 mcM (PM10) has been linked to an increased risk of thromboembolic events, but the mechanisms are not completely understood. The aim of this study was to evaluate the effect of PM10 phagocytosis on the release of procoagulant molecules in human differentiating macrophages, and that of PM10 inhalation in an experimental model in rats. Human monocytes were separated from the peripheral blood by the lymphoprep method, differentiated in vitro and treated with standard PM10 or vehicle. Sprague-Dawley rats were instilled intratracheally with PM10 or vehicle alone. The outcome was expression of proinflammatory genes and of tissue factor (TF). In human differentiating macrophages, PM10 exposure upregulated inflammatory genes, but most consistently induced TF mRNA and protein levels, but not TF protein inhibitor, resulting in increased TF membrane expression and a procoagulant phenotype. Differentiation towards the anti-inflammatory M2 phenotype inhibited PM10 -mediated TF expression. TF induction required phagocytosis of PM10 , whereas phagocytosis of inert particles was less effective. PM10 phagocytosis was associated with a gene expression profile consistent with intracellular retention of iron, inducing oxidative stress. Both PM10 and iron activated the stress kinases ERK1/2 pathway, involved in the induction of TF expression. In rats, alveolar exposure to PM10 was associated with pulmonary recruitment of inflammatory cells and resulted in local, but not systemic, induction of TF expression, which was sufficient to increase circulating TF levels. In conclusion, TF induction by differentiating lung macrophages, activated following phagocytosis, contributes to the increased risk of thromboembolic complications associated with PM10 exposure. PMID:25858758

  20. Troglitazone induces differentiation in Trypanosoma brucei

    SciTech Connect

    Denninger, Viola; Figarella, Katherine; Schoenfeld, Caroline; Brems, Stefanie; Busold, Christian; Lang, Florian; Hoheisel, Joerg; Duszenko, Michael . E-mail: michael.duszenko@uni-tuebingen.de

    2007-05-15

    Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor {gamma}. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.

  1. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs

    PubMed Central

    Yu, Huansha; Liu, Xing; Huang, Lulu; Wang, Qiang; Liu, Heng; Cui, Ye; Tang, Yijun; Zhang, Peng; Wang, Chen

    2016-01-01

    Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS) induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses. PMID:26900919

  2. Zeb2 recruits HDAC-NuRD to inhibit Notch and controls Schwann cell differentiation and remyelination.

    PubMed

    Wu, Lai Man Natalie; Wang, Jincheng; Conidi, Andrea; Zhao, Chuntao; Wang, Haibo; Ford, Zachary; Zhang, Liguo; Zweier, Christiane; Ayee, Brian G; Maurel, Patrice; Zwijsen, An; Chan, Jonah R; Jankowski, Michael P; Huylebroeck, Danny; Lu, Q Richard

    2016-08-01

    The mechanisms that coordinate and balance a complex network of opposing regulators to control Schwann cell (SC) differentiation remain elusive. Here we demonstrate that zinc-finger E-box-binding homeobox 2 (Zeb2, also called Sip1) transcription factor is a critical intrinsic timer that controls the onset of SC differentiation by recruiting histone deacetylases HDAC 1 and 2 (HDAC1/2) and nucleosome remodeling and deacetylase complex (NuRD) co-repressor complexes in mice. Zeb2 deletion arrests SCs at an undifferentiated state during peripheral nerve development and inhibits remyelination after injury. Zeb2 antagonizes inhibitory effectors including Notch and Sox2. Importantly, genome-wide transcriptome analysis reveals a Zeb2 target gene encoding the Notch effector Hey2 as a potent inhibitor for Schwann cell differentiation. Strikingly, a genetic Zeb2 variant associated with Mowat-Wilson syndrome disrupts the interaction with HDAC1/2-NuRD and abolishes Zeb2 activity for SC differentiation. Therefore, Zeb2 controls SC maturation by recruiting HDAC1/2-NuRD complexes and inhibiting a Notch-Hey2 signaling axis, pointing to the critical role of HDAC1/2-NuRD activity in peripheral neuropathies caused by ZEB2 mutations. PMID:27294509

  3. Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

    PubMed Central

    Elazar, Sharon; Gonen, Erez; Livneh-Kol, Ayala; Rosenshine, Ilan; Shpigel, Nahum Yehuda

    2009-01-01

    Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO. PMID:19828114

  4. Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

    PubMed

    Longo, Michele; Spinelli, Rosa; D'Esposito, Vittoria; Zatterale, Federica; Fiory, Francesca; Nigro, Cecilia; Raciti, Gregory A; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco; Di Jeso, Bruno

    2016-06-01

    Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. PMID:26940722

  5. Differential recruitment of introduced Pacific oysters and native mussels at the North Sea coast: coexistence possible?

    NASA Astrophysics Data System (ADS)

    Diederich, Susanne

    2005-04-01

    Pacific oysters ( Crassostrea gigas Thunberg 1793) have been introduced into the Wadden Sea (North Sea), where they settle on native mussel beds ( Mytilus edulis L.), which represent the only extensive insular hard substrata in this soft-sediment environment. As abundances of C. gigas rose, some mussel beds became increasingly overgrown with oysters, whereas others did not. Field experiments revealed that recruitment of C. gigas was higher in the lower intertidal than in the upper subtidal zone, that it was higher on conspecifics than on mussels, and that it was not affected by barnacle epigrowth except when settling on mussels. Mussel recruitment is known from inter- and subtidal zones. It occurred equally on oyster and mussel shells but showed a clear preference for barnacle epigrowth over clean shells. Assuming that settlement and recruitment are key processes for species abundances on the North Sea coast, it is predicted that the positive feedback in oyster settlement will lead to rapid reef formation of this invader at the expense of mussel beds. Mussels, however, may escape competitive exclusion by settling between or on the larger oysters especially when barnacles are abundant. Experimental patches with mussels were more often covered by fucoid algae ( Fucus vesiculosus forma mytili Nienburg) than patches with oysters, and oyster recruitment was poor underneath such algal canopies. Thus, fucoids may provide the native mussels with a refuge from the invading oysters and the two bivalves may coexist, provided food is not limiting.

  6. IQGAP1 controls tight junction formation through differential regulation of claudin recruitment

    PubMed Central

    Tanos, Barbara E.; Perez Bay, Andres E.; Salvarezza, Susana; Vivanco, Igor; Mellinghoff, Ingo; Osman, Mahasin; Sacks, David B.; Rodriguez-Boulan, Enrique

    2015-01-01

    ABSTRACT IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42–JNK pathway. PMID:25588839

  7. Induced differentiation inhibits sphere formation in neuroblastoma.

    PubMed

    Craig, Brian T; Rellinger, Eric J; Alvarez, Alexandra L; Dusek, Haley L; Qiao, Jingbo; Chung, Dai H

    2016-08-19

    Neuroblastoma arises from the neural crest, the precursor cells of the sympathoadrenal axis, and differentiation status is a key prognostic factor used for clinical risk group stratification and treatment strategies. Neuroblastoma tumor-initiating cells have been successfully isolated from patient tumor samples and bone marrow using sphere culture, which is well established to promote growth of neural crest stem cells. However, accurate quantification of sphere-forming frequency of commonly used neuroblastoma cell lines has not been reported. Here, we show that MYCN-amplified neuroblastoma cell lines form spheres more frequently than non-MYCN-amplified cell lines. We also show that sphere formation is directly sensitive to cellular differentiation status. 13-cis-retinoic acid is a clinically used differentiating agent that induces a neuronal phenotype in neuroblastoma cells. Induced differentiation nearly completely blocked sphere formation. Furthermore, sphere formation was specifically FGF-responsive and did not respond to increasing doses of EGF. Taken together, these data suggest that sphere formation is an accurate method of quantifying the stemness phenotype in neuroblastoma. PMID:27297102

  8. Twist1 induces CCL2 and recruits macrophages to promote angiogenesis

    PubMed Central

    Low-Marchelli, Janine M.; Ardi, Veronica C.; Vizcarra, Edward A.; van Rooijen, Nico; Quigley, James P.; Yang, Jing

    2012-01-01

    The transcription factor Twist1 induces Epithelial-Mesenchymal Transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common pro-angiogenic factors VEGF and bFGF, but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. Since Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers. PMID:23329645

  9. TLR2-induced IL-10 production impairs neutrophil recruitment to infected tissues during neonatal bacterial sepsis.

    PubMed

    Andrade, Elva B; Alves, Joana; Madureira, Pedro; Oliveira, Liliana; Ribeiro, Adília; Cordeiro-da-Silva, Anabela; Correia-Neves, Margarida; Trieu-Cuot, Patrick; Ferreira, Paula

    2013-11-01

    Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti-IL-10R mAb treatment or by using IL-10-deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis. PMID:24078699

  10. Hypoxia Inducible Factor 1 (HIF-1) Recruits Macrophage to Activate Pancreatic Stellate Cells in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Li, Na; Li, Yang; Li, Zengxun; Huang, Chongbiao; Yang, Yanhui; Lang, Mingxiao; Cao, Junli; Jiang, Wenna; Xu, Yu; Dong, Jie; Ren, He

    2016-01-01

    Hypoxia inducible factor 1 (HIF-1) is a transcription factor composed of two subunits, namely, HIF-1α and HIF-1β, in which HIF-1β is constitutively expressed. HIF-1 upregulates several hypoxia-responsive proteins, including angiogenesis factors, glycolysis solution enzymes, and cell survival proteins. HIF-1 is also associated with the degree of inflammation in the tumor region, but the exact mechanism remains unclear. This study aims to identify the molecular mechanism of recruiting monocytes/macrophages by HIF-1α in pancreatic ductal adenocarcinoma (PDAC) and the effects of macrophages on pancreatic stellate cells (PSCs). Immunohistochemistry (IHC) was performed for cluster of differentiation 68 (CD68), HIF-1α, and chemical chemokines 2 (CCL2). Western blot, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation assay, and The Cancer Genome Atlas (TCGA) were used to verify the correlation between HIF-1α and CCL2 at protein and nucleic acid levels. Monocytes/macrophages were co-cultured with PSCs to observe their interaction. Samples showed significant correlation between CD68 and HIF-1α (t-test, p < 0.05). HIF-1α recruited monocytes/macrophages by promoting CCL2 secretion. Moreover, macrophages could accelerate the activation of PSCs. HIF-1α might promote inflammation and fibrosis of PDAC through CCL2 secretion, which may provide a novel target to treat PDAC patients. PMID:27271610

  11. Subterranean Herbivore-induced Volatiles Released by Citrus Roots upon Feeding by Diaprepes abbreviatus Recruit Entomopathogenic Nematodes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Herbivore induced volatile emissions benefit plant hosts by recruiting natural enemies of herbivorous insects. Such tritrophic interactions have been thoroughly examined in the above-ground terrestrial environment. Recently, similar signals have also been described in the subterranean environment, ...

  12. Neotuberostemonine attenuates bleomycin-induced pulmonary fibrosis by suppressing the recruitment and activation of macrophages.

    PubMed

    Xiang, Juan; Cheng, Si; Feng, Tianlong; Wu, Yan; Xie, Weina; Zhang, Mian; Xu, Xianghong; Zhang, Chaofeng

    2016-07-01

    Neotuberostemonine (NTS) is one of the main antitussive alkaloids in the root of Stemona tuberosa Lour. This study aimed to investigate the effects of NTS on bleomycin (BLM)-induced pulmonary fibrosis in mice and the underlying mechanism. After BLM administration, NTS were orally administered to mice at 20 and 40mg/kg per day from days 8 to 21, with nintedanib as a positive control. The effect of NTS on BLM-induced mice was assessed via histopathological examination by HE and Masson's trichrome staining, TGF-β1 level and macrophage recruitment by immunohistochemical staining, expression of profibrotic media and M1/M2 polarization by western blot. RAW 264.7 cells were used to evaluate whether NTS (1, 10, 100μM) directly affected macrophages. The results revealed that NTS treatment significantly ameliorated lung histopathological changes and decreased inflammatory cell counts in the bronchoalveolar lavage fluid. The over-expression of collagen, α-SMA and TGF-β1 was reduced by NTS. Furthermore, NTS markedly lowered the expression of MMP-2 and TIMP-1 while raised the expression of MMP-9. A further analysis showed that NTS was able to decrease the recruitment of macrophages and to inhibit the M2 polarization in mice lung tissues. The experiment in vitro showed that NTS significantly reduced the arginase-1 (marker for M2) expression in a dose-dependent manner but down-regulated the iNOS (marker for M1) expression only at 100μM. In conclusion, our study demonstrated for the first time that NTS has a significant protective effect on BLM-induced pulmonary fibrosis through suppressing the recruitment and M2 polarization of macrophages. PMID:27144994

  13. The extinction differential induced virulence macroevolution

    NASA Astrophysics Data System (ADS)

    Zhang, Feng; Xu, Liufang; Wang, Jin

    2014-04-01

    We apply the potential-flux landscape theory to deal with the large fluctuation induced extinction phenomena. We quantify the most probable extinction pathway on the landscape and measure the extinction risk by the landscape topography. In this Letter, we investigate the disease extinction through an epidemic model described by a set of chemical reaction. We found the virulence-differential-dependent symbioses between mother and daughter pathogen species: mutualism and parasitism. The symbioses, whether mutualism or parasitism, benefit the higher virulence species. This implies that speciation towards lower virulence is an effective strategy for a pathogen species to reduce its extinction risk.

  14. Effect of boldine, secoboldine, and boldine methine on angiotensin II-induced neutrophil recruitment in vivo.

    PubMed

    Estellés, Rossana; Milian, Lara; Nabah, Yafa Naim Abu; Mateo, Teresa; Cerdá-Nicolás, Miguel; Losada, Mercedes; Ivorra, María Dolores; Issekutz, Andrew C; Cortijo, Julio; Morcillo, Esteban J; Blázquez, María Amparo; Sanz, María-Jesús

    2005-09-01

    Angiotensin-II (Ang-II) has inflammatory activity and is involved in different diseases associated with the cardiovascular system. This study has evaluated the effect of boldine (B), and two phenanthrene alkaloids semisynthesized by us, secoboldine (SB) and boldine methine (BM), on Ang-II-induced neutrophil recruitment. Intraperitoneal administration of 1 nM Ang-II induced significant neutrophil accumulation, which was maximal at 4-8 h. BM inhibited neutrophil infiltration into the peritoneal cavity at 4 h and 8 h by 73% and 77%, respectively, SB at 8 h by 55%, and B had no effect on this response. Although BM inhibited the release of cytokine-inducible neutrophil chemoattractant/keratinocyte-derived chemokine, macrophage inflammatory protein-2 (MIP-2), and platelet-activating factor (PAF) elicited by Ang-II, SB only reduced the release of MIP-2 after 4 h of its administration. Sixty-minute superfusion of the rat mesentery with 1 nM Ang-II induced a significant increase in the leukocyte-endothelial cell interactions and P-selectin up-regulation, which were inhibited by 1 microM BM and SB. The generation of reactive oxygen species (ROS) in endothelial cells stimulated with Ang-II was inhibited significantly by the three alkaloids tested. BM also diminished Ang-II-induced interleukin-8 release from endothelial cells and blocked the PAF receptor on human neutrophils (concentration of the compound needed to produce 50% inhibition value: 28.2 microM). Therefore, BM is a potent inhibitor of Ang-II-induced neutrophil accumulation in vivo. This effect appears to be mediated through inhibition of CXC chemokine and PAF release, ROS scavenging activity, and blockade of the PAF receptor. Thus, it may have potential therapeutic interest for the control of neutrophil recruitment that occurs in inflammation associated with elevated levels of Ang-II. PMID:15944212

  15. Orai1 controls C5a-induced neutrophil recruitment in inflammation.

    PubMed

    Sogkas, Georgios; Vögtle, Timo; Rau, Eduard; Gewecke, Britta; Stegner, David; Schmidt, Reinhold E; Nieswandt, Bernhard; Gessner, J Engelbert

    2015-07-01

    Stromal interaction molecule 1 (STIM1)-dependent store operated calcium-entry (SOCE) through Orai1-mediated calcium (Ca(2+) ) influx is considered a major pathway of Ca(2+) signaling, serving T-cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1-deficient neutrophils present defects in fMLP and complement C5a-induced Ca(2+) influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1-deficient neutrophils exhibit normal STIM1-dependent SOCE and STIM1-deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1(-/-) chimeric mice show impaired neutrophil recruitment in LPS-induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a-triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1(-/-) chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG-induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1-independent pathway of Ca(2+) -influx in neutrophils. PMID:25912155

  16. Differential Recruitment of Dentate Gyrus Interneuron Types by Commissural Versus Perforant Pathways.

    PubMed

    Hsu, Tsan-Ting; Lee, Cheng-Ta; Tai, Ming-Hong; Lien, Cheng-Chang

    2016-06-01

    Gamma-aminobutyric acidergic (GABAergic) interneurons (INs) in the dentate gyrus (DG) provide inhibitory control to granule cell (GC) activity and thus gate incoming signals to the hippocampus. However, how various IN subtypes inhibit GCs in response to different excitatory input pathways remains mostly unknown. By using electrophysiology and optogenetics, we investigated neurotransmission of the hilar commissural pathway (COM) and the medial perforant path (MPP) to the DG in acutely prepared mouse slices. We found that the short-term dynamics of excitatory COM-GC and MPP-GC synapses was similar, but that the dynamics of COM- and MPP-mediated inhibition measured in GCs was remarkably different, during theta-frequency stimulation. This resulted in the increased inhibition-excitation (I/E) ratios in single GCs for COM stimulation, but decreased I/E ratios for MPP stimulation. Further analysis of pathway-specific responses in identified INs revealed that basket cell-like INs, total molecular layer- and molecular layer-like cells, received greater excitation and were more reliably recruited by the COM than by the MPP inputs. In contrast, hilar perforant path-associated and hilar commissural-associational pathway-related-like cells were minimally activated by both inputs. These results demonstrate that distinct IN subtypes are preferentially recruited by different inputs to the DG, and reveal their relative contributions in COM-mediated feedforward inhibition. PMID:26045570

  17. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation

    PubMed Central

    Fan, Hong; Zeng, Qin; Pennypacker, Sally D.; Xie, Zhongjian

    2016-01-01

    Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. PMID:27340655

  18. Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation.

    PubMed

    Shrestha, Chandrama; Tang, Yuanyuan; Fan, Hong; Li, Lusha; Zeng, Qin; Pennypacker, Sally D; Bikle, Daniel D; Xie, Zhongjian

    2016-01-01

    Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-β-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation. PMID:27340655

  19. The involvement of platelet activating factor in endotoxin-induced pulmonary platelet recruitment in the guinea-pig.

    PubMed Central

    Beijer, L.; Botting, J.; Crook, P.; Oyekan, A. O.; Page, C. P.; Rylander, R.

    1987-01-01

    1 Exposure of conscious guinea-pigs to an aerosol of endotoxin (25-100 micrograms ml-1) resulted in a dose-related, progressive accumulation of platelets in the thoracic region. Accumulation of 111indium oxine labelled erythrocytes was not observed following exposure to an aerosol of endotoxin (50 micrograms ml-1). 2 Pretreatment of guinea-pigs with the selective platelet activating factor (Paf)-antagonists. CV-3988 or brotizolam resulted in a dose-related inhibition of endotoxin-induced pulmonary platelet recruitment. Pretreatment of guinea-pigs with the selective Paf-antagonist BN 52021 resulted in significant inhibition of endotoxin-induced pulmonary platelet recruitment, although the effects of BN 52021 were not dose-related. 3 Pretreatment of guinea-pigs with indomethacin at doses known to inhibit cyclo-oxygenase did not inhibit endotoxin-induced pulmonary platelet recruitment, whereas higher doses of indomethacin produced a reduction in platelet recruitment in the lung. 4 Pretreatment of guinea-pigs with the anticoagulant heparin and the prostacyclin analogue ZK 36374 inhibited endotoxin-induced platelet recruitment. 5 These observations suggest that endotoxin-induced pulmonary platelet recruitment in the guinea-pig is secondary to the release of platelet activating factor, but not to cyclo-oxygenase products of arachidonic acid and may also involve activation of the coagulation cascade. PMID:2447993

  20. Basolateral and central amygdala differentially recruit and maintain dorsolateral striatum-dependent cocaine-seeking habits

    PubMed Central

    Murray, Jennifer E.; Belin-Rauscent, Aude; Simon, Marine; Giuliano, Chiara; Benoit-Marand, Marianne; Everitt, Barry J.; Belin, David

    2015-01-01

    In the development of addiction, drug seeking becomes habitual and controlled by drug-associated cues, and the neural locus of control over behaviour shifts from the ventral to the dorsolateral striatum. The neural mechanisms underlying this functional transition from recreational drug use to drug-seeking habits are unknown. Here we combined functional disconnections and electrophysiological recordings of the amygdalo-striatal networks in rats trained to seek cocaine to demonstrate that functional shifts within the striatum are driven by transitions from the basolateral (BLA) to the central (CeN) amygdala. Thus, while the recruitment of dorsolateral striatum dopamine-dependent control over cocaine seeking is triggered by the BLA, its long-term maintenance depends instead on the CeN. These data demonstrate that limbic cortical areas both tune the function of cognitive territories of the striatum and thereby underpin maladaptive cocaine-seeking habits. PMID:26657320

  1. The Price Differential's Impact on Retention, Recruitment, and Quality in a Public University.

    ERIC Educational Resources Information Center

    Little, Michale W.; O'Toole, Dennis; Wetzel, James

    1997-01-01

    A survey of 467 business students at Virginia Commonwealth University investigated results of the business school's tuition differential pricing strategy, which charges a higher tuition in order to provide students with strong computer and technological instruction and support. Results indicated the additional services created added value for…

  2. GAP JUNCTION COMMUNICATION MEDIATES TRANSFORMING GROWTH FACTOR-BETA ACTIVATION AND ENDOTHELIAL-INDUCED MURAL CELL DIFFERENTIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recrui...

  3. Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment.

    PubMed

    Hu, Xiaoqian; Cifarelli, Vincenza; Sun, Shishuo; Kuda, Ondrej; Abumrad, Nada A; Su, Xiong

    2016-04-01

    Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E2(PGE2) and prostaglandin D2(PGD2), reflecting cytosolic phospholipase A2α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE2potently induced macrophage migration while different FFAs and PGD2had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE2levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE2with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis. PMID:26912395

  4. Pigment cell interactions and differential xanthophore recruitment underlying zebrafish stripe reiteration and Danio pattern evolution

    PubMed Central

    Patterson, Larissa B.; Bain, Emily J.; Parichy, David M.

    2014-01-01

    Fishes have diverse pigment patterns, yet mechanisms of pattern evolution remain poorly understood. In zebrafish, Danio rerio, pigment-cell autonomous interactions generate dark stripes of melanophores that alternate with light interstripes of xanthophores and iridophores. Here, we identify mechanisms underlying the evolution of a uniform pattern in D. albolineatus in which all three pigment cell classes are intermingled. We show that in this species xanthophores differentiate precociously over a wider area, and that cis regulatory evolution has increased expression of xanthogenic Colony Stimulating Factor-1 (Csf1). Expressing Csf1 similarly in D. rerio has cascading effects, driving the intermingling of all three pigment cell classes and resulting in the loss of stripes, as in D. albolineatus. Our results identify novel mechanisms of pattern development and illustrate how pattern diversity can be generated when a core network of pigment-cell autonomous interactions is coupled with changes in pigment cell differentiation. PMID:25374113

  5. Differential recruitment of executive control regions during phonological competition in monolinguals and bilinguals.

    PubMed

    Marian, Viorica; Chabal, Sarah; Bartolotti, James; Bradley, Kailyn; Hernandez, Arturo E

    2014-12-01

    Behavioral research suggests that monolinguals and bilinguals differ in how they manage within-language phonological competition when listening to language. The current study explored whether bilingual experience might also change the neural resources recruited to control spoken-word competition. Seventeen Spanish-English bilinguals and eighteen English monolinguals completed an fMRI task in which they searched for a picture representing an aurally presented word (e.g., "candy") from an array of four presented images. On competitor trials, one of the objects in the display shared initial phonological overlap with the target (e.g., candle). While both groups experienced competition and responded more slowly on competitor trials than on unrelated trials, fMRI data suggest that monolinguals, but not bilinguals, activated executive control regions (e.g., anterior cingulate, superior frontal gyrus) during within-language phonological competition. We conclude that differences in how monolinguals and bilinguals manage competition may result from bilinguals' more efficient deployment of neural resources. PMID:25463821

  6. Differential Recruitment of Executive Control Regions during Phonological Competition in Monolinguals and Bilinguals

    PubMed Central

    Marian, Viorica; Chabal, Sarah; Bartolotti, James; Bradley, Kailyn; Hernandez, Arturo E.

    2015-01-01

    Behavioral research suggests that monolinguals and bilinguals differ in how they manage within-language phonological competition when listening to language. The current study explored whether bilingual experience might also change the neural resources recruited to control spoken-word competition. Seventeen Spanish-English bilinguals and eighteen English monolinguals completed an fMRI task in which they searched for a picture representing an aurally presented word (e.g., “candy”) from an array of four presented images. On competitor trials, one of the objects in the display shared initial phonological overlap with the target (e.g., candle). While both groups experienced competition and responded more slowly on competitor trials than on unrelated trials, fMRI data suggest that monolinguals, but not bilinguals, activated executive control regions (e.g., anterior cingulate, superior frontal gyrus) during within-language phonological competition. We conclude that differences in how monolinguals and bilinguals manage competition may result from bilinguals’ more efficient deployment of neural resources. PMID:25463821

  7. Carboxy-Terminus Recruitment Induced by Substrate Binding in Eukaryotic Fructose Bis-phosphate Aldolases

    SciTech Connect

    Lafrance-Vanasse,J.; Sygusch, J.

    2007-01-01

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 {angstrom} resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P{sub 1}-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P{sub 1}-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P{sub 1}-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine.

  8. Carboxy-terminus recruitment induced by substrate binding in eukaryotic fructose bis-phosphate aldolases.

    PubMed

    Lafrance-Vanasse, Julien; Sygusch, Jurgen

    2007-08-21

    The crystal structures of Leishmania mexicana fructose-1,6-bis(phosphate) aldolase in complex with substrate and competitive inhibitor, mannitol-1,6-bis(phosphate), were solved to 2.2 A resolution. Crystallographic analysis revealed a Schiff base intermediate trapped in the native structure complexed with substrate while the inhibitor was trapped in a conformation mimicking the carbinolamine intermediate. Binding modes corroborated previous structures reported for rabbit muscle aldolase. Amino acid substitution of Gly-312 to Ala, adjacent to the P1-phosphate binding site and unique to trypanosomatids, did not perturb ligand binding in the active site. Ligand attachment ordered amino acid residues 359-367 of the C-terminal region (353-373) that was disordered beyond Asp-358 in the unbound structure, revealing a novel recruitment mechanism of this region by aldolases. C-Terminal peptide ordering is triggered by P1-phosphate binding that induces conformational changes whereby C-terminal Leu-364 contacts P1-phosphate binding residue Arg-313. C-Terminal region capture synergizes additional interactions with subunit surface residues, not perturbed by P1-phosphate binding, and stabilizes C-terminal attachment. Amino acid residues that participate in the capturing interaction are conserved among class I aldolases, indicating a general recruitment mechanism whereby C-terminal capture facilitates active site interactions in subsequent catalytic steps. Recruitment accelerates the enzymatic reaction by using binding energy to reduce configurational entropy during catalysis thereby localizing the conserved C-terminus tyrosine, which mediates proton transfer, proximal to the active site enamine. PMID:17661446

  9. Monocyte Recruitment to the Dermis and Differentiation to Dendritic Cells Increases the Targets for Dengue Virus Replication

    PubMed Central

    Schmid, Michael A.; Harris, Eva

    2014-01-01

    Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar–/–), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103+ classical dendritic cells (cDCs), Ly6C– CD11b+ cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6Chigh monocytes from wild-type and Ifnar–/– origin to the DENV-infected dermis and differentiation to Ly6C+ CD11b+ monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6Chigh monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination. PMID:25474197

  10. Asian sand dust enhances ovalbumin-induced eosinophil recruitment in the alveoli and airway of mice

    SciTech Connect

    Hiyoshi, Kyoko; Ichinose, Takamichi; Sadakane, Kaori; Takano, Hirohisa; Nishikawa, Masataka; Mori, Ikuko; Yanagisawa, Rie; Yoshida, Seiichi; Kumagai, Yoshito; Tomura, Shigeo; Shibamoto, Takayuki . E-mail: tshibamoto@ucdavis.edu

    2005-11-15

    Asian sand dust (ASD) containing sulfate (SO{sub 4} {sup 2-}) reportedly causes adverse respiratory health effects but there is no experimental study showing the effect of ASD toward allergic respiratory diseases. The effects of ASD and ASD plus SO{sub 4} {sup 2-} toward allergic lung inflammation induced by ovalbumin (OVA) were investigated in this study. ICR mice were administered intratracheally with saline; ASD alone (sample from Shapotou desert); and ASD plus SO{sub 4} {sup 2-} (ASD-SO{sub 4}); OVA+ASD; OVA+ASD-SO{sub 4}. ASD or ASD-SO{sub 4} alone caused mild nutrophilic inflammation in the bronchi and alveoli. ASD and ASD-SO{sub 4} increased pro-inflammatory mediators, such as Keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-1 alpha, in bronchoalveolar lavage fluids (BALF). ASD and ASD-SO{sub 4} enhanced eosinophil recruitment induced by OVA in the alveoli and in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. However, a further increase of eosinophils by addition of SO{sub 4} {sup 2-} was not observed. The two sand dusts synergistically increased interleukin-5 (IL-5) and monocyte chemotactic protein-1 (MCP-1), which were associated with OVA, in BALF. However, the increased levels of IL-5 were lower in the OVA+ASD-SO{sub 4} group than in the OVA+ASD group. ASD caused the adjuvant effects to specific-IgG1 production by OVA, but not to specific-IgE. These results suggest that the enhancement of eosinophil recruitment in the lung is mediated by synergistically increased IL-5 and MCP-1. IgG1 antibodies may play an important role in the enhancement of allergic reaction caused by OVA and sand dust. However, extra sulfate may not contribute to an increase of eosinophils.

  11. AF9 promotes hESC neural differentiation through recruiting TET2 to neurodevelopmental gene loci for methylcytosine hydroxylation

    PubMed Central

    Qiao, Yunbo; Wang, Xiongjun; Wang, Ran; Li, Yuanyuan; Yu, Fang; Yang, Xianfa; Song, Lu; Xu, Guoliang; Chin, Y Eugene; Jing, Naihe

    2015-01-01

    AF9 mutations have been implicated in human neurodevelopmental diseases and murine Af9 mediates histone methylation during cortical neuron generation. However, AF9 function and related mechanisms in human neurodevelopment remain unknown. Here we show that AF9 is necessary and sufficient for human embryonic stem cell (hESC) neural differentiation and neurodevelopmental gene activation. The 5-methylcytosine (5mC) dioxygenase TET2, which was identified in an AF9-associated protein complex, physically interacted with AF9. Both AF9 and TET2 co-localized in 5-hydroxymethylcytosine (5hmC)-positive hESC-derived neurons and were required for appropriate hESC neural differentiation. Upon binding to AAC-containing motifs, AF9 recruited TET2 to occupy the common neurodevelopmental gene loci to direct 5mC-to-5hmC conversion, which was followed by sequential activation of neural target genes and hESC neural commitment. These findings define an AF9–TET2 regulatory complex for modulating human neural development and reveal a novel mechanism by which the AF9 recognition specificity and TET2 hydroxylation activity cooperate to control neurodevelopmental gene activation. PMID:27462416

  12. Formation of oral and pharyngeal dentition in teleosts depends on differential recruitment of retinoic acid signaling.

    PubMed

    Gibert, Yann; Bernard, Laure; Debiais-Thibaud, Melanie; Bourrat, Franck; Joly, Jean-Stephane; Pottin, Karen; Meyer, Axel; Retaux, Sylvie; Stock, David W; Jackman, William R; Seritrakul, Pawat; Begemann, Gerrit; Laudet, Vincent

    2010-09-01

    One of the goals of evolutionary developmental biology is to link specific adaptations to changes in developmental pathways. The dentition of cypriniform fishes, which in contrast to many other teleost fish species possess pharyngeal teeth but lack oral teeth, provides a suitable model to study the development of feeding adaptations. Here, we have examined the involvement of retinoic acid (RA) in tooth development and show that RA is specifically required to induce the pharyngeal tooth developmental program in zebrafish. Perturbation of RA signaling at this stage abolished tooth induction without affecting the development of tooth-associated ceratobranchial bones. We show that this inductive event is dependent on RA synthesis from aldh1a2 in the ventral posterior pharynx. Fibroblast growth factor (FGF) signaling has been shown to be critical for tooth induction in zebrafish, and its loss has been associated with oral tooth loss in cypriniform fishes. Pharmacological treatments targeting the RA and FGF pathways revealed that both pathways act independently during tooth induction. In contrast, we find that in Mexican tetra and medaka, species that also possess oral teeth, both oral and pharyngeal teeth are induced independently of RA. Our analyses suggest an evolutionary scenario in which the gene network controlling tooth development obtained RA dependency in the lineage leading to the cypriniforms. The loss of pharyngeal teeth in this group was cancelled out through a shift in aldh1a2 expression, while oral teeth might have been lost ultimately due to deficient RA signaling in the oral cavity. PMID:20445074

  13. Differential recruitment of PKC isoforms in HeLa cells during redox stress

    PubMed Central

    Rimessi, Alessandro; Rizzuto, Rosario; Pinton, Paolo

    2007-01-01

    The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation. PMID:18229448

  14. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

    PubMed Central

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-01-01

    ABSTRACT Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUSR514S and FUSR521C patient fibroblasts, whereas in the case of the severe FUSP525L mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis. PMID:26035390

  15. Brain natriuretic peptide levels in six basic underwater demolitions/SEAL recruits presenting with swimming induced pulmonary edema (SIPE).

    PubMed

    Shearer, Damon; Mahon, Richard

    2009-01-01

    Swimming induced pulmonary edema (SIPE) is associated with both SCUBA diving and strenuous surface swimming; however, the majority of reported cases and clinically observed cases tend to occur during or after aggressive surface swimming. Capillary stress failure appears to be central to the pathophysiology of this disorder. Regional pulmonary capillaries are exposed to relatively high pressures secondary to increased vascular volume, elevation of pulmonary vascular resistance, and regional differences in perfusion secondary to forces of gravity and high cardiac output. Acute pulmonary edema can be classified as either cardiogenic or noncardiogenic or both. Cardiogenic pulmonary edema occurs when the pulmonary capillary hydrostatic pressure exceeds plasma oncotic pressure. Noncardiogenic pulmonary edema occurs when pulmonary capillary permeability is increased. Given the pathophysiology noted above, SIPE can be described as a cardiogenic pulmonary edema, at least in part, since an increased transalveolar pressure gradient has been implicated in the pathogenesis of SIPE. Brain natriuretic peptide (BNP) is used in the clinical setting to differentiate cardiac from pulmonary sources of dyspnea, specifically to diagnose cardiogenic pulmonary edema. During clinical management, BNP levels were drawn on six BUD/S recruits simultaneously presenting with pulmonary complaints consistent with SIPE, after an extended surface bay swim. This paper analyzes that data after de-identification and reviews the pathophysiology and clinical management of SIPE. PMID:19739476

  16. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons.

    PubMed

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-07-01

    Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis. PMID:26035390

  17. Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-β (TGF-β) Activation and Fibroblast Differentiation*

    PubMed Central

    Dayer, Cynthia; Stamenkovic, Ivan

    2015-01-01

    Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-β (TGF-β). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-β activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-β activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-β activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated. PMID:25825495

  18. Effects of lung recruitment maneuvers on splanchnic organ perfusion during endotoxin-induced pulmonary arterial hypertension.

    PubMed

    Daudel, Fritz; Gorrasi, José; Bracht, Hendrik; Brandt, Sebastian; Krejci, Vladimir; Jakob, Stephan M; Takala, Jukka; Rothen, Hans Ulrich

    2010-11-01

    Lung recruitment maneuvers (RMs), used to reopen atelectatic lung units and to improve oxygenation during mechanical ventilation, may result in hemodynamic impairment. We hypothesize that pulmonary arterial hypertension aggravates the consequences of RMs in the splanchnic circulation. Twelve anesthetized pigs underwent laparotomy and prolonged postoperative ventilation. Systemic, regional, and organ blood flows were monitored. After 6 h (= baseline), a recruitment maneuver was performed with sustained inflation of the lungs. Thereafter, the pigs were randomly assigned to group C (control, n = 6) or group E with endotoxin-induced pulmonary arterial hypertension (n = 6). Endotoxemia resulted in a normotensive and hyperdynamic state and a deterioration of the oxygenation index by 33%. The RM was then repeated in both groups. Pulmonary artery pressure increased during lipopolysaccharide infusion from 17 ± 2 mmHg (mean ± SD) to 31 ± 10 mmHg and remained unchanged in controls (P < 0.05). During endotoxemia, RM decreased aortic pulse pressure from 37 ± 14 mmHg to 27 ± 13 mmHg (mean ± SD, P = 0.024). The blood flows of the renal artery, hepatic artery, celiac trunk, superior mesenteric artery, and portal vein decreased to 71% ± 21%, 69% ± 20%, 76% ± 16%, 79% ± 18%, and 81% ± 12%, respectively, of baseline flows before RM (P < 0.05 all). Organ perfusion of kidney cortex, kidney medulla, liver, and jejunal mucosa in group E decreased to 65% ± 19%, 77% ± 13%, 66% ± 26%, and 71% ± 12%, respectively, of baseline flows (P < 0.05 all). The corresponding recovery to at least 90% of baseline regional blood flow and organ perfusion lasted 1 to 5 min. Importantly, the decreases in regional blood flows and organ perfusion and the time to recovery of these flows did not differ from the controls. In conclusion, lipopolysaccharide-induced pulmonary arterial hypertension does not aggravate the RM-induced significant but short-lasting decreases in systemic, regional, and

  19. Arsenic-induced SUMO-dependent recruitment of RNF4 into PML nuclear bodies.

    PubMed

    Geoffroy, Marie-Claude; Jaffray, Ellis G; Walker, Katherine J; Hay, Ronald T

    2010-12-01

    In acute promyelocytic leukemia (APL), the promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). Arsenic is an effective treatment for this disease as it induces SUMO-dependent ubiquitin-mediated proteasomal degradation of the PML-RAR fusion protein. Here we analyze the nuclear trafficking dynamics of PML and its SUMO-dependent ubiquitin E3 ligase, RNF4 in response to arsenic. After administration of arsenic, PML immediately transits into nuclear bodies where it undergoes SUMO modification. This initial recruitment of PML into nuclear bodies is not dependent on RNF4, but RNF4 quickly follows PML into the nuclear bodies where it is responsible for ubiquitylation of SUMO-modified PML and its degradation by the proteasome. While arsenic restricts the mobility of PML, FRAP analysis indicates that RNF4 continues to rapidly shuttle into PML nuclear bodies in a SUMO-dependent manner. Under these conditions FRET studies indicate that RNF4 interacts with SUMO in PML bodies but not directly with PML. These studies indicate that arsenic induces the rapid reorganization of the cell nucleus by SUMO modification of nuclear body-associated PML and uptake of the ubiquitin E3 ligase RNF4 leading to the ubiquitin-mediated degradation of PML. PMID:20943951

  20. Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo

    PubMed Central

    Westman, Johannes; Papareddy, Praveen; Dahlgren, Madelene W.; Chakrakodi, Bhavya; Norrby-Teglund, Anna; Smeds, Emanuel; Linder, Adam; Mörgelin, Matthias; Johansson-Lindbom, Bengt; Egesten, Arne; Herwald, Heiko

    2015-01-01

    The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection. PMID:26646682

  1. The LPS-induced neutrophil recruitment into rat air pouches is mediated by TNFα: likely macrophage origin

    PubMed Central

    Arreto, C-D.; Dumarey, C.; Nahori, M-A.; Vargaftig, B. B.

    1997-01-01

    The role of resident cells during the lipopolysaccharide (LPS)-induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2–2000 ng) induced a dose– and time-dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor-α (TNFα)-like activity. Dexamethasone (0.05–5 mug) and cycloheximide (6 ng), injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFα-like activity, while the H1-receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS) and the PAF-receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS) had no effect. Purified alveolar macrophages (AM) were used to replenish the pouches of cycloheximide-treated recipient rats. AM provided by PBS-treated animals led to the recovery of the LPS-induced neutrophil recruitment and of the TNFα-like formation contrasting with those from cycloheximide-treated animals (1 mg/kg, i.p.). When delivered in situ, liposome-encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFα-like activity. An anti-murine TNFα polyclonal antibody (0.5 h before LPS) was also effective. These results emphasize the pivotal role of macrophages for LPS-induced neutrophil recruitment via the formation of TNFα. PMID:18472868

  2. Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation

    PubMed Central

    Lee, Yu-Hui; Shynlova, Oksana; Lye, Stephen J

    2015-01-01

    Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte–endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum

  3. Hypervolemia induces and potentiates lung damage after recruitment maneuver in a model of sepsis-induced acute lung injury

    PubMed Central

    2010-01-01

    Introduction Recruitment maneuvers (RMs) seem to be more effective in extrapulmonary acute lung injury (ALI), caused mainly by sepsis, than in pulmonary ALI. Nevertheless, the maintenance of adequate volemic status is particularly challenging in sepsis. Since the interaction between volemic status and RMs is not well established, we investigated the effects of RMs on lung and distal organs in the presence of hypovolemia, normovolemia, and hypervolemia in a model of extrapulmonary lung injury induced by sepsis. Methods ALI was induced by cecal ligation and puncture surgery in 66 Wistar rats. After 48 h, animals were anesthetized, mechanically ventilated and randomly assigned to 3 volemic status (n = 22/group): 1) hypovolemia induced by blood drainage at mean arterial pressure (MAP)≈70 mmHg; 2) normovolemia (MAP≈100 mmHg), and 3) hypervolemia with colloid administration to achieve a MAP≈130 mmHg. In each group, animals were further randomized to be recruited (CPAP = 40 cm H2O for 40 s) or not (NR) (n = 11/group), followed by 1 h of protective mechanical ventilation. Echocardiography, arterial blood gases, static lung elastance (Est,L), histology (light and electron microscopy), lung wet-to-dry (W/D) ratio, interleukin (IL)-6, IL-1β, caspase-3, type III procollagen (PCIII), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) mRNA expressions in lung tissue, as well as lung and distal organ epithelial cell apoptosis were analyzed. Results We observed that: 1) hypervolemia increased lung W/D ratio with impairment of oxygenation and Est,L, and was associated with alveolar and endothelial cell damage and increased IL-6, VCAM-1, and ICAM-1 mRNA expressions; and 2) RM reduced alveolar collapse independent of volemic status. In hypervolemic animals, RM improved oxygenation above the levels observed with the use of positive-end expiratory pressure (PEEP), but increased lung injury and led to higher inflammatory and fibrogenetic

  4. TGFβ induces GDNF responsiveness in neurons by recruitment of GFRα1 to the plasma membrane

    PubMed Central

    Peterziel, H.; Unsicker, K.; Krieglstein, K.

    2002-01-01

    We have previously shown that the neurotrophic effect of glial cell line–derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)β. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFβ promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFβ, pretreatment with TGFβ confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFβ-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)α1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRα1 can promote survival in the absence of TGFβ. Our data suggest that TGFβ is involved in GFRα1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects. PMID:12370242

  5. Human breast cancer biopsies induce eosinophil recruitment and enhance adjacent cancer cell proliferation.

    PubMed

    Szalayova, Gabriela; Ogrodnik, Aleksandra; Spencer, Brianna; Wade, Jacqueline; Bunn, Janice; Ambaye, Abiy; James, Ted; Rincon, Mercedes

    2016-06-01

    Chronic inflammation is known to facilitate cancer progression and metastasis. Less is known about the effect of acute inflammation within the tumor microenvironment, resulting from standard invasive procedures. Recent studies in mouse models have shown that the acute inflammatory response triggered by a biopsy in mammary cancer increases the frequency of distal metastases. Although tumor biopsies are part of the standard clinical practice in breast cancer diagnosis, no studies have reported their effect on inflammatory response. The objective of this study is to (1) determine whether core needle biopsies in breast cancer patients trigger an inflammatory response, (2) characterize the type of inflammatory response present, and (3) evaluate the potential effect of any acute inflammatory response on residual tumor cells. The biopsy wound site was identified in the primary tumor resection tissue samples from breast cancer patients. The inflammatory response in areas adjacent (i.e., immediately around previous biopsy site) and distant to the wound biopsy was investigated by histology and immunohistochemistry analysis. Proliferation of tumor cells was also assayed. We demonstrate that diagnostic core needle biopsies trigger a selective recruitment of inflammatory cells at the site of the biopsy, and they persist for extended periods of time. While macrophages were part of the inflammatory response, an unexpected accumulation of eosinophils at the edge of the biopsy wound was also identified. Importantly, we show that biopsy causes an increase in the proliferation rate of tumor cells located in the area adjacent to the biopsy wound. Diagnostic core needle biopsies in breast cancer patients do induce a unique acute inflammatory response within the tumor microenvironment and have an effect on the surrounding tumor cells. Therefore, biopsy-induced inflammation could have an impact on residual tumor cell progression and/or metastasis in human breast cancer. These findings

  6. The citrus flavonone naringenin reduces lipopolysaccharide-induced inflammatory pain and leukocyte recruitment by inhibiting NF-κB activation.

    PubMed

    Pinho-Ribeiro, Felipe A; Zarpelon, Ana C; Mizokami, Sandra S; Borghi, Sergio M; Bordignon, Juliano; Silva, Rangel L; Cunha, Thiago M; Alves-Filho, Jose C; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2016-07-01

    Lipopolysaccharide (LPS) is the major structural component of Gram-negative bacteria cell wall and a highly pro-inflammatory toxin. Naringenin is found in Citrus fruits and exhibits antioxidant and anti-inflammatory properties through inhibition of NF-κB activation but its effects in LPS-induced inflammatory pain and leukocyte recruitment were not investigated yet. We investigated the effects of naringenin in mechanical hyperalgesia, thermal hyperalgesia and leukocyte recruitment induced by intraplantar injection of LPS in mice. We found that naringenin reduced hyperalgesia to mechanical and thermal stimuli, myeloperoxidase (MPO, a neutrophil and macrophage marker) and N-acetyl-β-D-glucosaminidase (NAG, a macrophage marker) activities, oxidative stress and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production in the paw skin. In the peritoneal cavity, naringenin reduced neutrophil and mononuclear cell recruitment, and abrogated MPO and NAG activity, cytokine and superoxide anion production, and lipid peroxidation. In vitro, pre-treatment with naringenin inhibited superoxide anion and cytokine (TNF-α, IL-1β, IL-6, and IL-12) production by LPS-stimulated RAW 264.7 macrophages. Finally, we demonstrated that naringenin inhibited NF-κB activation in vitro and in vivo. Therefore, naringenin is a promising compound to treat LPS-induced inflammatory pain and leukocyte recruitment. PMID:27260463

  7. Recruitment Maneuver Does not Increase the Risk of Ventilator Induced Lung Injury

    PubMed Central

    Akıncı, İbrahim Özkan; Atalan, Korkut; Tuğrul, Simru; Özcan, Perihan Ergin; Yılmazbayhan, Dilek; Kıran, Bayram; Basel, Ahmet; Telci, Lutfi; Çakar, Nahit

    2013-01-01

    Background: Mechanical ventilation (MV) may induce lung injury. Aims: To assess and evaluate the role of different mechanical ventilation strategies on ventilator-induced lung injury (VILI) in comparison to a strategy which includes recruitment manoeuvre (RM). Study design: Randomized animal experiment. Methods: Thirty male Sprague-Dawley rats were anaesthetised, tracheostomised and divided into 5 groups randomly according to driving pressures; these were mechanically ventilated with following peak alveolar opening (Pao) and positive end-expiratory pressures (PEEP) for 1 hour: Group 15-0: 15 cmH2O Pao and 0 cmH2O PEEP; Group 30-10: 30 cmH2O Pao and 10 cmH2O PEEP; Group 30-5: 30 cmH2O Pao and 5 cmH2O PEEP; Group 30-5&RM: 30 cmH2O Pao and 5 cmH2O PEEP with additional 45 cmH2O CPAP for 30 seconds in every 15 minutes; Group 45-0: 45 cmH2O Pao and 0 cmH2O PEEP Before rats were sacrificed, blood samples were obtained for the evaluation of cytokine and chemokine levels; then, the lungs were subsequently processed for morphologic evaluation. Results: Oxygenation results were similar in all groups; however, the groups were lined as follows according to the increasing severity of morphometric evaluation parameters: Group 15-0: (0±0.009) < Group 30-10: (0±0.14) < Group 30-5&RM: (1±0.12) < Group 30-5: (1±0.16) < Group 45-0: (2±0.16). Besides, inflammatory responses were the lowest in 30-5&RM group compared to all other groups. TNF-α, IL-1β, IL-6, MCP-1 levels were significantly different between group 30-5&RM and group 15-0 vs. group 45-0 in each group. Conclusion: RM with low PEEP reduces the risk of ventilator-induced lung injury with a lower release of systemic inflammatory mediators in response to mechanical ventilation. PMID:25207105

  8. Directed Myogenic Differentiation of Human Induced Pluripotent Stem Cells.

    PubMed

    Shoji, Emi; Woltjen, Knut; Sakurai, Hidetoshi

    2016-01-01

    Patient-derived induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. Yet for muscular diseases, a method for reproducible and efficient myogenic differentiation from human iPSCs is required for in vitro modeling. Here, we introduce a myogenic differentiation protocol mediated by inducible transcription factor expression that reproducibly and efficiently drives human iPSCs into myocytes. Delivering a tetracycline-inducible, myogenic differentiation 1 (MYOD1) piggyBac (PB) vector to human iPSCs enables the derivation of iPSCs that undergo uniform myogenic differentiation in a short period of time. This differentiation protocol yields a homogenous skeletal muscle cell population, reproducibly reaching efficiencies as high as 70-90 %. MYOD1-induced myocytes demonstrate characteristics of mature myocytes such as cell fusion and cell twitching in response to electric stimulation within 14 days of differentiation. This differentiation protocol can be applied widely in various types of patient-derived human iPSCs and has great prospects in disease modeling particularly with inherited diseases that require studies of early pathogenesis and drug screening. PMID:25971915

  9. Enhanced angiogenesis, hypoxia and neutrophil recruitment during Myc-induced liver tumorigenesis in zebrafish.

    PubMed

    Zhao, Ye; Huang, Xiaoqian; Ding, Tony Weixi; Gong, Zhiyuan

    2016-01-01

    Angiogenesis, hypoxia and immune cells are important components in tumor microenvironment affecting tumor growth. Here we employed a zebrafish liver tumor model to investigate the effect of Myc expression on angiogenesis, hypoxia and tumor-infiltrated neutrophils during the tumor initiation stage. We found that induced Myc expression in the liver caused a dramatic increase of liver size with neoplastic features. The tumorigenic liver was accompanied by enhanced angiogenesis and inhibition of angiogenesis by an inhibitor (SU5416 or sunitinib) hindered the tumorigenic growth, suggesting an essential role of angiogenesis in tumorigenic growth of liver tumor in this zebrafish model. Myc induction also caused hypoxia, which could be further enhanced by hypoxia activator, ML228, to lead to a further enlargement of tumorigenic liver. Furthermore, Myc overexpression incurred an increase of liver-infiltrated neutrophils and the increase could be suppressed by angiogenesis inhibitors or by morpholino knockdown inhibition of neutrophil differentiation, leading to a suppression of growth of tumorigenic livers. Finally, the enhanced angiogenesis, hypoxia and tumor-infiltrated neutrophils by Myc overexpression were validated by RT-qPCR examination of expression of relevant biomarker genes. In sum, the current study demonstrated that the Myc-induced liver tumor model in zebrafish provides an excellent platform for study of tumor microenvironment. PMID:27549025

  10. Enhanced angiogenesis, hypoxia and neutrophil recruitment during Myc-induced liver tumorigenesis in zebrafish

    PubMed Central

    Zhao, Ye; Huang, Xiaoqian; Ding, Tony Weixi; Gong, Zhiyuan

    2016-01-01

    Angiogenesis, hypoxia and immune cells are important components in tumor microenvironment affecting tumor growth. Here we employed a zebrafish liver tumor model to investigate the effect of Myc expression on angiogenesis, hypoxia and tumor-infiltrated neutrophils during the tumor initiation stage. We found that induced Myc expression in the liver caused a dramatic increase of liver size with neoplastic features. The tumorigenic liver was accompanied by enhanced angiogenesis and inhibition of angiogenesis by an inhibitor (SU5416 or sunitinib) hindered the tumorigenic growth, suggesting an essential role of angiogenesis in tumorigenic growth of liver tumor in this zebrafish model. Myc induction also caused hypoxia, which could be further enhanced by hypoxia activator, ML228, to lead to a further enlargement of tumorigenic liver. Furthermore, Myc overexpression incurred an increase of liver-infiltrated neutrophils and the increase could be suppressed by angiogenesis inhibitors or by morpholino knockdown inhibition of neutrophil differentiation, leading to a suppression of growth of tumorigenic livers. Finally, the enhanced angiogenesis, hypoxia and tumor-infiltrated neutrophils by Myc overexpression were validated by RT-qPCR examination of expression of relevant biomarker genes. In sum, the current study demonstrated that the Myc-induced liver tumor model in zebrafish provides an excellent platform for study of tumor microenvironment. PMID:27549025

  11. Progressive recruitment of cortical and striatal regions by inducible postsynaptic density transcripts after increasing doses of antipsychotics with different receptor profiles: insights for psychosis treatment.

    PubMed

    de Bartolomeis, Andrea; Iasevoli, Felice; Marmo, Federica; Buonaguro, Elisabetta F; Eramo, Anna; Rossi, Rodolfo; Avvisati, Livia; Latte, Gianmarco; Tomasetti, Carmine

    2015-04-01

    Antipsychotics may modulate the transcription of multiple gene programs, including those belonging to postsynaptic density (PSD) network, within cortical and subcortical brain regions. Understanding which brain region is activated progressively by increasing doses of antipsychotics and how their different receptor profiles may impact such an activation could be relevant to better correlate the mechanism of action of antipsychotics both with their efficacy and side effects. We analyzed the differential topography of PSD transcripts by incremental doses of two antipsychotics: haloperidol, the prototypical first generation antipsychotic with prevalent dopamine D2 receptors antagonism, and asenapine, a second generation antipsychotic characterized by multiple receptors occupancy. We investigated the expression of PSD genes involved in synaptic plasticity and previously demonstrated to be modulated by antipsychotics: Homer1a, and its related interacting constitutive genes Homer1b/c and PSD95, as well as Arc, C-fos and Zif-268, also known to be induced by antipsychotics administration. We found that increasing acute doses of haloperidol induced immediate-early genes (IEGs) expression in different striatal areas, which were progressively recruited by incremental doses with a dorsal-to-ventral gradient of expression. Conversely, increasing acute asenapine doses progressively de-recruited IEGs expression in cortical areas and increased striatal genes signal intensity. These effects were mirrored by a progressive reduction in locomotor animal activity by haloperidol, and an opposite increase by asenapine. Thus, we demonstrated for the first time that antipsychotics may progressively recruit PSD-related IEGs expression in cortical and subcortical areas when administered at incremental doses and these effects may reflect a fine-tuned dose-dependent modulation of the PSD. PMID:25649681

  12. Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

    PubMed Central

    Speers, W. C.; Birdwell, C. R.; Dixon, F. J.

    1979-01-01

    N,N-dimethylacetamide, hexamethylene bisacetamide, and Polybrene induced rapid and extensive differentiation in vitro in an otherwise slowly differentiating subline of embryonal carcinoma cells. The type of differentiated cell induced was dependent on the spatial organization of the stem cells during drug treatment. In monalayer culture "epithelial" cells were produced exclusively. However, treatment of aggregated suspension cultures yielded predominantly "fibroblast-like" cells. The undifferentiated embryonal carcinoma cells and the two differentiated cell types were morphologically distinct when examined by light microscopy, scanning electron microscopy, and transmission electron microscopy; and they had differences in cell surface antigens. Both differential cell types produced large amounts of fibronectin, whereas the embryonal carcinoma cells produced only minimal amounts. This system provides a convenient way to induce relatively synchronous differentiation of embryonal carcinoma cells into specific differentiated cell types. Images Figure 5 Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:507191

  13. HEXIM1 Induces Differentiation of Human Pluripotent Stem Cells

    PubMed Central

    Ding, Vanessa; Lew, Qiao Jing; Chu, Kai Ling; Natarajan, Subaashini; Rajasegaran, Vikneswari; Gurumurthy, Meera; Choo, Andre B. H.; Chao, Sheng-Hao

    2013-01-01

    Hexamethylene bisacetamide inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9)/cyclin T1. P-TEFb is an essential regulator for the transcriptional elongation by RNA polymerase II. A genome-wide study using human embryonic stem cells shows that most mRNA synthesis is regulated at the stage of transcription elongation, suggesting a possible role for P-TEFb/HEXIM1 in the gene regulation of stem cells. In this report, we detected a marked increase in HEXIM1 protein levels in the differentiated human pluripotent stem cells (hPSCs) induced by LY294002 treatment. Since no changes in CDK9 and cyclin T1 were observed in the LY294002-treated cells, increased levels of HEXIM1 might lead to inhibition of P-TEFb activity. However, treatment with a potent P-TEFb inhibiting compound, flavopiridol, failed to induce hPSC differentiation, ruling out the possible requirement for P-TEFb kinase activity in hPSC differentiation. Conversely, differentiation was observed when hPSCs were incubated with hexamethylene bisacetamide, a HEXIM1 inducing reagent. The involvement of HEXIM1 in the regulation of hPSCs was further supported when overexpression of HEXIM1 concomitantly induced hPSC differentiation. Collectively, our study demonstrates a novel role of HEXIM1 in regulating hPSC fate through a P-TEFb-independent pathway. PMID:23977357

  14. Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

    PubMed Central

    Nunes-Silva, Albená; Bernardes, Priscila T. T.; Rezende, Bárbara M.; Lopes, Fernando; Gomes, Elisa C.; Marques, Pedro E.; Lima, Paulo M. A.; Coimbra, Cândido C.; Menezes, Gustavo B.; Teixeira, Mauro M.; Pinho, Vanessa

    2014-01-01

    Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process. PMID:24798414

  15. Allergic pulmonary inflammation in mice is dependent on eosinophil-induced recruitment of effector T cells

    PubMed Central

    Jacobsen, Elizabeth A.; Ochkur, Sergei I.; Pero, Ralph S.; Taranova, Anna G.; Protheroe, Cheryl A.; Colbert, Dana C.; Lee, Nancy A.; Lee, James J.

    2008-01-01

    The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells. PMID:18316417

  16. The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

    PubMed

    Lazarou, Michael; Sliter, Danielle A; Kane, Lesley A; Sarraf, Shireen A; Wang, Chunxin; Burman, Jonathon L; Sideris, Dionisia P; Fogel, Adam I; Youle, Richard J

    2015-08-20

    Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways. PMID:26266977

  17. Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

    PubMed Central

    Son, Yonghae; Kim, Bo-Young; Eo, Seong-Kug; Park, Young Chul; Kim, Koanhoi

    2016-01-01

    Oxysterol like 27-hydroxycholesterol (27OHChol) has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx) affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells. PMID:27340507

  18. Biochemical changes induced by strontium ranelate in differentiating adipocytes.

    PubMed

    Vidal, Christopher; Gunaratnam, Krishanthi; Tong, Jessica; Duque, Gustavo

    2013-04-01

    Low bone formation in osteoporosis is associated with a shift from osteoblastic to adipogenic differentiation of mesenchymal stem cells (MSC) inducing a concomitant lipotoxic milieu within the bone marrow. Strontium ranelate (SrRN), a treatment for osteoporosis, has both anti-resorptive and anabolic effects on bone. The anabolic effect of SrRN has been associated with its effect on both osteoblastogenesis and adipogenesis. However, the effect of SrRN on the potentially lipotoxic factors produced by differentiating marrow adipocytes remains poorly understood. To expand the knowledge on the effect of SrRN treatment on the bone microenvironment, we assessed changes in adipogenic factors and adipokine expression in adipocytic differentiation of MSC in vitro. Primary human MSC were induced to differentiate in adipogenic conditions in the presence or absence of SrRN (1-2 mM). We tested the dose-dependent effects of SrRN on adipocyte differentiation including changes in the expression of adipogenic markers and adipokines. We report that adipogenesis was negatively affected in the presence of SrRN with a concomitant dose-dependent decrease in the expression of adipogenic markers and changes in adipokine profile. Taken together, our data suggests that SrRN induces biochemical changes in differentiating adipocytes that could generate a favorable osteogenic effect within the bone marrow milieu. PMID:23186800

  19. Retinoic Acid Induces Ubiquitination-Resistant RIP140/LSD1 Complex to Fine-Tune Pax6 Gene in Neuronal Differentiation.

    PubMed

    Wu, Cheng-Ying; Persaud, Shawna D; Wei, Li-Na

    2016-01-01

    Receptor-interacting protein 140 (RIP140) is a wide-spectrum coregulator for hormonal regulation of gene expression, but its activity in development/stem cell differentiation is unknown. Here, we identify RIP140 as an immediate retinoic acid (RA)-induced dual-function chaperone for LSD1 (lysine-specific demethylase 1). RIP140 protects LSD1's catalytic domain and antagonizes its Jade-2-mediated ubiquitination and degradation. In RA-induced neuronal differentiation, the increased RIP140/LSD1 complex is recruited by RA-elevated Pit-1 to specifically reduce H3K4me2 modification on the Pax6 promoter, thereby repressing RA-induction of Pax6. This study reveals a new RA-induced gene repressive mechanism that modulates the abundance, enzyme quality, and recruitment of histone modifier LSD1 to neuronal regulator Pax6, which provides a homeostatic control for RA induction of neuronal differentiation. PMID:26372689

  20. Gossypol-Induced Differentiation in Human Leukemia HL-60 Cells

    PubMed Central

    Wang, Wen-Qing; Li, Rong; Bai, Qing-Xian; Liu, Yu-Hong; Zhang, Wei-Ping; Wang, Juan-Hong; Wang, Zhe; Li, Yuan-Fei; Chen, Xie-Qun; Huang, Gao-Sheng

    2006-01-01

    The main treatment of leukemia is traditional radiochemotherapy, which is associated with serious side effects. In the past twenty years, differentiation was found as an important effective measure to treat leukemia with fewer side effects. Gossypol, a natural compound which has been used as an effective contraceptive drug, has been proposed to be a potent drug to treat leukemia, but the differentiation effect has not been studied. In the present study, we investigated the pro-differentiated effects, in vitro, of gossypol on the classic human myeloid leukemia HL-60 cell line. The effects of gossypol were investigated by using morphological changes, nitroblue tetrazolium (NBT) reduction, surface markers, cell-cycle analysis and Western blot analysis, etc. When HL-60 cells were incubated with low concentrations of gossypol (2-5μM) for 48hr, a prominent G0/G1 arrest was observed. At 96 hr of treatment, 90% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, and increase in cell surface expression of some molecules were detected. This study is the first to identify gossypol’s pro-differentiated effects on the leukemia cell line, and it induced differentiation through the PBK (PDZ-binding kinase)/TOPK (T-LAKcell-originated protein kinase) (PBK/TOPK) pathway. It is concluded that gossypol could induce differentiation in the leukemia HL-60 cells, and it may be a potential therapeutic agent, chemoprevention or chemotherapeutic adjuvant especially in combination drug therapy for leukemia. PMID:23675007

  1. Enhanced Noradrenergic Activity Potentiates Fear Memory Consolidation and Reconsolidation by Differentially Recruiting alpha1- and beta-Adrenergic Receptors

    ERIC Educational Resources Information Center

    Gazarini, Lucas; Stern, Cristina A. Jark; Carobrez, Antonio P.; Bertoglio, Leandro J.

    2013-01-01

    Consolidation and reconsolidation are phases of memory stabilization that diverge slightly. Noradrenaline is known to influence both processes, but the relative contribution of alpha1- and beta-adrenoceptors is unclear. The present study sought to investigate this matter by comparing their recruitment to consolidate and/or reconsolidate a…

  2. TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment

    PubMed Central

    Degryse, Amber L.; Tanjore, Harikrishna; Xu, Xiaochuan C.; Polosukhin, Vasiliy V.; Jones, Brittany R.; Boomershine, Chad S.; Ortiz, Camila; Sherrill, Taylor P.; McMahon, Frank B.; Gleaves, Linda A.; Blackwell, Timothy S.

    2011-01-01

    The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4+/β-gal+) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine. PMID:21441353

  3. TGFβ signaling in lung epithelium regulates bleomycin-induced alveolar injury and fibroblast recruitment.

    PubMed

    Degryse, Amber L; Tanjore, Harikrishna; Xu, Xiaochuan C; Polosukhin, Vasiliy V; Jones, Brittany R; Boomershine, Chad S; Ortiz, Camila; Sherrill, Taylor P; McMahon, Frank B; Gleaves, Linda A; Blackwell, Timothy S; Lawson, William E

    2011-06-01

    The response of alveolar epithelial cells (AECs) to lung injury plays a central role in the pathogenesis of pulmonary fibrosis, but the mechanisms by which AECs regulate fibrotic processes are not well defined. We aimed to elucidate how transforming growth factor-β (TGFβ) signaling in lung epithelium impacts lung fibrosis in the intratracheal bleomycin model. Mice with selective deficiency of TGFβ receptor 2 (TGFβR2) in lung epithelium were generated and crossed to cell fate reporter mice that express β-galactosidase (β-gal) in cells of lung epithelial lineage. Mice were given intratracheal bleomycin (0.08 U), and the following parameters were assessed: AEC death by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, inflammation by total and differential cell counts from bronchoalveolar lavage, fibrosis by scoring of trichrome-stained lung sections, and total lung collagen content. Mice with lung epithelial deficiency of TGFβR2 had improved AEC survival, despite greater lung inflammation, after bleomycin administration. At 3 wk after bleomycin administration, mice with epithelial TGFβR2 deficiency showed a significantly attenuated fibrotic response in the lungs, as determined by semiquantitatve scoring and total collagen content. The reduction in lung fibrosis in these mice was associated with a marked decrease in the lung fibroblast population, both total lung fibroblasts and epithelial-to-mesenchymal transition-derived (S100A4(+)/β-gal(+)) fibroblasts. Attenuation of TGFβ signaling in lung epithelium provides protection from bleomycin-induced fibrosis, indicating a critical role for the epithelium in transducing the profibrotic effects of this cytokine. PMID:21441353

  4. Fish Oil and Adjuvant-Induced Arthritis: Inhibitory Effect on Leukocyte Recruitment.

    PubMed

    Estevão-Silva, Camila Fernanda; Ames, Franciele Queiroz; Silva-Comar, Francielli Maria de Souza; Kummer, Raquel; Tronco, Rafael Prizon; Cuman, Roberto Kenji Nakamura; Bersani-Amado, Ciomar Aparecida

    2016-02-01

    Fish oil, a rich source of n-3 fatty acids, has been studied for its beneficial effects in many diseases. Recent studies have shown the robust anti-inflammatory activity of fish oil (FO), when administered orally to rats, in models of acute inflammation. Herein, we investigated if treatment with fish oil preparation (FOP) could interfere with the recruitment of leukocytes into the joint cavity of arthritic rats. We also evaluated the effect of treatment on rolling behavior and leukocyte adhesion in vivo and on leukocyte chemotaxis in vitro. Treatment with FOP (75, 150, and 300 mg/kg) initiated on the day of induction of arthritis (day 0) and maintained for 21 days reduced the total number of leukocytes recruited into the joint cavity, the number of rolling and adhered leukocytes in arthritic rats, and leukocyte migration in response to stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4). Together, our data provide evidence that FOP plays an important inhibitory role in the recruitment of leukocytes into the joint cavity of arthritic rats. PMID:26378008

  5. Electrostatically induced recruitment of membrane peptides into clusters requires ligand binding at both interfaces.

    PubMed

    Antonenko, Yuri N; Horner, Andreas; Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility D(PLL) and (ii) by an increased lifetime τ(PLL) of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither D(PLL) nor τ(P) changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  6. Electrostatically Induced Recruitment of Membrane Peptides into Clusters Requires Ligand Binding at Both Interfaces

    PubMed Central

    Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility DPLL and (ii) by an increased lifetime τPLL of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither DPLL nor τP changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  7. SOCS3 induces neurite differentiation and promotes neuronal cell survival.

    PubMed

    Mishra, Kanchan Kumar; Gupta, Sakshi; Banerjee, Kakoli

    2016-06-01

    Cytokines and growth factors play an important role in neuronal survival as well as cell death. The family of suppressors of cytokine signalling (SOCS) proteins, which includes SOCS1-7 and cytokine-induced suppressor (CIS), has been shown to act as negative regulators of cytokine-induced signalling. In this report, we highlight the role of SOCS3 in regulating neuronal differentiation and survival. We observed increased SOCS3 expression upon differentiation of PC12 cells as well as neural stem cells. SOCS3 overexpression upregulated differentiation of both neural stem cells and PC12 cells even in the absence of NGF, as evidenced by enhanced neurite outgrowth and upregulation of GAP43, marker associated with neurite outgrowth. siRNA-mediated silencing of SOCS3 confirmed the potential role of SOCS3 in neuritogenesis. We observed that, SOCS3-induced neurite differentiation was mediated via the PI3 kinase pathway. Another interesting observation was that SOCS3 overexpression promoted neuronal cell survival under H2 O2 -mediated stress indicating its fundamental role in cell survival. In conclusion, our results indicate that SOCS3 promotes differentiation and survival of neural cells and could be potentially useful in future therapy for treatment of neurodegenerative disorders. © 2016 IUBMB Life, 68(6):468-476, 2016. PMID:27118613

  8. Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption.

    PubMed

    Spazierer, Daniel; Raberger, Julia; Gross, Karin; Fuchs, Peter; Wiche, Gerhard

    2008-03-15

    Epiplakin is a large (>725 kDa) cytoskeletal protein exclusively expressed in epithelial tissues. It has a unique structure, consisting entirely of plakin repeat domains (PRDs), one of the hallmarks of spectraplakin protein family members. Previous studies, including the phenotypic analyses of knockout mice, failed to reveal the biological function of epiplakin. Using in vitro binding assays, we show here that all but one of the 16 PRDs of mouse epiplakin bind to keratins of basal keratinocytes. Nevertheless, in primary keratinocyte cell cultures, epiplakin only partially colocalized with keratin intermediate filament networks. However, upon application of cellular stress in the form of keratin hyperphosphorylation, osmotic shock or UV irradiation, the entire cytoplasmic epiplakin pool became associated with keratin. In response to such types of stress, epiplakin initially translocated to the still-intact keratin filament network and remained associated with keratin after its disruption and transformation into granular aggregates. Time-course experiments revealed that serine/threonine (okadaic acid) and tyrosine (orthovanadate) phosphatase inhibitor-induced filament disruption in differentiated keratinocytes proceeded faster in epiplakin-deficient cells compared with wild-type cells. Our data suggest that epiplakin plays a role in keratin filament reorganization in response to stress, probably by protecting keratin filaments against disruption in a chaperone-like fashion. PMID:18285451

  9. Protein kinase A mediates glucagon-like peptide 1-induced nitric oxide production and muscle microvascular recruitment

    PubMed Central

    Dong, Zhenhua; Chai, Weidong; Wang, Wenhui; Zhao, Lina; Fu, Zhuo; Cao, Wenhong

    2013-01-01

    Glucagon-like peptide-1 (GLP-1) causes vasodilation and increases muscle glucose uptake independent of insulin. Recently, we have shown that GLP-1 recruits muscle microvasculature and increases muscle glucose use via a nitric oxide (NO)-dependent mechanism. Protein kinase A (PKA) is a major signaling intermediate downstream of GLP-1 receptors. To examine whether PKA mediates GLP-1's microvascular action in muscle, GLP-1 was infused to overnight-fasted male rats for 120 min in the presence or absence of H89, a PKA inhibitor. Hindleg muscle microvascular recruitment and glucose use were determined. GLP-1 infusion acutely increased muscle microvascular blood volume within 30 min without altering microvascular blood flow velocity or blood pressure. This effect persisted throughout the 120-min infusion period, leading to a significant increase in muscle microvascular blood flow. These changes were paralleled with an approximately twofold increase in plasma NO levels and hindleg glucose extraction. Systemic infusion of H89 completely blocked GLP-1-mediated muscle microvascular recruitment and increases in NO production and muscle glucose extraction. In cultured endothelial cells, GLP-1 acutely increased PKA activity and stimulated endothelial NO synthase phosphorylation at Ser1177 and NO production. PKA inhibition abolished these effects. In ex vivo studies, perfusion of the distal saphenous artery with GLP-1 induced significant vasorelaxation that was also abolished by pretreatment of the vessels with PKA inhibitor H89. We conclude that GLP-1 recruits muscle microvasculature by expanding microvascular volume and increases glucose extraction in muscle via a PKA/NO-dependent pathway in the vascular endothelium. This may contribute to postprandial glycemic control and complication prevention in diabetes. PMID:23193054

  10. Cholesterol starvation induces differentiation of human leukemia HL-60 cells.

    PubMed

    Sánchez-Martín, Carolina C; Dávalos, Alberto; Martín-Sánchez, Covadonga; de la Peña, Gema; Fernández-Hernando, Carlos; Lasunción, Miguel A

    2007-04-01

    Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy. PMID:17409448

  11. Differential effects of glucagon-like peptide-1 on microvascular recruitment and glucose metabolism in short- and long-term insulin resistance

    PubMed Central

    Sjøberg, Kim A; Rattigan, Stephen; Jeppesen, Jacob F; Lundsgaard, Anne-Marie; Holst, Jens J; Kiens, Bente

    2015-01-01

    Abstract Acute infusion of glucagon-like peptide-1 (GLP-1) has potent effects on blood flow distribution through the microcirculation in healthy humans and rats. A high fat diet induces impairments in insulin-mediated microvascular recruitment (MVR) and muscle glucose uptake, and here we examined whether this could be reversed by GLP-1. Using contrast-enhanced ultrasound, microvascular recruitment was assessed by continuous real-time imaging of gas-filled microbubbles in the microcirculation after acute (5 days) and prolonged (8 weeks) high fat diet (HF)-induced insulin resistance in rats. A euglycaemic hyperinsulinaemic clamp (3 mU min−1 kg−1), with or without a co-infusion of GLP-1 (100 pmol l−1), was performed in anaesthetized rats. Consumption of HF attenuated the insulin-mediated MVR in both 5 day and 8 week HF interventions which was associated with a 50% reduction in insulin-mediated glucose uptake compared to controls. Acute administration of GLP-1 restored the normal microvascular response by increasing the MVR after both 5 days and 8 weeks of HF intervention (P < 0.05). This effect of GLP-1 was associated with a restoration of both whole body insulin sensitivity and increased insulin-mediated glucose uptake in skeletal muscle by 90% (P < 0.05) after 5 days of HF but not after 8 weeks of HF. The present study demonstrates that GLP-1 increases MVR in rat skeletal muscle and can reverse early stages of high fat diet-induced insulin resistance in vivo. Key points Acute glucagon-like peptide-1 (GLP-1) infusion reversed the high fat diet-induced microvascular insulin resistance that occurred after both 5 days and 8 weeks of a high fat diet intervention. When GLP-1 was co-infused with insulin it had overt effects on whole body insulin sensitivity as well as insulin-mediated skeletal muscle glucose uptake after 5 days of a high fat diet, but not after 8 weeks of high fat diet intervention. Acute GLP-1 infusion did not have an additive

  12. Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation

    PubMed Central

    Schoenfeld, Robert; Wong, Alice; Silva, Jillian; Li, Ming; Itoh, Aki; Horiuchi, Makoto; Itoh, Takayuki; Pleasure, David; Cortopassi, Gino

    2011-01-01

    Demyelination occurs in multiple inherited mitochondrial diseases. We studied which genes were induced as a consequence of differentiation in rodent and human oligodendroglia. Cholesterol, myelin and mitochondrial genes were significantly increased with oligodendroglial differentiation. Mitochondrial DNA content per cell and acetyl CoA-related transcripts increased significantly; thus, the large buildup of cholesterol necessary for myelination appears to require mitochondrial production of acetyl-CoA. Oligodendroglia were treated with low doses of the mitochondrial inhibitor rotenone to test the dependence of differentiation on mitochondrial function. Undifferentiated cells were resistant to rotenone, whereas differentiating cells were much more sensitive. Very low doses of rotenone that did not affect viability or ATP synthesis still inhibited differentiation, as measured by reduced levels of the myelin transcripts 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase and Myelin Basic Protein. Thus, mitochondrial transcripts and mtDNA are amplified during oligodendroglial differentiation, and differentiating oligodendroglia are especially sensitive to mitochondrial inhibition, suggesting mechanisms for demyelination observed in mitochondrial disease. PMID:20005986

  13. Role of stress-inducible protein-1 in recruitment of bone marrow derived cells into the ischemic brains

    PubMed Central

    Lee, Shin-Da; Lai, Ted Weita; Lin, Shinn-Zong; Lin, Chen-Huan; Hsu, Yung-Hsiang; Li, Chi-Yuan; Wang, Hsiao-Jung; Lee, Wei; Su, Ching-Yuan; Yu, Yung-Luen; Shyu, Woei-Cherng

    2013-01-01

    Stress-inducible protein-1 (STI-1) is the proposed ligand for the cellular prion protein (PrPC), which is thought to facilitate recovery following stroke. Whether STI-1 expression is affected by stroke and how its signalling facilitates recovery remain elusive. Brain slices from patients that died of ischemic stroke were collected for STI-1 immunohistochemistry. These findings were compared to results from cell cultures, mice with or without the PrPC knockout, and rats. Based on these findings, molecular and pharmacological interventions were administered to investigate the underlying mechanisms and to test the possibility for therapy in experimental stroke models. STI-1 was upregulated in the ischemic brains from humans and rodents. The increase in STI-1 expression in vivo was not cell-type specific, as it was found in neurons, glia and endothelial cells. Likewise, this increase in STI-1 expression can be mimicked by sublethal hypoxia in primary cortical cultures (PCCs) in vitro, and appear to have resulted from the direct binding of the hypoxia inducible factor-1α (HIF-1α) to the STI-1 promoter. Importantly, this STI-1 signalling promoted bone marrow derived cells (BMDCs) proliferation and migration in vitro and recruitment to the ischemic brain in vivo, and augmenting its signalling facilitated neurological recovery in part by recruiting BMDCs to the ischemic brain. Our results thus identified a novel mechanism by which ischemic insults can trigger a self-protective mechanism to facilitate recovery. This work identifies HIF-1α-mediated transcription of STI-1 and PrPc interaction as leading to BMDCs recruitment into ischemic brains following stroke in both patients and animal models of stroke, highlighting novel neuroprotective possibilities. PMID:23836498

  14. Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

    PubMed

    Hettich, Michael; Lahoti, Jayashree; Prasad, Shruthi; Niedermann, Gabriele

    2016-08-15

    T cell-recruiting bispecific antibodies (bsAb) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133×CD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by BIM and FAS as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers, and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor regrowth, reminiscent of tolerance induction by CD3 mAb-mediated T-cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors. Cancer Res; 76(16); 4673-83. ©2016 AACR. PMID:27302161

  15. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  16. Differential Incorporation of β-actin as A Component of RNA Polymerase II into Regulatory Regions of Stemness/Differentiation Genes in Retinoic Acid-Induced Differentiated Human Embryonic Carcinoma Cells

    PubMed Central

    Falahzadeh, Khadijeh; Shahhoseini, Maryam; Afsharian, Parvaneh

    2016-01-01

    Objective Nuclear actin is involved in transcription regulation by recruitment of histone modifiers and chromatin remodelers to the regulatory regions of active genes. In recent years, further attention has been focused on the role of actin as a nuclear protein in transcriptional processes. In the current study, the epigenetic role of nuclear actin on transcription regulation of two stemness (OCT4 and NANOG) and two differentiation) NESTIN and PAX6) marker genes was evaluated in a human embryonal carcinoma cell line (NT2) before and after differentiation induction. Materials and Methods In this experimental study, differentiation of embryonal cells was induced by retinoic acid (RA), and quantitative real-time polymerase chain reaction (PCR) was used to evaluate differential expression of marker genes before and 3 days after RA- induced differentiation. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR was then undertaken to monitor the incorporation of β-actin, as a functional component of RNA polymerase II, in the regulatory regions of marker genes. Results Data showed significant change in nuclear actin incorporation into the promoter regions of NESTIN and PAX6 after RA-induction. Conclusion We emphasize the dynamic functional role of nuclear actin in differentiation of embryonal cells and its role as a subunit of RNA polymerase II. PMID:27540526

  17. PER1 prevents excessive innate immune response during endotoxin-induced liver injury through regulation of macrophage recruitment in mice

    PubMed Central

    Wang, T; Wang, Z; Yang, P; Xia, L; Zhou, M; Wang, S; Du, Jie; Zhang, J

    2016-01-01

    The severity of acute liver failure (ALF) induced by bacterial lipopolysaccharide (LPS) is associated with the hepatic innate immune response. The core circadian molecular clock modulates the innate immune response by controlling rhythmic pathogen recognition by the innate immune system and daily variations in cytokine gene expression. However, the molecular link between circadian genes and the innate immune system has remained unclear. Here, we showed that mice lacking the clock gene Per1 (Period1) are more susceptible to LPS/d-galactosamine (LPS/GalN)-induced macrophage-dependent ALF compared with wild-type (WT) mice. Per1 deletion caused a remarkable increase in the number of Kupffer cells (KCs) in the liver, resulting in an elevation of the levels of pro-inflammatory cytokines after LPS treatment. Loss of Per1 had no effect on the proliferation or apoptosis of macrophages; however, it enhanced the recruitment of macrophages, which was associated with an increase in CC chemokine receptor 2 (Ccr2) expression levels in monocytes/macrophages. Deletion of Ccr2 rescued d-GalN/LPS-induced liver injury in Per1−/− mice. We demonstrated that the upregulation of Ccr2 expression by Per1 deletion could be reversed by the synthetic peroxisome proliferator-activated receptor gamma (PPAR-γ) antagonist GW9662. Further analysis indicated that PER1 binds to PPAR-γ on the Ccr2 promoter and enhanced the inhibitory effect of PPAR-γ on Ccr2 expression. These results reveal that Per1 reduces hepatic macrophage recruitment through interaction with PPAR-γ and prevents an excessive innate immune response in endotoxin-induced liver injury. PMID:27054331

  18. Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

    PubMed

    Tao, Le; Zeng, Yigang; Wang, Jun; Liu, Zhihong; Shen, Bing; Ge, Jifu; Liu, Yong; Guo, Yifeng; Qiu, Jianxin

    2015-11-01

    Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA‑containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA‑induced upper urothelial tract cancer (AAN‑UUC) and non‑AAN‑UUC tissues. Patients with AAN‑UUC and non‑AAN‑UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues (P<0.05). TargenScan and Gene ontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa‑miR‑4795‑5p, Hsa‑miR‑488, Hsa‑miR‑4784, Hsa‑miR‑330, Hsa‑miR‑3916, Hsa‑miR‑4274 and Hsa‑miR‑181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively. PMID:26397152

  19. Testicular acid phosphatase induces odontoblast differentiation and mineralization.

    PubMed

    Choi, Hwajung; Kim, Tak-Heun; Yun, Chi-Young; Kim, Jung-Wook; Cho, Eui-Sic

    2016-04-01

    Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration. PMID:26547858

  20. The role of endothelial cell adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 in leucocyte recruitment induced by exogenous methylglyoxal

    PubMed Central

    Su, Yang; Lei, Xi; Wu, Lingyun; Liu, Lixin

    2012-01-01

    Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In hyperglycaemic conditions, increased MG level has been linked to the development of diabetes and its vascular complications at the macrovascular and microvascular levels where inflammation plays a role. To study the mechanism of MG-induced inflammation in vivo, we applied MG locally to healthy mice and used intravital microscopy to investigate the role of endothelial cell adhesion molecules in MG-induced leucocyte recruitment in cremasteric microvasculature. Administration of MG (25 and 50 mg/kg) to the tissue dose-dependently induced leucocyte recruitment at 4·0–5·5 hr, with 84–92% recruited cells being neutrophils. Such MG treatment up-regulated the expression of endothelial cell adhesion molecules P-selectin, E-selectin, intercellular adhesion molecule-1, but not vascular cell adhesion molecule-1. Activation of the nuclear factor-κB signalling pathway contributed to MG-induced up-regulation of these adhesion molecules and leucocyte recruitment. The role of the up-regulated endothelial cell adhesion molecules in MG-induced leucocyte recruitment was determined by applying specific functional blocking antibodies to MG-treated animals and observing changes in leucocyte recruitment parameters. Our data demonstrate that the up-regulation of P-selectin, E-selectin and intercellular adhesion molecule-1 contributes to the increased leucocyte rolling flux, reduced leucocyte rolling velocity, and increased leucocyte adhesion, respectively. Our results reveal the role of endothelial cell adhesion molecules in MG-induced leucocyte recruitment in microvasculature, an inflammatory condition related to diabetic vascular complications. PMID:22681228

  1. Muramyl Dipeptide Induces NOD2-Dependent Ly6Chigh Monocyte Recruitment to the Lungs and Protects Against Influenza Virus Infection

    PubMed Central

    Coulombe, François; Fiola, Stéphanie; Akira, Shizuo; Cormier, Yvon; Gosselin, Jean

    2012-01-01

    Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6Chigh “inflammatory” monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6Chigh monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6Chigh monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV. PMID:22590599

  2. Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

    PubMed

    Imamura, Osamu; Arai, Masaaki; Dateki, Minori; Ogata, Toru; Uchida, Ryuji; Tomoda, Hiroshi; Takishima, Kunio

    2015-12-01

    Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil

  3. Magnetic field induced differential neutron phase contrast imaging

    SciTech Connect

    Strobl, M.; Treimer, W.; Walter, P.; Keil, S.; Manke, I.

    2007-12-17

    Besides the attenuation of a neutron beam penetrating an object, induced phase changes have been utilized to provide contrast in neutron and x-ray imaging. In analogy to differential phase contrast imaging of bulk samples, the refraction of neutrons by magnetic fields yields image contrast. Here, it will be reported how double crystal setups can provide quantitative tomographic images of magnetic fields. The use of magnetic air prisms adequate to split the neutron spin states enables a distinction of field induced phase shifts and these introduced by interaction with matter.

  4. Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

    PubMed Central

    Jones-Villeneuve, E M; Rudnicki, M A; Harris, J F; McBurney, M W

    1983-01-01

    We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs. Images PMID:6656766

  5. Employee recruitment.

    PubMed

    Breaugh, James A

    2013-01-01

    The way an organization recruits can influence the type of employees it hires, how they perform, and their retention rate. This article provides a selective review of research that has addressed recruitment targeting, recruitment methods, the recruitment message, recruiters, the organizational site visit, the job offer, and the timing of recruitment actions. These and other topics (e.g., the job applicant's perspective) are discussed in terms of their potential influence on prehire (e.g., the quality of job applicants) and posthire (e.g., new employee retention) recruitment outcomes. In reviewing research, attention is given to the current state of scientific knowledge, limitations of previous research, and important issues meriting future investigation. PMID:23121331

  6. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

    PubMed

    Parameswaran, Sreejit; Sharma, Rajendra K

    2016-09-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  7. Induced differentiation of adipose-derived stromal cells into myoblasts.

    PubMed

    Wu, Guizhu; Zheng, Xiu; Jiang, Zhongqing; Wang, Jinhua; Song, Yanfeng

    2010-06-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no

  8. BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling

    PubMed Central

    Tang, Ni; Song, Wen‐Xin; Luo, Jinyong; Luo, Xiaoji; Chen, Jin; Sharff, Katie A.; Bi, Yang; He, Bai‐Cheng; Huang, Jia‐Yi; Zhu, Gao‐Hui; Su, Yu‐Xi; Jiang, Wei; Tang, Min; He, Yun; Wang, Yi; Chen, Liang; Zuo, Guo‐Wei; Shen, Jikun; Pan, Xiaochuan; Reid, Russell R.; Luu, Hue H.; Haydon, Rex C.

    2008-01-01

    Abstract Bone morphogenetic protein 9 (BMP‐9) is a member of the transforming growth factor (TGF)‐β/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/β‐catenin signalling plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. Wnt3A and BMP‐9 enhanced each other’s ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP‐9‐induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR‐5 or low‐density lipoprotein receptor‐related protein (LRP)‐6 exerted no inhibitory effect on BMP‐9‐induced ALP activity. β‐Catenin knockdown in MSCs and MEFs diminished BMP‐9‐induced ALP activity, and led to a decrease in BMP‐9‐induced osteocalcin reporter activity and BMP‐9‐induced expression of late osteogenic markers. Furthermore, β‐catenin knockdown or FrzB overexpression inhibited BMP‐9‐induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP‐9 induced recruitment of both Runx2 and β‐catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between β‐catenin and Runx2, plays an important role in BMP‐9‐induced osteogenic differentiation of MSCs. PMID:19175684

  9. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes

    PubMed Central

    Choi, Si Ho; Gearhart, Micah D.; Cui, Ziyou; Bosnakovski, Darko; Kim, Minjee; Schennum, Natalie; Kyba, Michael

    2016-01-01

    Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation. PMID:26951377

  10. DUX4 recruits p300/CBP through its C-terminus and induces global H3K27 acetylation changes.

    PubMed

    Choi, Si Ho; Gearhart, Micah D; Cui, Ziyou; Bosnakovski, Darko; Kim, Minjee; Schennum, Natalie; Kyba, Michael

    2016-06-20

    Ectopic expression of the double homeodomain transcription factor DUX4 causes facioscapulohumeral muscular dystrophy (FSHD). Mechanisms of action of DUX4 are currently unknown. Using immortalized human myoblasts with a titratable DUX4 transgene, we identify by mass spectrometry an interaction between the DUX4 C-terminus and the histone acetyltransferases p300/CBP. Chromatin immunoprecipitation shows that DUX4 recruits p300 to its target gene, ZSCAN4, displaces histone H3 from the center of its binding site, and induces H3K27Ac in its vicinity, but C-terminal deleted DUX4 does not. We show that a DUX4 minigene, bearing only the homeodomains and C-terminus, is transcriptionally functional and cytotoxic, and that overexpression of a nuclear targeted C-terminus impairs the ability of WT DUX4 to interact with p300 and to regulate target genes. Genomic profiling of DUX4, histone H3, and H3 modifications reveals that DUX4 binds two classes of loci: DNase accessible H3K27Ac-rich chromatin and inaccessible H3K27Ac-depleted MaLR-enriched chromatin. At this latter class, it acts as a pioneer factor, recruiting H3K27 acetyltransferase activity and opening the locus for transcription. In concert with local increased H3K27Ac, the strong H3K27Ac peaks at distant sites are significantly depleted of H3K27Ac, thus DUX4 uses its C-terminus to induce a global reorganization of H3K27 acetylation. PMID:26951377

  11. Laser-induced differential normalized fluorescence method for cancer diagnosis

    DOEpatents

    Vo-Dinh, T.; Panjehpour, M.; Overholt, B.F.

    1996-12-03

    An apparatus and method for cancer diagnosis are disclosed. The diagnostic method includes the steps of irradiating a tissue sample with monochromatic excitation light, producing a laser-induced fluorescence spectrum from emission radiation generated by interaction of the excitation light with the tissue sample, and dividing the intensity at each wavelength of the laser-induced fluorescence spectrum by the integrated area under the laser-induced fluorescence spectrum to produce a normalized spectrum. A mathematical difference between the normalized spectrum and an average value of a reference set of normalized spectra which correspond to normal tissues is calculated, which provides for amplifying small changes in weak signals from malignant tissues for improved analysis. The calculated differential normalized spectrum is correlated to a specific condition of a tissue sample. 5 figs.

  12. Laser-induced differential normalized fluorescence method for cancer diagnosis

    DOEpatents

    Vo-Dinh, Tuan; Panjehpour, Masoud; Overholt, Bergein F.

    1996-01-01

    An apparatus and method for cancer diagnosis are disclosed. The diagnostic method includes the steps of irradiating a tissue sample with monochromatic excitation light, producing a laser-induced fluorescence spectrum from emission radiation generated by interaction of the excitation light with the tissue sample, and dividing the intensity at each wavelength of the laser-induced fluorescence spectrum by the integrated area under the laser-induced fluorescence spectrum to produce a normalized spectrum. A mathematical difference between the normalized spectrum and an average value of a reference set of normalized spectra which correspond to normal tissues is calculated, which provides for amplifying small changes in weak signals from malignant tissues for improved analysis. The calculated differential normalized spectrum is correlated to a specific condition of a tissue sample.

  13. Differential Binding Partners of the Mis18α/β YIPPEE Domains Regulate Mis18 Complex Recruitment to Centromeres.

    PubMed

    Stellfox, Madison E; Nardi, Isaac K; Knippler, Christina M; Foltz, Daniel R

    2016-06-01

    The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A-specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β, and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N terminus of Mis18BP1, whereas Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition. PMID:27239045

  14. Differential binding partners of the Mis18α/β YIPPEE domains regulates the Mis18 complex recruitment to centromeres

    PubMed Central

    Knippler, Christina M.; Foltz, Daniel R.

    2016-01-01

    The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N-terminus of Mis18BP1; whereas, Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N-terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition. PMID:27239045

  15. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

    PubMed

    Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

    2014-01-01

    Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species. PMID:25199961

  16. Baicalin, a Flavone, Induces the Differentiation of Cultured Osteoblasts

    PubMed Central

    Guo, Ava J. Y.; Choi, Roy C. Y.; Cheung, Anna W. H.; Chen, Vicky P.; Xu, Sherry L.; Dong, Tina T. X.; Chen, Ji J.; Tsim, Karl W. K.

    2011-01-01

    Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/β-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3β and, subsequently, induced the nuclear accumulation of the β-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/β-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/β-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis. PMID:21652696

  17. Bile acids induce hepatic differentiation of mesenchymal stem cells

    PubMed Central

    Sawitza, Iris; Kordes, Claus; Götze, Silke; Herebian, Diran; Häussinger, Dieter

    2015-01-01

    Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-β/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas β-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration. PMID:26304833

  18. Changes in polysialic acid expression on myeloid cells during differentiation and recruitment to sites of inflammation: Role in phagocytosis

    PubMed Central

    Stamatos, Nicholas M; Zhang, Lei; Jokilammi, Anne; Finne, Jukka; Chen, Wilbur H; El-Maarouf, Abderrahman; Cross, Alan S; Hankey, Kim G

    2014-01-01

    Polysialic acid (polySia) is a unique linear homopolymer of α2,8-linked sialic acid that has been studied extensively as a posttranslational modification of neural cell adhesion molecule in the central nervous system. Only two proteins are known to be polysialylated in cells of the immune system: CD56 on human natural killer cells and murine bone marrow (BM) leukocytes, and neuropilin-2 (NRP-2) on dendritic cells (DCs). We tested the hypothesis that polySia expression is regulated during maturation and migration of leukocytes and plays a role in functional activity. Using wild-type and NCAM−/− mice, we show that BM neutrophils express only polysialylated CD56, whereas a subset of BM monocytes expresses polysialylated CD56 and/or another polysialylated protein(s). We demonstrate that polysialylated CD56 expression is progressively down-regulated in wild-type monocytes and monocyte-derived cells during migration from BM through peripheral blood to pulmonary and peritoneal sites of inflammation. Freshly isolated monocyte-derived peritoneal macrophages are devoid of polySia yet re-express polySia on NRP-2 and an additional protein(s) after maintenance in culture. Removal of polySia from these cells enhances phagocytosis of Klebsiella pneumoniae, suggesting that down-regulation of polySia on macrophages facilitates bacterial clearance. Using wild-type and NRP-2−/− mice, we demonstrate that NRP-2 and an additional protein(s) are polysialylated by ST8 SiaIV in BM-derived DCs. We conclude that polySia expression in monocyte-derived cells is dynamically regulated by ST8 SiaIV activity and by expression of carrier proteins during recruitment to sites of inflammation and influences cellular interactions with microbes, contributing to innate and adaptive immune responses. PMID:24865221

  19. N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis

    SciTech Connect

    Tillet, Emmanuelle . E-mail: emmanuelle.tillet@cea.fr; Vittet, Daniel; Feraud, Olivier; Moore, Robert; Kemler, Rolf; Huber, Philippe

    2005-11-01

    Endothelial cells express two classical cadherins, VE-cadherin and N-cadherin. VE-cadherin is absolutely required for vascular morphogenesis, but N-cadherin is thought to participate in vessel stabilization by interacting with periendothelial cells during vessel formation. However, recent data suggest a more critical role for N-cadherin in endothelium that would regulate angiogenesis, in part by controlling VE-cadherin expression. In this study, we have assessed N-cadherin function in vascular development using an in vitro model derived from embryonic stem (ES) cell differentiation. We show that pluripotent ES cells genetically null for N-cadherin can differentiate normally into endothelial cells. In addition, sprouting angiogenesis was unaltered, suggesting that N-cadherin is not essential for the early events of angiogenesis. However, the lack of N-cadherin led to an impairment in pericyte covering of endothelial outgrowths. We conclude that N-cadherin is necessary neither for vasculogenesis nor proliferation and migration of endothelial cells but is required for the subsequent maturation of endothelial sprouts by interacting with pericytes.

  20. Differential diagnosis of food protein-induced enterocolitis syndrome

    PubMed Central

    Fiocchi, Alessandro; Claps, Alessia; Dahdah, Lamia; Brindisi, Giulia; Dionisi-Vici, Carlo; Martelli, Alberto

    2014-01-01

    Purpose of review To assess all the possible differential diagnosis of food protein-induced enterocolitis syndrome (FPIES), both in acute and chronic presentation, reviewing the data reported in published studies. Recent findings There is an increase of reported cases of FPIES in recent years. As the disease presents with nonspecific symptoms, it can be misunderstood in many ways. The differential diagnosis includes, in acute presentations, the following: sepsis, other infectious diseases, acute gastrointestinal episodes, surgical emergencies, food allergies. In its chronic forms, FPIES may mimic malabsorption syndromes, metabolic disorders, primary immunodeficiencies, neurological conditions, coagulation defects, and other types of non-IgE-mediated food allergy. Summary A thorough clinical evaluation, including symptoms, signs, and laboratory findings, is necessary to lead the clinicians toward the diagnosis of FPIES. The major reason for delayed diagnosis appears to be the lack of knowledge of the disease. PMID:24739227

  1. Pattern formation induced by a differential Poiseuille flow

    NASA Astrophysics Data System (ADS)

    Stucchi, L.; Vasquez, D. A.

    2014-12-01

    Differential advection, where a reactant is advected while another one is immobilized, leads to instabilities in reaction-advection-diffusion systems. In particular, a homogeneous steady state looses stability for strong enough flows, leading to chemical patterns moving in the direction of the flow. In this paper we study the effects of differential advection due to a two-dimensional Poiseuille flow. We carry out a linear stability analysis on a homogeneous state using an activator-inhibitor reaction. We find that shear dispersion induced by the Poiseuille flow may lead to instabilities at slower flow rates. We find that contrary to the one-dimensional system, the instability depends on which substance is advected. We find a critical average flow speed for instability depending on tube size. Numerical solutions of the nonlinear reaction-advection-diffusion result in patterns of constant shape propagating along the tube.

  2. Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes.

    PubMed

    Fujihara, Masatoshi; Obara, Hisato; Watanabe, Yusaku; Ono, Hisaya K; Sasaki, Jun; Goryo, Masanobu; Harasawa, Ryô

    2011-07-01

    Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine. PMID:21707738

  3. SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis

    PubMed Central

    Watson, Neva B.; Schneider, Karin M.; Massa, Paul T.

    2015-01-01

    Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the central nervous system causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator SHP-1, which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler’s murine encephalomyelitis virus infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification with loss of ambulation in wild type mice. Surprisingly, although similar extensive myofiber infection and inflammation is observed in SHP-1-deficient (SHP-1−/−) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1−/− muscle, and an increased infiltration of immature monocytes/macrophages correlated with absence of clinical disease in SHP-1−/− mice, while mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus-induced myopathy. PMID:25681345

  4. Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF

    PubMed Central

    Finsterbusch, Michaela; Voisin, Mathieu-Benoit; Beyrau, Martina; Williams, Timothy John

    2014-01-01

    Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB4, C5a, and KC. Further, in TNFR−/− mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema. PMID:24913232

  5. Oxidized Low Density Lipoprotein Induces Differentiation and Adhesion of Human Monocytes and the Monocytic Cell Line U937

    NASA Astrophysics Data System (ADS)

    Frostegard, Johan; Nilsson, Jan; Haegerstrand, Anders; Hamsten, Anders; Wigzell, Hans; Gidlund, Magnus

    1990-02-01

    Hypercholesterolemia is a major risk factor for development of atherosclerosis. In experimental animals fed a high-cholesterol diet, monocytes adhere to the arterial endothelium and penetrate into the intima where they differentiate into macrophages and ingest lipids thus giving rise to fatty streaks, the earliest type of atherosclerotic plaque. Macrophages express few receptors for normal low density lipo-protein (LDL) but can take up oxidized LDL by way of a scavenger receptor. The present study was designed to investigate the possible role of oxidized LDL in recruitment of resident intimal macrophages. We found that oxidized LDL induced enhanced expression of major histocompatibility complex class II molecules on human monocytes and U937 cells, a well-established system for studies of monocytic differentiation. Oxidized LDL also induced enhanced expression of the surface antigen LEuM3 but caused decreased expression of CD4 antigen, a pattern compatible with expression of a more differentiated macrophage-like phenotype. Oxidized LDL also initiated aggregation of monocytes and U937 cells and stimulated adhesion of U937 cells to cultured endothelial cells. The results indicate that oxidized LDL may contribute to development of atherosclerosis by inducing adhesion of monocytes to the arterial intima and by stimulating intimal monocytes to differentiate into resident macrophages.

  6. β-Carotene Attenuates Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Apoe−/− Mice

    PubMed Central

    Gopal, Kaliappan; Nagarajan, Perumal; Jedy, Jose; Raj, Avinash T.; Gnanaselvi, S. Kalai; Jahan, Parveen; Sharma, Yogendra; Shankar, Esaki M.; Kumar, Jerald M.

    2013-01-01

    Abdominal aortic aneurysm (AAA) is a common chronic degenerative disease characterized by progressive aortic dilation and rupture. The mechanisms underlying the role of α-tocopherol and β-carotene on AAA have not been comprehensively assessed. We investigated if α-tocopherol and β-carotene supplementation could attenuate AAA, and studied the underlying mechanisms utilized by the antioxidants to alleviate AAA. Four-months-old Apoe−/− mice were used in the induction of aneurysm by infusion of angiotensin II (Ang II), and were orally administered with α-tocopherol and β-carotene enriched diet for 60 days. Significant increase of LDL, cholesterol, triglycerides and circulating inflammatory cells was observed in the Ang II-treated animals, and gene expression studies showed that ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9 and MMP-12 were upregulated in the aorta of aneurysm-induced mice. Extensive plaques, aneurysm and diffusion of inflammatory cells into the tunica intima were also noticed. The size of aorta was significantly (P = 0.0002) increased (2.24±0.20 mm) in the aneurysm-induced animals as compared to control mice (1.17±0.06 mm). Interestingly, β-carotene dramatically controlled the diffusion of macrophages into the aortic tunica intima, and circulation. It also dissolved the formation of atheromatous plaque. Further, β-carotene significantly decreased the aortic diameter (1.33±0.12 mm) in the aneurysm-induced mice (β-carotene, P = 0.0002). It also downregulated ICAM-1, VCAM-1, MCP-1, M-CSF, MMP-2, MMP-9, MMP-12, PPAR-α and PPAR-γ following treatment. Hence, dietary supplementation of β-carotene may have a protective function against Ang II-induced AAA by ameliorating macrophage recruitment in Apoe−/− mice. PMID:23826202

  7. Asenapine-Induced Restless Legs Syndrome: Differentiation from Akathisia

    PubMed Central

    McCall, W. Vaughn; Riley, Mary Anne; Hodges, Chelsea; McCloud, Laryssa; Phillips, Marjorie; Rosenquist, Peter B.

    2014-01-01

    Akathisia and restless legs syndrome (RLS) share some common clinical features and a common relationship with dopamine dysfunction. However, the underlying causes and appropriate treatments for akathisia and RLS are different. Herein we describe a case of RLS that was precipitated by a single dose of asenapine, which is an atypical antipsychotic, and dissect the features that support the contention that this was indeed a case of RLS and not akathisia. Citation: McCall WV, Riley MA, Hodges C, McCloud L, Phillips M, Rosenquist PB. Asenapine-induced restless legs syndrome: differentiation from akathisia. J Clin Sleep Med 2014;10(12):1341-1342. PMID:25325577

  8. Substrate Induced Osteoblast-Like Differentiation of Stromal Stem Cells

    NASA Astrophysics Data System (ADS)

    Belizar, Jacqueline; Glaser, Reena; Hung, Matthew; Simon, Marcia; Jurukovski, Vladimir; Rafailovich, Miriam; Shih, Alice

    2009-03-01

    We have demonstrated that Adipose-derived stem cells (ASCs) can be induced to biomineralize on a polybutadiene (PB) coated Si substrate. The cells began to generate calcium phosphate deposits after a five-day incubation period in the absence of dexamethasone. Control cells plated on tissue culture PS culture dish (TCP) did not biomineralize. In addition, the biomineralizing culture retained proliferative cells In order to determine whether the induction was transient, we transferred the cells exposed to polybutadiene after 14 and 28-day incubation periods to TCP dishes. These cells continued to biominerlize. Genetic testing is underway which will determine whether differentiation is maintained after transfer.

  9. Spinal GABA-B receptor modulates neutrophil recruitment to the knee joint in zymosan-induced arthritis.

    PubMed

    Bassi, Gabriel S; do C Malvar, David; Cunha, Thiago M; Cunha, Fernando Q; Kanashiro, Alexandre

    2016-08-01

    Recent studies have demonstrated that the central nervous system controls inflammatory responses by activating complex efferent neuroimmune pathways. The present study was designed to evaluate the role that central gamma-aminobutyric acid type B (GABA-B) receptor plays in neutrophil migration in a murine model of zymosan-induced arthritis by using different pharmacological tools. We observed that intrathecal administration of baclofen, a selective GABA-B agonist, exacerbated the inflammatory response in the knee after zymosan administration characterized by an increase in the neutrophil recruitment and knee joint edema, whereas saclofen, a GABA-B antagonist, exerted the opposite effect. Intrathecal pretreatment of the animals with SB203580 (an inhibitor of p38 mitogen-activated protein kinase) blocked the pro-inflammatory effect of baclofen. On the other hand, systemic administration of guanethidine, a sympatholytic drug that inhibits catecholamine release, and nadolol, a beta-adrenergic receptor antagonist, reversed the effect of saclofen. Moreover, saclofen suppressed the release of the pro-inflammatory cytokines into the knee joint (ELISA) and pain-related behaviors (open field test). Since the anti-inflammatory effect of saclofen depends on the sympathetic nervous system integrity, we observed that isoproterenol, a beta-adrenergic receptor agonist, mimics the central GABA-B blockade decreasing knee joint neutrophil recruitment. Together, these results demonstrate that the pharmacological manipulation of spinal GABAergic transmission aids control of neutrophil migration to the inflamed joint by modulating the activation of the knee joint-innervating sympathetic terminal fibers through a mechanism dependent on peripheral beta-adrenergic receptors and central components, such as p38 MAPK. PMID:27106212

  10. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

    PubMed Central

    Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-01-01

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  11. Inhibition of bacterial cell wall-induced leukocyte recruitment and hepatic granuloma formation by TGF-beta gene transfer.

    PubMed

    Song, X; Zeng, L; Pilo, C M; Zagorski, J; Wahl, S M

    1999-10-01

    Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-beta 1 i.m. to the SCW animals to determine the effect of TGF-beta 1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-beta 1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-beta 1 DNA not only reduced key proinflammatory cytokines including TNF-alpha, IL-1 beta, IFN-gamma, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-beta 1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-beta 1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases. PMID:10491005

  12. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

    PubMed

    Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-11-24

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  13. Recruitment of 99m-technetium- or 111-indium-labelled polymorphonuclear leucocytes in experimentally induced pyogranulomas in lambs

    SciTech Connect

    Guilloteau, L.; Pepin, M.; Pardon, P.; Le Pape, A. )

    1990-10-01

    The recruitment of polymorphonuclear leucocytes (PMNs) during the development of experimental pyogranulomas induced by Corynebacterium pseudotuberculosis was followed in nine male lambs by scintigraphic examination. Autologous blood PMNs were labelled with 99m-technetium or 111-indium and were re-injected intravenously into infected lambs. The functional properties of the labelled cells were monitored (1) in vitro by measuring their phagocytic and bactericidal activity against C. pseudotuberculosis and their chemotaxis under agarose, and (2) in vivo by following scintigraphically their capacity to accumulate in an inflammatory focus induced by intradermal injection of latex beads coated with Salmonella abortus equi lipopolysaccharide. Following inoculation of corynebacteria into the right ear of lambs, radioactive foci were observed to be localized in the right ear and in the draining lymph nodes during the 4 days following inoculation. Histopathological examination performed 32 h after inoculation confirmed the intense accumulation of PMNs at these sites. With the exception of one animal, which presented visible foci in the neck 14 days postinoculation, no radioactive foci were observed during the later phases of experimental infection, despite the presence of multiple pyogranulomas which were confirmed by bacteriological examination after necropsy of the lambs. Histopathological examination of these lesions revealed layers of fibroblasts, lymphocytes, and macrophages surrounding a necrotic centre. The results of these studies suggest that the contribution of PMNs during the chronic phase of inflammation is considerably reduced in comparison with the acute inflammatory phase of the infectious process.

  14. Vestibular recruitment

    NASA Technical Reports Server (NTRS)

    Tsemakhov, S. G.

    1980-01-01

    Vestibular recruitment is defined through the analysis of several references. It is concluded that vestibular recruitment is an objective phenomenon which manifests itself during the affection of the vestibular receptor and thus serves as a diagnostic tool during affection of the vestibular system.

  15. OCT4 Acts as an Integrator of Pluripotency and Signal-Induced Differentiation.

    PubMed

    Simandi, Zoltan; Horvath, Attila; Wright, Lyndsey C; Cuaranta-Monroy, Ixchelt; De Luca, Isabella; Karolyi, Katalin; Sauer, Sascha; Deleuze, Jean-Francois; Gudas, Lorraine J; Cowley, Shaun M; Nagy, Laszlo

    2016-08-18

    Cell type specification relies on the capacity of undifferentiated cells to properly respond to specific differentiation-inducing signals. Using genomic approaches along with loss- and gain-of-function genetic models, we identified OCT4-dependent mechanisms that provide embryonic stem cells with the means to customize their response to external cues. OCT4 binds a large set of low-accessible genomic regions. At these sites, OCT4 is required for proper enhancer and gene activation by recruiting co-regulators and RAR:RXR or β-catenin, suggesting an unexpected collaboration between the lineage-determining transcription factor and these differentiation-initiating, signal-dependent transcription factors. As a proof of concept, we demonstrate that overexpression of OCT4 in a kidney cell line is sufficient for signal-dependent activation of otherwise unresponsive genes in these cells. Our results uncover OCT4 as an integral and necessary component of signal-regulated transcriptional processes required for tissue-specific responses. PMID:27499297

  16. Resident recruitment.

    PubMed

    Longmaid, H Esterbrook

    2003-02-01

    This article has introduced the reader to the critical components of successful recruitment of radiology residents. With particular attention to the ACGME institutional and program requirements regarding resident recruitment, and an explanation of the support systems (ERAS and NRMP) currently available to those involved in applicant review and selection, the article has sought to delineate a sensible approach to recruitment. Successful recruiters have mastered the essentials of these programs and have learned to adapt the programs to their needs. As new program directors work with their departments' resident selection committees, they will identify the factors that faculty and current residents cite as most important in the successful selection of new residents. By structuring the application review process, exploiting the power of the ERAS, and crafting a purposeful and friendly interview process, radiology residency directors can find and recruit the residents who best match their programs. PMID:12585436

  17. Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

    PubMed

    Casas, Sandra M; La Peyre, Jerome F

    2013-05-01

    Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species. PMID:22999495

  18. IL-4 Induces Cholinergic Differentiation of Retinal Cells In Vitro.

    PubMed

    Granja, Marcelo Gomes; Braga, Luis Eduardo Gomes; Carpi-Santos, Raul; de Araujo-Martins, Leandro; Nunes-Tavares, Nilson; Calaza, Karin C; Dos Santos, Aline Araujo; Giestal-de-Araujo, Elizabeth

    2015-07-01

    Interleukin-4 (IL-4) is a pleiotropic cytokine that regulates several phenomena, among them survival and differentiation of neuronal and glial cells. The aim of this work was to investigate the effect of IL-4 on the cholinergic differentiation of neonatal rat retinal cells in vitro, evaluating its effect on the levels of cholinergic markers (CHT1-high-affinity choline transporter; VAChT-vesicular acetylcholine transporter, ChAT-choline acetyltransferase, AChE-acetylcholinesterase), muscarinic receptors, and on the signaling pathways involved. Lister Hooded rat pups were used in postnatal days 0-2 (P0-P2). Our results show that IL-4 treatment (50 U/mL) for 48 h increases the levels of the cholinergic transporters VAChT and CHT1, the acetylcholinesterase activity, and the number of ChAT-positive cells. It also induces changes in muscarinic receptor levels, leading to a small decrease in M1 levels and a significant increase in M3 and M5 levels after 48 h of treatment. We also showed that IL-4 effect on M3 receptors is dependent on type I IL-4 receptor and on an increase in NFκB phosphorylation. These results indicate that IL-4 stimulates cholinergic differentiation of retinal cells. PMID:25682112

  19. Increase of cellular recruitment, phagocytosis ability and nitric oxide production induced by hydroalcoholic extract from Chenopodium ambrosioides leaves.

    PubMed

    Cruz, Gustavo V B; Pereira, Paulo Vitor S; Patrício, Fernando J; Costa, Graciomar C; Sousa, Sanara M; Frazão, Josias B; Aragão-Filho, Walmir C; Maciel, Márcia C G; Silva, Lucilene A; Amaral, Flávia M M; Barroqueiro, Elizabeth S B; Guerra, Rosane N M; Nascimento, Flávia R F

    2007-04-20

    The leaves and the oil from the seeds of Chenopodium ambrosioides L. (Chenopodiaceae), a plant known in Brazil as 'mastruz', have been used by native people to treat parasitic diseases. Experimentally it was shown that Chenopodium ambrosioides inhibits the Ehrlich tumor growth, what could be due to an immunomodulatory effect of this product. The aim of this study was to investigate the effect of hydroalcoholic crude extract (HCE) from leaves of Chenopodium ambrosioides on macrophage activity and on lymphoid organs cellularity. C3H/HePas mice received the HCE (5mg/kg) by intraperitoneal via and were sacrificed 2 days later. HCE treatment did not alter the cell number in bone marrow, but it increased the cell number in peritoneal cavity, spleen and lymph node. The spreading and phagocytosis activity, the PMA-induced hydrogen peroxide (H(2)O(2)) release and the nitric oxide (NO) production were also increased when compared to control group. Similar results were obtained with concanavalin A (Con A), used as a positive control, with exception of the NO production that was only detected in HCE-derived macrophages. The in vitro treatment with HCE induced a dose-dependent NO production by resident macrophages, but did not enhance the NO production by HCE-derived macrophage, which however, was enhanced by Con A, suggesting that HCE and Con A induce NO production by different routes. In conclusion, HCE-treatment was able to increase the macrophages activity and also the cellular recruitment to secondary lymphoid organs, what could explain the previously related anti-tumor activity of Chenopodium ambrosioides. PMID:17156956

  20. Early Macrophage Recruitment and Alternative Activation Are Critical for the Later Development of Hypoxia-induced Pulmonary Hypertension

    PubMed Central

    Vergadi, Eleni; Chang, Mun Seog; Lee, Changjin; Liang, Olin; Liu, Xianlan; Fernandez-Gonzalez, Angeles; Mitsialis, S. Alex; Kourembanas, Stella

    2011-01-01

    Background Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, is protective in HPH. Methods and Results We generated bitransgenic mice that overexpress human HO-1 under doxycycline (dox) control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of dox resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of Found in Inflammatory Zone-1, Arginase-1 and Chitinase-3-like-3. A brief, two-day pulse of dox delayed but did not prevent the peak of hypoxic inflammation, and could not protect from HPH. In contrast, a seven-day dox treatment sustained high HO-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage IL-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted proliferation of pulmonary artery smooth muscle cells while treatment with carbon monoxide, a HO-1 enzymatic product, abrogated this effect. Conclusions Early recruitment and alternative activation of macrophages in hypoxic lungs is critical for the later development of HPH. HO-1 may confer protection from HPH by effectively modifing macrophage activation state in hypoxia. PMID:21518986

  1. Mineralized gelatin methacrylate-based matrices induce osteogenic differentiation of human induced pluripotent stem cells

    PubMed Central

    Kang, Heemin; Shih, Yu-Ru V.; Hwang, Yongsung; Wen, Cai; Rao, Vikram; Seo, Timothy; Varghese, Shyni

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source with pluripotency and self-renewal properties. Design of simple and robust biomaterials with an innate ability to induce lineage-specificity of hiPSCs is desirable to realize their applications in regenerative medicine. In this study, we investigated the potential of biomaterials containing calcium phosphate minerals to induce osteogenic differentiation of hiPSCs. hiPSCs cultured using mineralized gelatin methacrylate-based matrices underwent osteogenic differentiation ex vivo, both in two- dimensional (2-D) and three-dimensional (3-D) cultures, in growth medium devoid of any osteogenic-inducing chemical components or growth factors. Our findings that osteogenic differentiation of hiPSCs can be achieved through biomaterial-based cues alone present new avenues for personalized regenerative medicine. Such biomaterials that could not only act as structural scaffolds, but could also provide tissue-specific functions such as directing stem cell differentiation commitment, have great potential in bone tissue engineering. PMID:25153779

  2. Dextran induces differentiation of circulating endothelial progenitor cells

    PubMed Central

    Obi, Syotaro; Masuda, Haruchika; Akimaru, Hiroshi; Shizuno, Tomoko; Yamamoto, Kimiko; Ando, Joji; Asahara, Takayuki

    2014-01-01

    Abstract Endothelial progenitor cells (EPCs) have been demonstrated to be effective for the treatment of cardiovascular diseases. However, the differentiation process from circulation to adhesion has not been clarified because circulating EPCs rarely attached to dishes in EPC cultures previously. Here we investigated whether immature circulating EPCs differentiate into mature adhesive EPCs in response to dextran. When floating‐circulating EPCs derived from ex vivo expanded human cord blood were cultured with 5% and 10% dextran, they attached to fibronectin‐coated dishes and grew exponentially. The bioactivities of adhesion, proliferation, migration, tube formation, and differentiated type of EPC colony formation increased in EPCs exposed to dextran. The surface protein expression rate of the endothelial markers vascular endothelial growth factor (VEGF)‐R1/2, VE‐cadherin, Tie2, ICAM1, VCAM1, and integrin αv/β3 increased in EPCs exposed to dextran. The mRNA levels of VEGF‐R1/2, VE‐cadherin, Tie2, endothelial nitric oxide synthase, MMP9, and VEGF increased in EPCs treated with dextran. Those of endothelium‐related transcription factors ID1/2, FOXM1, HEY1, SMAD1, FOSL1, NFkB1, NRF2, HIF1A, EPAS1 increased in dextran‐treated EPCs; however, those of hematopoietic‐ and antiangiogenic‐related transcription factors TAL1, RUNX1, c‐MYB, GATA1/2, ERG, FOXH1, HHEX, SMAD2/3 decreased in dextran‐exposed EPCs. Inhibitor analysis showed that PI3K/Akt, ERK1/2, JNK, and p38 signal transduction pathways are involved in the differentiation in response to dextran. In conclusion, dextran induces differentiation of circulating EPCs in terms of adhesion, migration, proliferation, and vasculogenesis. The differentiation mechanism in response to dextran is regulated by multiple signal transductions including PI3K/Akt, ERK1/2, JNK, and p38. These findings indicate that dextran is an effective treatment for EPCs in regenerative medicines. PMID:24760515

  3. Human Papillomaviruses Activate and Recruit SMC1 Cohesin Proteins for the Differentiation-Dependent Life Cycle through Association with CTCF Insulators

    PubMed Central

    Satsuka, Ayano; Laimins, Laimonis A.

    2015-01-01

    Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses

  4. Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF-induced neutrophil recruitment.

    PubMed

    Phan, Vernon T; Wu, Xiumin; Cheng, Jason H; Sheng, Rebecca X; Chung, Alicia S; Zhuang, Guanglei; Tran, Christopher; Song, Qinghua; Kowanetz, Marcin; Sambrone, Amy; Tan, Martha; Meng, Y Gloria; Jackson, Erica L; Peale, Franklin V; Junttila, Melissa R; Ferrara, Napoleone

    2013-04-01

    Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b(+)Gr1(+) myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b(+)Ly6G(+) neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies. PMID:23530240

  5. Oncogenic RAS pathway activation promotes resistance to anti-VEGF therapy through G-CSF–induced neutrophil recruitment

    PubMed Central

    Phan, Vernon T.; Wu, Xiumin; Cheng, Jason H.; Sheng, Rebecca X.; Chung, Alicia S.; Zhuang, Guanglei; Tran, Christopher; Song, Qinghua; Kowanetz, Marcin; Sambrone, Amy; Tan, Martha; Meng, Y. Gloria; Jackson, Erica L.; Peale, Franklin V.; Junttila, Melissa R.; Ferrara, Napoleone

    2013-01-01

    Granulocyte-colony stimulating factor (G-CSF) promotes mobilization of CD11b+Gr1+ myeloid cells and has been implicated in resistance to anti-VEGF therapy in mouse models. High G-CSF production has been associated with a poor prognosis in cancer patients. Here we show that activation of the RAS/MEK/ERK pathway regulates G-CSF expression through the Ets transcription factor. Several growth factors induced G-CSF expression by a MEK-dependent mechanism. Inhibition of G-CSF release with a MEK inhibitor markedly reduced G-CSF production in vitro and synergized with anti-VEGF antibodies to reduce CD11b+Ly6G+ neutrophil mobilization and tumor growth and led to increased survival in animal models of cancer, including a genetically engineered mouse model of pancreatic adenocarcinoma. Analysis of biopsies from pancreatic cancer patients revealed increased phospho-MEK, G-CSF, and Ets expression and enhanced neutrophil recruitment compared with normal pancreata. These results provide insights into G-CSF regulation and on the mechanism of action of MEK inhibitors and point to unique anticancer strategies. PMID:23530240

  6. pDC therapy induces recovery from EAE by recruiting endogenous pDC to sites of CNS inflammation

    PubMed Central

    Duraes, Fernanda V.; Lippens, Carla; Steinbach, Karin; Dubrot, Juan; Brighouse, Dale; Bendriss-Vermare, Nathalie; Issazadeh-Navikas, Shohreh; Merkler, Doron; Hugues, Stephanie

    2016-01-01

    Plasmacytoid dendritic cells (pDCs) exhibit both innate and adaptive functions. In particular they are the main source of type I IFNs and directly impact T cell responses through antigen presentation. We have previously demonstrated that during experimental autoimmune encephalomyelitis (EAE) initiation, myelin-antigen presentation by pDCs is associated with suppressive Treg development and results in attenuated EAE. Here, we show that pDCs transferred during acute disease phase confer recovery from EAE. Clinical improvement is associated with migration of injected pDCs into inflamed CNS and is dependent on the subsequent and selective chemerin-mediated recruitment of endogenous pDCs to the CNS. The protective effect requires pDC pre-loading with myelin antigen, and is associated with the modulation of CNS-infiltrating pDC phenotype and inhibition of CNS encephalitogenic T cells. This study may pave the way for novel pDC-based cell therapies in autoimmune diseases, aiming at specifically modulating pathogenic cells that induce and sustain autoimmune inflammation. PMID:26341385

  7. Architectural factor HMGA induces promoter bending and recruits C/EBP and GATA during silkmoth chorion gene regulation.

    PubMed

    Papantonis, Argyris; Vanden Broeck, Josef; Lecanidou, Rena

    2008-11-15

    A protein displaying significant similarity to mammalian HMGA (high-mobility group A) proteins, but also bearing unique structural features, was isolated from silkmoth (Bombyx mori) follicular cells. This factor, named BmHMGA, exhibits specific binding preference for chorion gene promoter elements and induces DNA bending thereon. BmHMGA deploys temporal-specific interaction with transcription factors BmC/EBP (C/EBP is CCAAT/enhancer-binding protein) and BmGATAbeta during follicle maturation. The respective protein complexes can be detected on chorion gene promoters in vivo, with different developmental profiles each time. Analogous interaction takes place on the putative promoter of the BmC/EBP gene, hinting towards a transcriptional circuit that is responsible for the progress of choriogenesis as a whole. Finally, transient suppression of BmHMGA expression led to down-regulation of chorion genes and the BmC/EBP gene, and revealed recruitment of BmC/EBP, BmGATAbeta and TFIID (transcription factor IID)/TBP (TATA-box-binding protein) by BmHMGA. A revised model for chorion gene regulation is discussed in view of these findings. PMID:18636971

  8. AhR modulates the IL-22-producing cell proliferation/recruitment in imiquimod-induced psoriasis mouse model.

    PubMed

    Cochez, Perrine M; Michiels, Camille; Hendrickx, Emilie; Van Belle, Astrid B; Lemaire, Muriel M; Dauguet, Nicolas; Warnier, Guy; de Heusch, Magali; Togbe, Dieudonnée; Ryffel, Bernhard; Coulie, Pierre G; Renauld, Jean-Christophe; Dumoutier, Laure

    2016-06-01

    IL-22 has a detrimental role in skin inflammatory processes, for example in psoriasis. As transcription factor, AhR controls the IL-22 production by several cell types (i.e. Th17 cells). Here, we analyzed the role of Ahr in IL-22 production by immune cells in the inflamed skin, using an imiquimod-induced psoriasis mouse model. Our results indicate that IL-22 is expressed in the ear of imiquimod-treated Ahr(-/-) mice but less than in wild-type mice. We then studied the role of AhR on three cell populations known to produce IL-22 in the skin: γδ T cells, Th17 cells, and ILC3, and a novel IL-22-producing cell type identified in this setting: CD4(-) CD8(-) TCRβ(+) T cells. We showed that AhR is required for IL-22 production by Th17, but not by the three other cell types, in the imiquimod-treated ears. Moreover, AhR has a role in the recruitment of γδ T cells, ILC3, and CD4(-) CD8(-) TCRβ(+) T cells into the inflamed skin or in their local proliferation. Taken together, AhR has a direct role in IL-22 production by Th17 cells in the mouse ear skin, but not by γδ T cells, CD4(-) CD8(-) TCRβ(+) T cells and ILCs. PMID:27000947

  9. Olig2-expressing progenitor cells preferentially differentiate into oligodendrocytes in cuprizone-induced demyelinated lesions.

    PubMed

    Islam, Mohammad Shyful; Tatsumi, Kouko; Okuda, Hiroaki; Shiosaka, Sadao; Wanaka, Akio

    2009-01-01

    Many oligodendrocyte progenitor cells (OPCs) are found in acute or chronic demyelinated area, but not all of them differentiate efficiently into mature oligodendrocytes in the demyelinated central nervous system (CNS). Recent studies have shown that the basic helix-loop-helix transcription factor Olig2, which stimulates OPCs to differentiate into oligodendrocyte, is strongly up-regulated in many pathological conditions including acute or chronic demyelinating lesions in the adult CNS. Despite their potential role in the treatment of demyelinating diseases, the long-term fate of these up-regulated Olig2 cells has not been identified due to the lack of stable labeling methods. To trace their fate we have used double-transgenic mice, in which we were able to label Olig2-positive cells conditionally with green fluorescent protein (GFP). Demyelination was induced in these mice by feeding cuprizone, a copper chelator. After 6 weeks of cuprizone exposure, GFP-positive (GFP(+)) cells were processed for a second labeling with antibodies to major neural cell markers APC (mature oligodendrocyte marker), GFAP (astrocyte marker), NeuN (neuron marker), Iba1 (microglia marker) and NG2 proteoglycan (oligodendrocyte progenitor marker). More than half of the GFP(+) cells in the external capsule showed co-localization with NG2 proteoglycan. While the percentages of NG2-positive (NG2(+)) and APC-positive (APC(+)) oligodendrocyte lineage cells in cuprizone-treated mice were significantly higher than those in the normal diet group, no significant difference was observed for GFAP-positive (GFAP(+)) astrocytic lineage cells. Our data therefore provide direct evidence that proliferation and differentiation of local and/or recruited Olig2 progenitors contribute to remyelination in demyelinated lesions. PMID:19070638

  10. Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats.

    PubMed

    Rocha, Juraci Aparecida; Ribeiro, Susan Pereira; França, Cristiane Miranda; Coelho, Otávio; Alves, Gisele; Lacchini, Silvia; Kallás, Esper Georges; Irigoyen, Maria Cláudia; Consolim-Colombo, Fernanda M

    2016-04-15

    We tested the hypothesis that an increase in the anti-inflammatory cholinergic pathway, when induced by pyridostigmine (PY), may modulate subtypes of lymphocytes (CD4+, CD8+, FOXP3+) and macrophages (M1/M2) soon after myocardial infarction (MI) in rats. Wistar rats, randomly allocated to receive PY (40 mg·kg(-1)·day(-1)) in drinking water or to stay without treatment, were followed for 4 days and then were subjected to ligation of the left coronary artery. The groups-denominated as the pyridostigmine-treated infarcted (IP) and infarcted control (I) groups-were submitted to euthanasia 3 days after MI; the heart was removed for immunohistochemistry, and the peripheral blood and spleen were collected for flow cytometry analysis. Noninfarcted and untreated rats were used as controls (C Group). Echocardiographic measurements were registered on the second day after MI, and heart rate variability was measured on the third day after MI. The infarcted groups had similar MI areas, degrees of systolic dysfunction, blood pressures, and heart rates. Compared with the I Group, the IP Group showed a significant higher parasympathetic modulation and a lower sympathetic modulation, which were associated with a small, but significant, increase in diastolic function. The IP Group showed a significant increase in M2 macrophages and FOXP3(+)cells in the infarcted and peri-infarcted areas, a significantly higher frequency of circulating Treg cells (CD4(+)CD25(+)FOXP3(+)), and a less extreme decrease in conventional T cells (CD25(+)FOXP3(-)) compared with the I Group. Therefore, increasing cholinergic modulation with PY induces greater anti-inflammatory cell recruitment soon after MY in rats. PMID:26791829

  11. Type I interferons induced by radiation therapy mediate recruitment and effector function of CD8(+) T cells.

    PubMed

    Lim, Joanne Y H; Gerber, Scott A; Murphy, Shawn P; Lord, Edith M

    2014-03-01

    The need for an intact immune system for cancer radiation therapy to be effective suggests that radiation not only acts directly on the tumor but also indirectly, through the activation of host immune components. Recent studies demonstrated that endogenous type I interferons (type I IFNs) play a role in radiation-mediated anti-tumor immunity by enhancing the ability of dendritic cells to cross-prime CD8(+) T cells. However, it is still unclear to what extent endogenous type I IFNs contribute to the recruitment and function of CD8(+) T cells. Little is also known about the effects of type I IFNs on myeloid cells. In the current study, we demonstrate that type I and type II IFNs (IFN-γ) are both required for the increased production of CXCL10 (IP-10) chemokine by myeloid cells within the tumor after radiation treatment. Radiation-induced intratumoral IP-10 levels in turn correlate with tumor-infiltrating CD8(+) T cell numbers. Moreover, type I IFNs promote potent tumor-reactive CD8(+) T cells by directly affecting the phenotype, effector molecule production, and enhancing cytolytic activity. Using a unique inducible expression system to increase local levels of IFN-α exogenously, we show here that the capacity of radiation therapy to result in tumor control can be enhanced. Our preclinical approach to study the effects of local increase in IFN-α levels can be used to further optimize the combination therapy strategy in terms of dosing and scheduling, which may lead to better clinical outcome. PMID:24357146

  12. Laboratory-induced hyperventilation differentiates female sexual arousal disorder subtypes.

    PubMed

    Brotto, Lori A; Klein, Carolin; Gorzalka, Boris B

    2009-08-01

    The effects of heightened sympathetic nervous system (SNS) activity via laboratory-induced hyperventilation (LIH) on subjective and physiological sexual arousal were examined in a heterogeneous group of women with Sexual Arousal Disorder (SAD; n = 60), as well as across subtypes of SAD, in comparison to a control group of women without sexual difficulties (n = 42). Participants took part in 2 min of rapid breathing, a technique previously found to increase SNS activity, immediately prior to viewing erotic stimuli. Physiological arousal (i.e., vaginal pulse amplitude; VPA) was measured via the vaginal photoplethysmograph and subjective arousal was measured via self-report questionnaires. LIH differentiated women with SAD from those in the control group, with LIH increasing VPA in the latter, but having no significant effect in the heterogeneous SAD group. However, among subtypes of SAD, LIH differentiated women with genital (n = 16) and subjective (n = 16) subtypes of SAD from women with combined SAD (n = 28) and women without sexual difficulties. Specifically, women in the control group and those with combined SAD had a significant increase in VPA whereas women with genital or subjective SAD had a significant decrease in VPA following LIH. There was no significant effect of LIH on any self-report measure of sexual arousal following erotic stimuli. Implications of the results for the conceptualization, diagnosis, and treatment of SAD are discussed. PMID:18343989

  13. Tumour necrosis factor-α expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A

    PubMed Central

    Williams, Cara M M; Smith, Lance; Flanagan, Brian F; Steve Clegg, L; Coleman, John W

    1997-01-01

    Tumour necrosis factor-α (TNF-α) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-α is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-α expression influences recruitment of specific inflammatory cell types. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24–72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. Messenger RNA encoding TNF-α was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-α protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-α mRNA expression by BAL cells. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-α, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-α expression, whereas mobilization of

  14. A fetal human heart cardiac-inducing RNA (CIR) promotes the differentiation of stem cells into cardiomyocytes.

    PubMed

    Kochegarov, Andrei; Moses-Arms, Ashley; Lemanski, Larry F

    2015-08-01

    A specific human fetal heart RNA has been discovered, which has the ability to induce myocardial cell formation from mouse embryonic and human-induced pluripotent stem cells in culture. In this study, commercially obtained RNA from human fetal heart was cloned, sequenced, and synthesized using standard laboratory approaches. Molecular analyses of the specific fetal cardiac-inducing RNA (CIR), revealed that it is a fragment of N-sulfoglucosaminesulfohydrolase and the caspase recruitment domain family member 14 precursor. Stem cells transfected with CIRs often form into spindle-shaped cells characteristic of cardiomyocytes,and express the cardiac-specific contractile protein marker, troponin-T, in addition to tropomyosin and α-actinin as detected by immunohistochemical staining. Expression of these contractile proteins showed organization into sarcomeric myofibrils characteristic of striated cardiac muscle cells. Computer analyses of the RNA secondary structures of the active CIR show significant similarities to a RNA from salamander or myofibril-inducing RNA (MIR), which also promotes non-muscle cells to differentiate into cardiac muscle. Thus, these two RNAs, salamander MIR and the newly discovered human-cloned CIR reported here, appear to have evolutionarily conserved secondary structures suggesting that both play major roles in vertebrate heart development and, particularly, in the differentiation of cardiomyocytes from non-muscle cells during development. PMID:25761723

  15. Interleukin 17A promotes pneumococcal clearance by recruiting neutrophils and inducing apoptosis through a p38 mitogen-activated protein kinase-dependent mechanism in acute otitis media.

    PubMed

    Wang, Wei; Zhou, Aie; Zhang, Xuemei; Xiang, Yun; Huang, Yifei; Wang, Lei; Zhang, Shuai; Liu, Yusi; Yin, Yibing; He, Yujuan

    2014-06-01

    Streptococcus pneumoniae is a Gram-positive and human-restricted pathogen colonizing the nasopharynx with an absence of clinical symptoms as well as a major pathogen causing otitis media (OM), one of the most common childhood infections. Upon bacterial infection, neutrophils are rapidly activated and recruited to the infected site, acting as the frontline defender against emerging microbial pathogens via different ways. Evidence shows that interleukin 17A (IL-17A), a neutrophil-inducing factor, plays important roles in the immune responses in several diseases. However, its function in response to S. pneumoniae OM remains unclear. In this study, the function of IL-17A in response to S. pneumoniae OM was examined using an in vivo model. We developed a model of acute OM (AOM) in C57BL/6 mice and found that neutrophils were the dominant immune cells that infiltrated to the middle ear cavity (MEC) and contributed to bacterial clearance. Using IL-17A knockout (KO) mice, we found that IL-17A boosted neutrophil recruitment to the MEC and afterwards induced apoptosis, which was identified to be conducive to bacterial clearance. In addition, our observation suggested that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in the recruitment and apoptosis of neutrophils mediated by IL-17A. These data support the conclusion that IL-17A contributes to the host immune response against S. pneumoniae by promoting neutrophil recruitment and apoptosis through the p38 MAPK signaling pathway. PMID:24664502

  16. Preferential macrophage recruitment and polarization in LPS-induced animal model for COPD: noninvasive tracking using MRI.

    PubMed

    Al Faraj, Achraf; Sultana Shaik, Asma; Pureza, Mary Angeline; Alnafea, Mohammad; Halwani, Rabih

    2014-01-01

    Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of chronic obstructive respiratory diseases (COPD), which make them attractive vehicles to deliver contrast agents for diagnostic or drugs for therapeutic purposes. This study was designed to monitor and evaluate the migration of differently polarized M1 and M2 iron labeled macrophage subsets to the lung of a LPS-induced COPD animal model and to assess their polarization state once they have reached the inflammatory sites in the lung after intravenous injection. Ex vivo polarized bone marrow derived M1 or M2 macrophages were first efficiently and safely labeled with amine-modified PEGylated dextran-coated SPIO nanoparticles and without altering their polarization profile. Their biodistribution in abdominal organs and their homing to the site of inflammation in the lung was tracked for the first time using a free-breathing non-invasive MR imaging protocol on a 4.7T magnet after their intravenous administration. This imaging protocol was optimized to allow both detection of iron labeled macrophages and visualization of inflammation in the lung. M1 and M2 macrophages were successfully detected in the lung starting from 2 hours post injection with no variation in their migration profile. Quantification of cytokines release, analysis of surface membrane expression using flow cytometry and immunohistochemistry investigations confirmed the successful recruitment of injected iron labeled macrophages in the lung of COPD mice and revealed that even with a continuum switch in the polarization profile of M1 and M2 macrophages during the time course of inflammation a balanced number of macrophage subsets predominate. PMID:24598763

  17. Analysis of Differentially Expressed Genes Associated with Coronatine-Induced Laticifer Differentiation in the Rubber Tree by Subtractive Hybridization Suppression.

    PubMed

    Zhang, Shi-Xin; Wu, Shao-Hua; Chen, Yue-Yi; Tian, Wei-Min

    2015-01-01

    The secondary laticifer in the secondary phloem is differentiated from the vascular cambia of the rubber tree (Hevea brasiliensis Muell. Arg.). The number of secondary laticifers is closely related to the rubber yield potential of Hevea. Pharmacological data show that jasmonic acid and its precursor linolenic acid are effective in inducing secondary laticifer differentiation in epicormic shoots of the rubber tree. In the present study, an experimental system of coronatine-induced laticifer differentiation was developed to perform SSH identification of genes with differential expression. A total of 528 positive clones were obtained by blue-white screening, of which 248 clones came from the forward SSH library while 280 clones came from the reverse SSH library. Approximately 215 of the 248 clones and 171 of the 280 clones contained cDNA inserts by colony PCR screening. A total of 286 of the 386 ESTs were detected to be differentially expressed by reverse northern blot and sequenced. Approximately 147 unigenes with an average length of 497 bp from the forward and 109 unigenes with an average length of 514 bp from the reverse SSH libraries were assembled and annotated. The unigenes were associated with the stress/defense response, plant hormone signal transduction and structure development. It is suggested that Ca2+ signal transduction and redox seem to be involved in differentiation, while PGA and EIF are associated with the division of cambium initials for COR-induced secondary laticifer differentiation in the rubber tree. PMID:26147807

  18. Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

    PubMed

    Pagkalos, Joseph; Cha, Jae Min; Kang, Yunyi; Heliotis, Manolis; Tsiridis, Eleftherios; Mantalaris, Athanasios

    2010-11-01

    Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications. PMID:20564244

  19. Hepatitis B Virus-Induced Parkin-Dependent Recruitment of Linear Ubiquitin Assembly Complex (LUBAC) to Mitochondria and Attenuation of Innate Immunity

    PubMed Central

    Khan, Mohsin; Syed, Gulam Hussain; Kim, Seong-Jun; Siddiqui, Aleem

    2016-01-01

    Hepatitis B virus (HBV) suppresses innate immune signaling to establish persistent infection. Although HBV is a DNA virus, its pre-genomic RNA (pgRNA) can be sensed by RIG-I and activates MAVS to mediate interferon (IFN) λ synthesis. Despite of the activation of RIG-I-MAVS axis by pgRNA, the underlying mechanism explaining how HBV infection fails to induce interferon-αβ (IFN) synthesis remained uncharacterized. We demonstrate that HBV induced parkin is able to recruit the linear ubiquitin assembly complex (LUBAC) to mitochondria and abrogates IFN β synthesis. Parkin interacts with MAVS, accumulates unanchored linear polyubiquitin chains on MAVS via LUBAC, to disrupt MAVS signalosome and attenuate IRF3 activation. This study highlights the novel role of parkin in antiviral signaling which involves LUBAC being recruited to the mitochondria. These results provide avenues of investigations on the role of mitochondrial dynamics in innate immunity. PMID:27348524

  20. Retinoic acid‐induced glandular differentiation of the oesophagus

    PubMed Central

    Chang, Chih‐Long; Lao‐Sirieix, Pierre; Save, Vicki; De La Cueva Mendez, Guillermo; Laskey, Ron; Fitzgerald, Rebecca C

    2007-01-01

    Background Retinoic acid (RA) is a powerful differentiation agent. Barrett's oesophagus occurs when duodeno‐gastro‐oesophageal reflux causes squamous epithelium (SE) tissue to become columnar epithelium tissue by an unknown mechanism. The bile acid lithocholic acid (LCA) competes for the retinoid X receptor retinoid binding site. Hence, RA pathways may be implicated in Barrett's oesophagus. Methods RA activity in tissues and cell lines treated with all‐trans retinoic acid (ATRA) with or without LCA was assessed using a reporter. Expression of p21 was determined by real‐time PCR in Barrett's oesophagus cell lines with or without LCA. SE and Barrett's oesophagus biopsy specimens were exposed to 100 μM of ATRA or 20 mM of a RA inhibitor, citral, in organ culture for >72 h. Characteristics of treated specimens, compared with untreated controls, were analysed by immunohistochemical analysis (cytokeratins (CKs), vimentin) and RT‐PCR (CKs). Confocal microscopy assessed temporal changes in co‐localisation of CK8/18 and vimentin. Cell proliferation was assessed by bromo‐deoxyuridine incorporation and immunohistochemical analysis for Ki67 and p21. Results RA biosynthesis was increased in Barrett's oesophagus compared with SE (p<0.001). LCA and ATRA caused a synergistic increase in RA signalling as shown by increased p21 (p<0.01). Morphological and molecular analysis of SE exposed to ATRA showed columnar differentiation independent of proliferation. Metaplasia could be induced from the stromal compartment alone and vimentin expression co‐localised with CK8/18 at 24 h, which separated into CK8/18‐positive glands and vimentin‐positive stroma by 48 h. Citral‐treated Barrett's oesophagus led to phenotypic and immunohistochemical characteristics of SE, which was independent of proliferation. Conclusion RA activity is increased in Barrett's oesophagus and is induced by LCA. Under conditions of altered RA activity and an intact stroma, the

  1. Derivation, characterization and retinal differentiation of induced pluripotent stem cells.

    PubMed

    Mekala, Subba Rao; Vauhini, Vasundhara; Nagarajan, Usha; Maddileti, Savitri; Gaddipati, Subhash; Mariappan, Indumathi

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads. PMID:23385820

  2. Changes in morphology and spatial position of coiled bodies during NGF-induced neuronal differentiation of PC12 cells.

    PubMed

    Janevski, J; Park, P C; De Boni, U

    1997-11-01

    Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleo-proteins (snRNPs) indicates that it functions in RNA processing. In cycling cells, coiled bodies are round structures not associated with nucleoli. In contrast, in neurons, they frequently present as nucleolar "caps." To test the hypothesis that neuronal differentiation is accompanied by changes in the spatial association of coiled bodies with nucleoli and in their morphology, PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) and coiled bodies detected by immunocytochemical localization of p80-coilin and snRNPs. The fraction of cells that showed coiled bodies as nucleolar caps increased from 1.6 +/- 0.9% (mean +/- SEM) in controls to 16.5 +/- 1.6% in NGF-differentiated cultures. The fraction of cells with ring-like coiled bodies increased from 17.2 +/- 5.0% in controls to 57.8 +/- 4.4% in differentiated cells. This was accompanied by a decrease, from 81.2 +/- 5.7% to 25.7 +/- 3.1%, in the fraction of cells with small, round coiled bodies. SnRNPs remained associated with typical coiled bodies and with ring-like coiled bodies during NGF-induced recruitment of snRNPs to the nuclear periphery. Together with the observation that coiled bodies are also present as nucleolar caps in sensory neurons, the results indicate that coiled bodies alter their morphology and increase their association with nucleoli during NGF-induced neuronal differentiation. PMID:9358854

  3. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  4. High-dose cyclophosphamide induces specific tumor immunity with concomitant recruitment of LAMP1/CD107a-expressing CD4-positive T cells into tumor sites.

    PubMed

    Naito, Tatsushi; Baba, Tomohisa; Takeda, Kazuyoshi; Sasaki, Soichiro; Nakamoto, Yasunari; Mukaida, Naofumi

    2015-09-28

    Cancer chemotherapy regimens, particularly those employing high-dose cytotoxic drugs such as cyclophosphamide (CTX), have been considered to be immune suppressive. However, we observed that a single administration of high-dose CTX abolished tumors arising from subcutaneous injection of a mouse hepatoma cell line and subsequently induced specific tumor immunity. Depletion of T cells, specifically CD4(+) T cells, abrogated the CTX-mediated tumor regression. CTX treatment induced the rapid recruitment of CD4(+) T cells into the tumors, and these recruited cells initiated expression of LAMP1/CD107a, a cytotoxic granule molecule, and granzyme B in the absence of antigen presentation at draining lymph nodes and proliferation in the tumor tissues. Moreover, CTX enhanced the expression of a CC chemokine, CCL3, in tumor tissues, and CTX-mediated tumor regression was attenuated in mice deficient in CCR5, the receptor for this chemokine. Consistently, less CTX-induced accumulation of intratumoral LAMP1/CD107a-expressing CD4(+) T cells was observed in mice receiving splenocytes derived from CCR5-deficient mice than in those receiving splenocytes derived from WT mice. Thus, CTX induces the expression of CCL3, which induces the intratumoral migration of CD4(+) T cells expressing cytotoxic molecules, leading to tumor eradication and subsequent specific tumor immunity. PMID:26116901

  5. Proposed Recruit Training Topic: Rape Awareness.

    ERIC Educational Resources Information Center

    Thompson, Janet R.

    This guide, prepared by the United States Navy Recruit Training Command, was designed to address the differential training needs of male and female recruits in the area of personal security, i.e., rape awareness/prevention. In section I, the role of the Recruit Training Command is noted, and information on the incidence of sexual assault is…

  6. Low-Threshold Exocytosis Induced by cAMP-Recruited CaV3.2 (α1H) Channels in Rat Chromaffin Cells

    PubMed Central

    Giancippoli, A.; Novara, M.; de Luca, A.; Baldelli, P.; Marcantoni, A.; Carbone, E.; Carabelli, V.

    2006-01-01

    We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (−50, −40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 μM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the “low-threshold exocytosis” induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the α1H (CaV3.2) channel isoform. PMID:16361341

  7. Low-threshold exocytosis induced by cAMP-recruited CaV3.2 (alpha1H) channels in rat chromaffin cells.

    PubMed

    Giancippoli, A; Novara, M; de Luca, A; Baldelli, P; Marcantoni, A; Carbone, E; Carabelli, V

    2006-03-01

    We have studied the functional role of CaV3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). CaV3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (-50, -40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 microM Ni2+, which selectively blocks T-type currents in RCCs. This suggests that the "low-threshold exocytosis" induced by cAMP is due to increased Ca2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited CaV3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca2+ efficacy similar to that of already existing high-threshold Ca2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca2+ current component recruited by cAMP is selectively associated to the alpha1H (CaV3.2) channel isoform. PMID:16361341

  8. Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells

    PubMed Central

    Tan, Sik-Loo; Ahmad, Tunku Sara; Ng, Wuey-Min; Azlina, Amir Abbas; Azhar, Mahmood Merican; Selvaratnam, Lakshmi; Kamarul, Tunku

    2015-01-01

    To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and

  9. Muscle Recruitment and Coordination following Constraint-Induced Movement Therapy with Electrical Stimulation on Children with Hemiplegic Cerebral Palsy: A Randomized Controlled Trial

    PubMed Central

    Xu, Kaishou; He, Lu; Mai, Jianning; Yan, Xiaohua; Chen, Ying

    2015-01-01

    Objective To investigate changes of muscle recruitment and coordination following constraint-induced movement therapy, constraint-induced movement therapy plus electrical stimulation, and traditional occupational therapy in treating hand dysfunction. Methods In a randomized, single-blind, controlled trial, children with hemiplegic cerebral palsy were randomly assigned to receive constraint-induced movement therapy (n = 22), constraint-induced movement therapy plus electrical stimulation (n = 23), or traditional occupational therapy (n = 23). Three groups received a 2-week hospital-based intervention and a 6-month home-based exercise program following hospital-based intervention. Constraint-induced movement therapy involved intensive functional training of the involved hand during which the uninvolved hand was constrained. Electrical stimulation was applied on wrist extensors of the involved hand. Traditional occupational therapy involved functional unimanual and bimanual training. All children underwent clinical assessments and surface electromyography (EMG) at baseline, 2 weeks, 3 and 6 months after treatment. Surface myoelectric signals were integrated EMG, root mean square and cocontraction ratio. Clinical measures were grip strength and upper extremity functional test. Results Constraint-induced movement therapy plus electrical stimulation group showed both a greater rate of improvement in integrated EMG of the involved wrist extensors and cocontraction ratio compared to the other two groups at 3 and 6 months, as well as improving in root mean square of the involved wrist extensors than traditional occupational therapy group (p<0.05). Positive correlations were found between both upper extremity functional test scores and integrated EMG of the involved wrist as well as grip strength and integrated EMG of the involved wrist extensors (p<0.05). Conclusions Constraint-induced movement therapy plus electrical stimulation is likely to produce the best outcome in

  10. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    PubMed

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  11. Leukotrienes are involved in leukocyte recruitment induced by live Histoplasma capsulatum or by the β-glucan present in their cell wall

    PubMed Central

    Medeiros, Alexandra I; Silva, Célio L; Malheiro, Adriana; Maffei, Cláudia M L; Faccioli, Lúcia H

    1999-01-01

    The inflammatory cell influx towards the peritoneal cavity in mice inoculated i.p. with live or dead Histoplasma capsulatum or with its subcellular preparations was studied. We also evaluated the effects of dexamethasone (Dexa) or MK886, an inhibitor of leukotriene (LT) biosynthesis, on the recruitment of leukocytes.Live yeast form of fungus (LYH) induced an increase in neutrophils (NE) which was highest 4 to 24 h after inoculation. Mononuclear cell (MN) migration beginning at 24 h with a gradual increase over 48 and 168 h, and an eosinophil (EO) recruitment occurs between 24 and 48 h.NE and EO recruitment induced by dead mycelial form of fungus (DMH) was greater than that observed for dead yeast form of fungus (DYH). A similar leukocyte migration pattern was seen after i.p. injection of the alkali-insoluble fraction (F1) from DYH (F1Y) and F1 from DMH (F1M) this being more active than former. The difference in concentration of β-glucan in DYH and DMH could explain the different inflammatory capacity exhibited by the two forms of H. capsulatum.LT seems to be the principal mediator of leukocyte migration in response to LYH, DYH or DMH or to β-glucan. However, other mediators appear to contribute to NE and EO migration since the treatment with Dexa was more effective in inhibiting cell migration than MK886. Complement dependent leukocyte migration may participate in this recruitment. Treatment with MK886 completely abolished MN cell migration, indicating its dependence on the presence of LT. PMID:10602333

  12. Identification of H7 as a novel peroxiredoxin I inhibitor to induce differentiation of leukemia cells

    PubMed Central

    Qin, Dongjun; Chen, Yingyi; Liu, Chuanxu; Xia, Li; Wang, Tongdan; Lei, Hu; Yu, Yun; Huang, Min; Tong, Yin; Xu, Hanzhang; Gao, Fenghou

    2016-01-01

    Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirment. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II–V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPβ axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition. PMID:26716647

  13. MYC accelerates p21CIP-induced megakaryocytic differentiation involving early mitosis arrest in leukemia cells.

    PubMed

    Muñoz-Alonso, María J; Ceballos, Laura; Bretones, Gabriel; Frade, Pilar; León, Javier; Gandarillas, Alberto

    2012-05-01

    p21(CIP) is a potent cell cycle inhibitor often up-regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21(CIP). However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21(CIP) in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21(CIP) to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over-expression studies showed that p21(CIP) is required to induce differentiation. However, the expression of p21(CIP) needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21(CIP) caused early down-regulation of mitotic Cyclins and up-regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over-activation of MYC did not release the proliferative block imposed by p21(CIP) and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21(CIP) takes part in a mitosis control driving MYC-induced cellular growth into differentiation. PMID:21769863

  14. Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

    PubMed

    Liu, Ting; Men, Qiuxu; Wu, Guixian; Yu, Chunrong; Huang, Zan; Liu, Xin; Li, Wenhua

    2015-04-10

    All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells. PMID:25797266

  15. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    SciTech Connect

    Qiao, Jingbo; Paul, Pritha; Lee, Sora; Qiao, Lan; Josifi, Erlena; Tiao, Joshua R.; Chung, Dai H.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  16. Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production

    PubMed Central

    Casagrande, Rubia; Verri, Waldiceu A.

    2016-01-01

    Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation. PMID:26895409

  17. Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production.

    PubMed

    Mizokami, Sandra S; Hohmann, Miriam S N; Staurengo-Ferrari, Larissa; Carvalho, Thacyana T; Zarpelon, Ana C; Possebon, Maria I; de Souza, Anderson R; Veneziani, Rodrigo C S; Arakawa, Nilton S; Casagrande, Rubia; Verri, Waldiceu A

    2016-01-01

    Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation. PMID:26895409

  18. Reduction of eotaxin production and eosinophil recruitment by pulmonary autologous macrophage transfer in a cockroach allergen-induced asthma model.

    PubMed

    Beal, Dominic R; Stepien, David M; Natarajan, Sudha; Kim, Jiyoun; Remick, Daniel G

    2013-12-01

    We sought to investigate the effects of cockroach allergen (CRA) exposure on the lung macrophage population to determine how different macrophage phenotypes influence exacerbation of disease. CRA exposure caused significantly reduced expression of CD86 on lung macrophages. These effects were not systemic, as peritoneal macrophage CD86 expression was not altered. To investigate whether naïve macrophages could reduce asthma-like pulmonary inflammation, autologous peritoneal macrophages were instilled into the airways 24 h before the final CRA challenge. Pulmonary inflammation was assessed by measurement of airway hyperresponsiveness, mucin production, inflammatory cell recruitment, and cytokine production. Cell transfer did not have significant effects in control mice, nor did it affect pulmonary mucin production or airway hyperresponsiveness in control or CRA-exposed mice. However, there was significant reduction in the number of eosinophils recovered in the bronchoalveolar lavage (BAL) (5.8 × 10⁵ vs. 0.88 × 10⁵), and total cell recruitment to the airways of CRA-exposed mice was markedly reduced (1.1 × 10⁶ vs. 0.57 × 10⁶). The reduced eosinophil recruitment was reflected by substantially lower levels of eosinophil peroxidase in the lung and significantly lower concentrations of eotaxins in BAL (eotaxin 1: 3 pg/ml vs. undetectable; eotaxin 2: 2,383 vs. 131 pg/ml) and lung homogenate (eotaxin 1: 1,043 vs. 218 pg/ml; eotaxin 2: 10 vs. 1.5 ng/ml). We conclude that CRA decreases lung macrophage CD86 expression. Furthermore, supplementation of the lung cell population with peritoneal macrophages inhibits eosinophil recruitment, achieved through reduction of eotaxin production. These data demonstrate that transfer of naïve macrophages will reduce some aspects of asthma-like pulmonary inflammation in response to CRA. PMID:24077949

  19. Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH▿

    PubMed Central

    Zotter, Angelika; Luijsterburg, Martijn S.; Warmerdam, Daniël O.; Ibrahim, Shehu; Nigg, Alex; van Cappellen, Wiggert A.; Hoeijmakers, Jan H. J.; van Driel, Roel; Vermeulen, Wim; Houtsmuller, Adriaan B.

    2006-01-01

    The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process. PMID:17000769

  20. Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein.

    PubMed

    Yoo, S; Dynan, W S

    1999-12-15

    Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested. PMID:10572166

  1. Hypoxia induces adipocyte differentiation of adipose-derived stem cells by triggering reactive oxygen species generation.

    PubMed

    Kim, Ji Hye; Kim, Seok-Ho; Song, Seung Yong; Kim, Won-Serk; Song, Sun U; Yi, TacGhee; Jeon, Myung-Shin; Chung, Hyung-Min; Xia, Ying; Sung, Jong-Hyuk

    2014-01-01

    Generation of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) induces the proliferation and migration of adipose-derived stem cells (ASCs). However, the functional role of mitochondrial ROS (mtROS) generation in ASCs is unknown. Therefore, we have investigated whether hypoxia induces the differentiation of ASCs via ROS generation. We also have tried to identify the cellular mechanisms of ROS generation underlying adipocyte differentiation. Hypoxia (2%) and ROS generators, such as antimycin and rotenone, induced adipocyte differentiation, which was attenuated by an ROS scavenger. Although Nox4 generates ROS and regulates proliferation of ASCs, Nox4 inhibition or Nox4 silencing did not inhibit adipocyte differentiation; indeed fluorescence intensity of mito-SOX increased in hypoxia, and treatment with mito-CP, a mtROS scavenger, significantly reduced hypoxia-induced adipocyte differentiation. Phosphorylation of Akt and mTOR was induced by hypoxia, while inhibition of these molecules prevented adipocyte differentiation. Thus hypoxia induces adipocyte differentiation by mtROS generation, and the PI3K/Akt/mTOR pathway is involved. PMID:23956071

  2. Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation

    PubMed Central

    Cauchy, Pierre; Maqbool, Muhammad A.; Zacarias-Cabeza, Joaquin; Vanhille, Laurent; Koch, Frederic; Fenouil, Romain; Gut, Marta; Gut, Ivo; Santana, Maria A.; Griffon, Aurélien; Imbert, Jean; Moraes-Cabé, Carolina; Bories, Jean-Christophe; Ferrier, Pierre; Spicuglia, Salvatore; Andrau, Jean-Christophe

    2016-01-01

    Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4−/CD8− double negative (DN) to CD4+/CD8+ double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1−/− thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity. PMID:26673693

  3. Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation.

    PubMed

    Cauchy, Pierre; Maqbool, Muhammad A; Zacarias-Cabeza, Joaquin; Vanhille, Laurent; Koch, Frederic; Fenouil, Romain; Gut, Marta; Gut, Ivo; Santana, Maria A; Griffon, Aurélien; Imbert, Jean; Moraes-Cabé, Carolina; Bories, Jean-Christophe; Ferrier, Pierre; Spicuglia, Salvatore; Andrau, Jean-Christophe

    2016-05-01

    Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity. PMID:26673693

  4. COMPOUND-INDUCED ALTERATIONS OF SEXUAL DIFFERENTIATION: A REVIEW OF EFFECTS IN HUMANS AND RODENTS

    EPA Science Inventory

    During sexual differentiation there are a number of critical periods when the reproductive system is uniquely susceptible to chemically-induced perturbations. At these times an inappropriate chemical signal can result in irreversible lesions that often result in infertility, wher...

  5. Polar/apolar compounds induce leukemia cell differentiation by modulating cell-surface potential.

    PubMed Central

    Arcangeli, A; Carlà, M; Del Bene, M R; Becchetti, A; Wanke, E; Olivotto, M

    1993-01-01

    The mechanism of action of polar/apolar inducers of cell differentiation, such as dimethyl sulfoxide and hexamethylene-bisacetamide, is still obscure. In this paper evidence is provided that their effects on murine erythroleukemia cells are modulated by various extracellular cations as a precise function of the cation effects on membrane surface potential. The interfacial effects of the inducers were directly measured on the charged electrode, showing that both dimethyl sulfoxide and hexamethylene-bisacetamide, at the effective concentrations for cell differentiation and within the physiological range of charge density, adsorb at the charged surface and produce a potential shift. A linear correlation was found between this shift and the inducer effects on cell differentiation. Besides offering a different interpretation of the mechanism of action of the inducers, these findings indicate that surface potential has a signaling function. They may also be relevant to cancer treatments based on tumor-cell commitment to terminal differentiation. Images Fig. 1 PMID:8516337

  6. High salt promotes autoimmunity by TET2-induced DNA demethylation and driving the differentiation of Tfh cells

    PubMed Central

    Wu, Haijing; Huang, Xin; Qiu, Hong; Zhao, Ming; Liao, Wei; Yuan, Shuguang; Xie, Yubing; Dai, Yong; Chang, Christopher; Yoshimura, Akihiko; Lu, Qianjin

    2016-01-01

    Follicular helper T cells (Tfh) have been well documented to play a critical role in autoimmunity, such as systemic lupus erythematosus (SLE), by helping B cells. In this study, high salt (sodium chloride, NaCl), under physiological conditions, was demonstrated to increase the differentiation of Tfh. A high-salt diet markedly increased lupus features in MRL/lpr mice. The mechanism is NaCl-induced DNA demethylation via the recruitment of the hydroxytransferase Ten-Eleven Translocation 2 (TET2). Gene silencing of TET2 obviously diminished NaCl-induced Tfh cell polarization in vitro. In addition, the gene expression of sh2d1a, map3k1, spn and stat5b was enhanced after NaCl treatment, consistent with the findings in lupus CD4+T cells. However, only spn was directly regulated by TET2, and spn was not the sole target for NaCl. Our findings not only explain the epigenetic mechanisms of high-salt induced autoimmunity but also provide an attractive molecular target for intervention strategies of patients. PMID:27325182

  7. High salt promotes autoimmunity by TET2-induced DNA demethylation and driving the differentiation of Tfh cells.

    PubMed

    Wu, Haijing; Huang, Xin; Qiu, Hong; Zhao, Ming; Liao, Wei; Yuan, Shuguang; Xie, Yubing; Dai, Yong; Chang, Christopher; Yoshimura, Akihiko; Lu, Qianjin

    2016-01-01

    Follicular helper T cells (Tfh) have been well documented to play a critical role in autoimmunity, such as systemic lupus erythematosus (SLE), by helping B cells. In this study, high salt (sodium chloride, NaCl), under physiological conditions, was demonstrated to increase the differentiation of Tfh. A high-salt diet markedly increased lupus features in MRL/lpr mice. The mechanism is NaCl-induced DNA demethylation via the recruitment of the hydroxytransferase Ten-Eleven Translocation 2 (TET2). Gene silencing of TET2 obviously diminished NaCl-induced Tfh cell polarization in vitro. In addition, the gene expression of sh2d1a, map3k1, spn and stat5b was enhanced after NaCl treatment, consistent with the findings in lupus CD4(+)T cells. However, only spn was directly regulated by TET2, and spn was not the sole target for NaCl. Our findings not only explain the epigenetic mechanisms of high-salt induced autoimmunity but also provide an attractive molecular target for intervention strategies of patients. PMID:27325182

  8. Inducing endoderm differentiation by modulating mechanical properties of soft substrates.

    PubMed

    Jaramillo, Maria; Singh, Satish S; Velankar, Sachin; Kumta, Prashant N; Banerjee, Ipsita

    2015-01-01

    Early embryonic stem cell (ESC) differentiation is marked by the formation of three germ layers from which all tissues types arise. Conventionally, ESCs are differentiated by altering their chemical microenvironment. Recently however, it was established that a mechanical microenvironment can also contribute towards cellular phenotype commitment. In this study, we report how the cellular mechanical microenvironment of soft substrates affects the differentiation and phenotypic commitment of ESCs. Mouse ESCs were cultured in a fibrin hydrogel matrix in 2D and 3D cultures. The gelation characteristics of the substrates were modulated by systematically altering the fibrinogen concentration and the fibrinogen-thrombin crosslinking ratio. Analysis of the ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate physical characteristics assert on cellular behaviours. Specifically, it was found that ESCs had a higher proliferation rate in gels of lower stiffness. Early differentiation events were studied by analyzing the gene and protein expression levels of early germ layer markers. Our results revealed that lower substrate stiffness elicited stronger upregulation of endoderm related genes Sox17, Afp and Hnf4 compared to stiffer substrates. While both 2D and 3D cultures showed a similar response, the effects were much stronger in 3D culture. These results suggest that physical cues can be used to modulate ESC differentiation into clinically relevant tissues such as liver and pancreas. PMID:23008262

  9. In vitro differentiation of rhabdomyosarcomas induced by nickel or by Moloney murine sarcoma virus.

    PubMed Central

    Nanni, P.; Azzarello, G.; Tessarollo, L.; De Giovanni, C.; Lollini, P. L.; Nicoletti, G.; Scotlandi, K.; Landuzzi, L.; Panozzo, M.; D'Andrea, E.

    1991-01-01

    In vitro cultures and clonal derivatives have been established from rat rhabdomyosarcomas induced by Moloney-Murine Sarcoma Virus (MSV) or by nickel sulfide; differentiation ability has been studied as expression of desmin, embryonic and adult myosin isoforms, alpha-actin isoforms and cellular fusion. The two rhabdomyosarcoma models showed different levels of myogenic differentiation. Multinucleated myotube-like structures were frequently observed in cultures derived from nickel-induced tumours. Desmin was present in 50-80% of cells and embryonic myosin in up to 10%. In MSV-tumour-derived cultures and in their metastases or clonal derivatives two cell types are present in different ratios: spindle-shaped cells, adherent to plastic surfaces, and rounded cells, loosely attached or floating free in the medium. These cultures showed features of myogenic differentiation (10-80% desmin-positive cells), but embryonic myosin expression and production of multinucleated myotube-like structures were very rare events. Cultures from autochthonous lymph node and lung metastatic cells showed similar patterns of differentiation. Retinoic acid increased differentiated features (myotube formation and embryonic myosin expression) only in nickel-induced rhabdomyosarcoma cells. The two models described here mimic the heterogeneity in differentiation pattern found among human rhabdomyosarcomas. Myogenic differentiation ability was retained at a good level by nickel-induced tumours, whereas it was strongly impaired in MSV-induced tumours. Images Figure 1 Figure 2 Figure 4 Figure 7 Figure 8 PMID:2039698

  10. Analogue modelling of salt diapirism induced by differential loading

    NASA Astrophysics Data System (ADS)

    Warsitzka, Michael; Kley, Jonas; Kukowski, Nina; Jähne, Fabian

    2010-05-01

    In salt tectonics, two general concepts exist to explain salt diapirism. First, the theory of active piercement by Trusheim (1960) states that salt rises up and pierces its overburden autonomously by buoyancy forces. Second, the theory of reactive piercement by Vendeville and Jackson (1992) considers a tectonic stress field responsible for initiation of salt uplift and has been tested in many analogue experiments. In this study, we investigated the hypothesis in which salt diapir formation is activated by sedimentary processes alone, i.e. without a tectonic trigger. Our models consisted of a viscous silicone layer simulating rock salt overlain by layers of sand that mimic brittle behaviour in natural overburden sediments. The experiments were monitored with a high-resolution strain analysis tool based on digital image correlation (particle image velocimetry, PIV). Deformation in the silicone was initiated by a lateral variation in the thickness or density of the overburden, which established a differential loading on the silicone layer. Subsequent sedimentation in certain time intervals forced the silicone to rise up and break through the initial sand layer by buoyancy forces. The model results support the hypothesis of active piercement of diapirs. Uplift of the silicone and creation of a pillow structure with a significant elevation can be achieved if the overburden does not exceed a critical thickness and if the load gradient in the overburden reaches a minimum value. Then, ongoing sedimentation in adjacent areas increases the lateral load gradient until the buoyancy force in the silicone is high enough to overcome the shear strength of the sand. Synkinematic sedimentation produces some typical strata geometries in the sand layer that can also be observed in nature, e.g. drag folds bordering the diapirs and layer thickening in the peripherical rim synclines. The creation of one diapir and its peripherical sinks induces a lateral migration of the deformation to

  11. Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

    PubMed

    Mishra, Rachana; Kaur, Gurcharan

    2015-08-01

    Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells. PMID:25280667

  12. Vinpocetine reduces lipopolysaccharide-induced inflammatory pain and neutrophil recruitment in mice by targeting oxidative stress, cytokines and NF-κB.

    PubMed

    Ruiz-Miyazawa, Kenji W; Pinho-Ribeiro, Felipe A; Zarpelon, Ana C; Staurengo-Ferrari, Larissa; Silva, Rangel L; Alves-Filho, Jose C; Cunha, Thiago M; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2015-07-25

    In response to lipopolysaccharide (LPS), tissue resident macrophages and recruited neutrophils produce inflammatory mediators through activation of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway. These mediators include inflammatory cytokines and reactive oxygen species that, in turn, sensitize nociceptors and lead to inflammatory pain. Vinpocetine is a nootropic drug widely used to treat cognitive and neurovascular disorders, and more recently its anti-inflammatory properties through inhibition of NF-κB activation have been described. In the present study, we used the intraplantar and intraperitoneal LPS stimulus in mice to investigate the effects of vinpocetine pre-treatment (3, 10, or 30mg/kg by gavage) in hyperalgesia, leukocyte recruitment, oxidative stress, and pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-33). LPS-induced NF-κB activation and cytokine production were investigated using RAW 264.7 macrophage cell in vitro. Vinpocetine (30mg/kg) significantly reduces hyperalgesia to mechanical and thermal stimuli, and myeloperoxidase (MPO) activity (a neutrophil marker) in the plantar paw skin, and also inhibits neutrophil and mononuclear cell recruitment, superoxide anion and nitric oxide production, oxidative stress, and cytokine production (TNF-α, IL-1β and IL-33) in the peritoneal cavity. At least in part, these effects seem to be mediated by direct effects of vinpocetine on macrophages, since it inhibited the production of the same cytokines (TNF-α, IL-1β and IL-33) and the NF-κB activation in LPS-stimulated RAW 264.7 macrophages. Our results suggest that vinpocetine represents an important therapeutic approach to treat inflammation and pain induced by a gram-negative bacterial component by targeting NF-κB activation and NF-κB-related cytokine production in macrophages. PMID:25980587

  13. Graphene induces spontaneous cardiac differentiation in embryoid bodies

    NASA Astrophysics Data System (ADS)

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-03-01

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac

  14. Activation of PPAR{gamma} is not involved in butyrate-induced epithelial cell differentiation

    SciTech Connect

    Ulrich, S.; Waechtershaeuser, A.; Loitsch, S.; Knethen, A. von; Bruene, B.; Stein, J. . E-mail: j.stein@em.uni-frankfurt.de

    2005-10-15

    Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPAR{gamma} in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPAR{gamma} in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPAR{gamma} ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPAR{gamma} ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPAR{gamma} mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPAR{gamma} is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation.

  15. Induced myelomonocytic differentiation in leukemia cells is accompanied by noncanonical transcription factor expression

    PubMed Central

    Jensen, Holly A.; Yourish, Harmony B.; Bunaciu, Rodica P.; Varner, Jeffrey D.; Yen, Andrew

    2015-01-01

    Transcription factors that drive non-neoplastic myelomonocytic differentiation are well characterized but have not been systematically analyzed in the leukemic context. We investigated widely used, patient-derived myeloid leukemia cell lines with proclivity for differentiation into granulocytes by retinoic acid (RA) and/or monocytes by 1,25-dihyrdroxyvitamin D3 (D3). Using K562 (FAB M1), HL60 (FAB M2), RA-resistant HL60 sublines, NB4 (FAB M3), and U937 (FAB M5), we correlated nuclear transcription factor expression to immunophenotype, G1/G0 cell cycle arrest and functional inducible oxidative metabolism. We found that myelomonocytic transcription factors are aberrantly expressed in these cell lines. Monocytic-lineage factor EGR1 was not induced by D3 (the monocytic inducer) but instead by RA (the granulocytic inducer) in lineage bipotent myeloblastic HL60. In promyelocytic NB4 cells, EGR1 levels were increased by D3, while Gfi-1 expression (which promotes the granulocytic lineage) was upregulated during D3-induced monocytic differentiation in HL60, and by RA treatment in monocytic U937 cells. Furthermore, RARα and VDR expression were not strongly correlated to differentiation. In response to different differentiation inducers, U937 exhibited the most distinct transcription factor expression profile, while similarly mature NB4 and HL60 were better coupled. Overall, the differentiation induction agents RA and D3 elicited cell-specific responses across these common FAB M1-M5 cell lines. PMID:26566473

  16. Polycan suppresses osteoclast differentiation and titanium particle-induced osteolysis in mice.

    PubMed

    Lee, Young-Eun; Park, Kwang-Soo; Park, Eui-Kyun; Im, Sang-Uk; Choi, Youn-Hee; Song, Keun-Bae

    2016-08-01

    Particle-induced osteolysis is a major issue, and it is most likely the result of enhanced osteoclast activation in the pathogenesis of various skeletal diseases. This study investigated whether the inhibitory effect that Polycan has on osteoclast differentiation can be used to treat osteolysis induced by titanium (Ti) particles. To this end, the effects of Polycan were examined in terms of the cytotoxicity, osteoclast differentiation, cytokine expression, and Ti-induced calvarial osteolysis. Polycan had no significant cytotoxic effects on bone marrow macrophages (BMMs) but instead increased BMM proliferation. High levels of interleukin (IL)-6, IL-12, and macrophage colony-stimulating factor (M-CSF) were expressed in BMM cells in the presence of Polycan, suggesting that Polycan drives the differentiation of BMMs into M1 macrophages. Polycan significantly inhibited osteoclast differentiation induced by M-CSF and the receptor activator of nuclear factor kappa-B ligand (RANKL). The expression levels of the osteoclast marker genes significantly decreased, and Polycan induced and maintained the expression of IL-12, which suppressed osteoclast differentiation. In contrast, the RANKL signaling pathway was not inhibited by Polycan. An in vivo calvarial osteolysis model revealed that Polycan significantly decreased the osteoclast numbers and suppressed osteolysis. Our results suggest that the natural compound Polycan is a good candidate for therapeutic intervention against enhanced osteoclast differentiation and Ti particle-induced osteolysis. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1170-1175, 2016. PMID:26097144

  17. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

    PubMed

    Maza, Paloma K; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  18. Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

    PubMed Central

    Maza, Paloma K.; Suzuki, Erika

    2016-01-01

    Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation. PMID:27148251

  19. Human MAF1 targets and represses active RNA polymerase III genes by preventing recruitment rather than inducing long-term transcriptional arrest

    PubMed Central

    Orioli, Andrea; Praz, Viviane; Lhôte, Philippe; Hernandez, Nouria

    2016-01-01

    RNA polymerase III (Pol III) is tightly controlled in response to environmental cues, yet a genomic-scale picture of Pol III regulation and the role played by its repressor MAF1 is lacking. Here, we describe genome-wide studies in human fibroblasts that reveal a dynamic and gene-specific adaptation of Pol III recruitment to extracellular signals in an mTORC1-dependent manner. Repression of Pol III recruitment and transcription are tightly linked to MAF1, which selectively localizes at Pol III loci, even under serum-replete conditions, and increasingly targets transcribing Pol III in response to serum starvation. Combining Pol III binding profiles with EU-labeling and high-throughput sequencing of newly synthesized small RNAs, we show that Pol III occupancy closely reflects ongoing transcription. Our results exclude the long-term, unproductive arrest of Pol III on the DNA as a major regulatory mechanism and identify previously uncharacterized, differential coordination in Pol III binding and transcription under different growth conditions. PMID:26941251

  20. EGF Inhibits Wnt/β-Catenin-Induced Osteoblast Differentiation by Promoting β-Catenin Degradation.

    PubMed

    Boonanantanasarn, Kanitsak; Lee, Hye-Lim; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa; Kim, Gwan-Shik

    2015-12-01

    Bone morphogenetic protein (BMP) and canonical Wnts are representative developmental signals that enhance osteoblast differentiation and bone formation. Previously, we demonstrated that epidermal growth factor (EGF) inhibits BMP2-induced osteoblast differentiation by inducing Smurf1 expression. However, the regulatory role of EGF in Wnt/β-catenin-induced osteoblast differentiation has not been elucidated. In this study, we investigated the effect of EGF on Wnt/β-catenin signaling-induced osteoblast differentiation using the C2C12 cell line. EGF significantly suppressed the expression of osteoblast marker genes, which were induced by Wnt3a and a GSK-3β inhibitor. EGF increased the expression levels of Smurf1 mRNA and protein. Smurf1 knockdown rescued Wnt/β-catenin-induced osteogenic marker gene expression in the presence of EGF. EGF treatment or Smurf1 overexpression did not affect β-catenin mRNA expression levels, but reduced β-catenin protein levels and TOP-Flash activity. EGF and Smurf1 promoted β-catenin ubiquitination. Co-immunoprecipitation and GST pull-down assays showed that Smurf1 associates with β-catenin. These results suggest that EGF/Smurf1 inhibits Wnt/β-catenin-induced osteogenic differentiation and that Smurf1 downregulates Wnt/β-catenin signaling by enhancing proteasomal degradation of β-catenin. PMID:26015066

  1. Beige differentiation of adipose depots in mice lacking prolactin receptor protects against high-fat-diet-induced obesity.

    PubMed

    Auffret, Julien; Viengchareun, Say; Carré, Nadège; Denis, Raphaël G P; Magnan, Christophe; Marie, Pierre-Yves; Muscat, Adeline; Fève, Bruno; Lombès, Marc; Binart, Nadine

    2012-09-01

    Stimulating conversion of white fat to metabolically active adipocytes (beige fat) constitutes a promising strategy against weight gain and its deleterious associated-disorders. We provide direct evidence that prolactin (PRL), best known for its actions on the mammary gland, plays a pivotal role in energy balance through the control of adipocyte differentiation and fate. Here we show that lack of prolactin receptor (PRLR) causes resistance to high-fat-diet-induced obesity due to enhanced energy expenditure and increased metabolic rate. Mutant mice displayed reduced fat mass associated with appearance of massive brown-like adipocyte foci in perirenal and subcutaneous but not in gonadal fat depots under a high-fat diet. Positron emission tomography imaging further demonstrated the occurrence of these thermogenic brown fat depots in adult mice, providing additional support for recruitable brown-like adipocytes (beigeing) in white fat depots. Consistent with the activation of brown adipose tissue, PRLR inactivation increases expression of master genes controlling brown adipocyte fate (PRDM16) and mitochondrial function (PGC1α, UCP1). Altered pRb/Foxc2 expression suggests that this PRL-regulated pathway may contribute to beige cell commitment. Together, these results provide direct genetic evidence that PRLR affects energy balance and metabolic adaptation in rodents via effects on brown adipose tissue differentiation and function. PMID:22637534

  2. Hypoxia induced the differentiation of Tbx18-positive epicardial cells to CoSMCs

    PubMed Central

    Jing, Xiaodong; Gao, Yulin; Xiao, Songlin; Qin, Qin; Wei, Xiaoming; Yan, Yuling; Wu, Ling; Deng, Songbai; Du, Jianlin; Liu, Yajie; She, Qiang

    2016-01-01

    Understanding the origin and differentiation mechanism of coronary vascular smooth muscle cells (CoSMCs) is very important to cardiovascular biology. The early cardiovascular system is formed in a hypoxic microenvironment, and Tbx18-positive epicardial cells are a source of CoSMCs. However, the effects of hypoxia on the differentiation of Tbx18-positive epicardial cells to CoSMCs and the primary regulatory mechanism are insufficiently understood. Using Tbx18:Cre/R26REYFP/LacZ fate-tracing mice, we cultured highly purified Tbx18-positive epicardial cells. We further showed that hypoxia induced Tbx18-positive epicardial cells to differentiate into CoSMCs and promoted the epithelial-mesenchymal transition (EMT) process of the cells in vitro. The induction of differentiation was primarily achieved via the hypoxia inducible factor-1α (HIF-1α)-mediated effects exerted on Snail. Using a cell migration assay, we showed that hypoxia enhanced the motility of Tbx18-positive epicardial cells. By constructing a hypoxic model of the embryonic epicardium in vivo, we showed that hypoxia led to premature in situ differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, hypoxia was sufficient to induce Snail expression in Tbx18-positive epicardial cells in vivo. Our study suggests that hypoxia intervention was sufficient to induce the differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, this differentiation was achieved primarily via HIF-1α-mediated regulation of Snail. PMID:27456656

  3. Hypoxia induced the differentiation of Tbx18-positive epicardial cells to CoSMCs.

    PubMed

    Jing, Xiaodong; Gao, Yulin; Xiao, Songlin; Qin, Qin; Wei, Xiaoming; Yan, Yuling; Wu, Ling; Deng, Songbai; Du, Jianlin; Liu, Yajie; She, Qiang

    2016-01-01

    Understanding the origin and differentiation mechanism of coronary vascular smooth muscle cells (CoSMCs) is very important to cardiovascular biology. The early cardiovascular system is formed in a hypoxic microenvironment, and Tbx18-positive epicardial cells are a source of CoSMCs. However, the effects of hypoxia on the differentiation of Tbx18-positive epicardial cells to CoSMCs and the primary regulatory mechanism are insufficiently understood. Using Tbx18:Cre/R26R(EYFP/LacZ) fate-tracing mice, we cultured highly purified Tbx18-positive epicardial cells. We further showed that hypoxia induced Tbx18-positive epicardial cells to differentiate into CoSMCs and promoted the epithelial-mesenchymal transition (EMT) process of the cells in vitro. The induction of differentiation was primarily achieved via the hypoxia inducible factor-1α (HIF-1α)-mediated effects exerted on Snail. Using a cell migration assay, we showed that hypoxia enhanced the motility of Tbx18-positive epicardial cells. By constructing a hypoxic model of the embryonic epicardium in vivo, we showed that hypoxia led to premature in situ differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, hypoxia was sufficient to induce Snail expression in Tbx18-positive epicardial cells in vivo. Our study suggests that hypoxia intervention was sufficient to induce the differentiation of Tbx18-positive epicardial cells to CoSMCs. Furthermore, this differentiation was achieved primarily via HIF-1α-mediated regulation of Snail. PMID:27456656

  4. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    PubMed Central

    Wang, Li; Liu, Yuan; Li, Sen; Long, Zai-Yun; Wu, Ya-Min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway. PMID:25755748

  5. High glucose induces adipogenic differentiation of muscle-derived stem cells

    PubMed Central

    Aguiari, Paola; Leo, Sara; Zavan, Barbara; Vindigni, Vincenzo; Rimessi, Alessandro; Bianchi, Katiuscia; Franzin, Chiara; Cortivo, Roberta; Rossato, Marco; Vettor, Roberto; Abatangelo, Giovanni; Pozzan, Tullio; Pinton, Paolo; Rizzuto, Rosario

    2008-01-01

    Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus. PMID:18212116

  6. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  7. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    PubMed

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. PMID:26984756

  8. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

    PubMed

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

  9. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

    PubMed Central

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

  10. Muramyl dipeptide induces NOD2-dependent Ly6C(high) monocyte recruitment to the lungs and protects against influenza virus infection.

    PubMed

    Coulombe, François; Fiola, Stéphanie; Akira, Shizuo; Cormier, Yvon; Gosselin, Jean

    2012-01-01

    Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6C(high) "inflammatory" monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6C(high) monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6C(high) monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV. PMID:22590599

  11. Covalent immobilization of stem cell inducing/recruiting factor and heparin on cell-free small-diameter vascular graft for accelerated in situ tissue regeneration.

    PubMed

    Shafiq, Muhammad; Jung, Youngmee; Kim, Soo Hyun

    2016-06-01

    The development of cell-free vascular grafts has tremendous potential for tissue engineering. However, thrombus formation, less-than-ideal cell infiltration, and a lack of growth potential limit the application of electrospun scaffolds for in situ tissue-engineered vasculature. To overcome these challenges, here we present development of an acellular tissue-engineered vessel based on electrospun poly(L-lactide-co-ɛ-caprolactone) scaffolds. Heparin was conjugated to suppress thrombogenic responses, and substance P (SP) was immobilized to recruit host cells. SP was released in a sustained manner from scaffolds and recruited human bone marrow-derived mesenchymal stem cells. The biocompatibility and biological performance of the grafts were evaluated by in vivo experiments involving subcutaneous scaffold implantation in Sprague-Dawley rats (n = 12) for up to 4 weeks. Histological analysis revealed a higher extent of accumulative host cell infiltration, neotissue formation, collagen deposition, and elastin deposition in scaffolds containing either SP or heparin/SP than in the control groups. We also observed the presence of a large number of laminin-positive blood vessels, von Willebrand factor (vWF(+) ) cells, and alpha smooth muscle actin-positive cells in the explants containing SP and heparin/SP. Additionally, SP and heparin/SP grafts showed the existence of CD90(+) and CD105(+) MSCs and induced a large number of M2 macrophages to infiltrate the graft wall compared with that observed with the control group. Our cell-free grafts could enhance vascular regeneration by endogenous cell recruitment and by mediating macrophage polarization into the M2 phenotype, suggesting that these constructs may be a promising cell-free graft candidate and are worthy of further in vivo evaluation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1352-1371, 2016. PMID:26822178

  12. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    PubMed Central

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J.; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F.; Psathaki, Olympia E.; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R.; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. PMID:25326431

  13. Azithromycin differentially affects the IL-13-induced expression profile in human bronchial epithelial cells.

    PubMed

    Mertens, Tinne C J; Hiemstra, Pieter S; Taube, Christian

    2016-08-01

    The T helper 2 (Th2) cytokine interleukin(IL)-13 is a central regulator in goblet cell metaplasia and induces the recently described Th2 gene signature consisting of periostin (POSTN), chloride channel regulator 1 (CLCA1) and serpin B2 (SERPINB2) in airway epithelial cells. This Th2 gene signature has been proposed as a biomarker to classify asthma into Th2-high and Th2-low phenotypes. Clinical studies have shown that the macrolide antibiotic azithromycin reduced clinical symptoms in neutrophilic asthma, but not in the classical Th2-mediated asthma despite the ability of azithromycin to reduce IL-13-induced mucus production. We therefore hypothesize that azithromycin differentially affects the IL-13-induced expression profile. To investigate this, we focus on IL-13-induced mucin and Th2-signature expression in human bronchial epithelial cells and how this combined expression profile is affected by azithromycin treatment. Primary bronchial epithelial cells were differentiated at air liquid interface in presence of IL-13 with or without azithromycin. Azithromycin inhibited IL-13-induced MUC5AC, which was accompanied by inhibition of IL-13-induced CLCA1 and SERPINB2 expression. In contrast, IL-13-induced expression of POSTN was further increased in cells treated with azithromycin. This indicates that azithromycin has a differential effect on the IL-13-induced Th2 gene signature. Furthermore, the ability of azithromycin to decrease IL-13-induced MUC5AC expression may be mediated by a reduction in CLCA1. PMID:27246785

  14. Recruiting Strategies for Women's Colleges.

    ERIC Educational Resources Information Center

    Ricci, Ronald J.

    1994-01-01

    Methods for combating declining applicant pools at women's colleges are discussed. Research suggests that effective student recruitment can be facilitated by the use of single-gender market niche as a means for differentiation and parent influence for promotion. Review of strategies currently used indicate these marketing methods are underused and…

  15. Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip*S⃞

    PubMed Central

    Stensrud, Kenneth F.; Adam, Philip R.; La Mar, Cassandra D.; Olive, Andrew J.; Lushington, Gerald H.; Sudharsan, Raghavi; Shelton, Naomi L.; Givens, Richard S.; Picking, Wendy L.; Picking, William D.

    2008-01-01

    Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a “tip complex” composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells. PMID:18450744

  16. Brucella CβG induces a dual pro- and anti-inflammatory response leading to a transient neutrophil recruitment

    PubMed Central

    Degos, Clara; Gagnaire, Aurélie; Banchereau, Romain; Moriyón, Ignacio; Gorvel, Jean-Pierre

    2015-01-01

    Brucella is the causing agent of a chronic zoonosis called brucellosis. The Brucella β-1,2 cyclic glucan (CβG) is a virulence factor, which has been described as a potent immune stimulator, albeit with no toxicity for cells and animals. We first used a genome-wide approach to characterize human myeloid dendritic cell (mDC) responses to CβG. Transcripts related to inflammation (IL-6, IL2RA, PTGS2), chemokine (CXCR7, CXCL2) and anti-inflammatory pathways (TNFAIP6, SOCS3) were highly expressed in CβG-treated mDC. In mouse GMCSF-derived DC, CβG triggered the expression of both activation (CXCL2, KC) and inhibition (SOCS3 and TNFAIP6) molecules. We then characterized the inflammatory infiltrates at the level of mouse ear when injected with CβG or LPS. CβG yielded a lower and transient recruitment of neutrophils compared to LPS. The consequence of these dual pro- and anti-inflammatory signals triggered by CβG corresponds to the induction of a controlled local inflammation. PMID:25654761

  17. Carbachol-induced MUC17 endocytosis is concomitant with NHE3 internalization and CFTR membrane recruitment in enterocytes.

    PubMed

    Pelaseyed, Thaher; Gustafsson, Jenny K; Gustafsson, Ida J; Ermund, Anna; Hansson, Gunnar C

    2013-08-15

    We have reported that transmembrane mucin MUC17 binds PDZ protein PDZK1, which retains MUC17 apically in enterocytes. MUC17 and transmembrane mucins MUC3 and MUC12 are suggested to build the enterocyte apical glycocalyx. Carbachol (CCh) stimulation of the small intestine results in gel-forming mucin secretion from goblet cells, something that requires adjacent enterocytes to secrete chloride and bicarbonate for proper mucin formation. Surface labeling and confocal imaging demonstrated that apically expressed MUC17 in Caco-2 cells and Muc3(17) in murine enterocytes were endocytosed upon stimulation with CCh. Relocation of MUC17 in response to CCh was specific as MUC3 and MUC12 did not relocate following CCh stimulation. MUC17 colocalized with PDZK1 under basal conditions, while MUC17 relocated to the terminal web and into early endosomes after CCh stimulation. CCh stimulation concomitantly internalized the Na(+/)H(+) exchanger 3 (NHE3) and recruited cystic fibrosis transmembrane conductance regulator (CFTR) to the apical membranes, a process that was important for CFTR-mediated bicarbonate secretion necessary for proper gel-forming mucin unfolding. The reason for the specific internalization of MUC17 is not understood, but it could limit the diffusion barrier for ion secretion caused by the apical enterocyte glycocalyx or alternatively act to sample luminal bacteria. Our results reveal well-orchestrated mucus secretion and trafficking of ion channels and the MUC17 mucin. PMID:23784542

  18. Interleukin-34 induces monocytic-like differentiation in leukemia cell lines

    PubMed Central

    Booker, Burthia E; Clark, Ryan S; Pellom, Samuel T; Adunyah, Samuel E

    2015-01-01

    Interleukin-34 (IL-34) is a cytokine consisting of a 39kD homodimer, shown to be a ligand for both the Macrophage Colony Stimulating Factor (M-CSF/CSF-1) receptor and the Receptor-like protein tyrosine phosphatase-zeta (RPTP-ƺ). IL-34 has been shown to promote monocyte viability and proliferation as well as the differentiation of bone marrow cells into macrophage progenitors. Published work on IL-34 involves its effects on normal hematopoietic and osteoclast progenitors. However, it is not known whether IL-34 has biologic effects in cancer, including leukemia. Here we report that the biological effects of IL-34 include induction of differential expression of Interleukins-1α and -1β as well as induction of differentiation of U937, HL-60 and THP-1 leukemia cell lines demonstrating monocyte-like characteristics. The ability of IL-34 to induce monocytic-like differentiation is supported by strong morphological and functional evidence. Cell surface markers of myeloid lineage, CD64 and CD86, remain constant while the levels of CD11b and CD71 decline with IL-34 treatment. IL-34 also induced increases in CD14 and CD68 expression, further supporting maturation toward monocytic character. IL-34-induced differentiated U937 and THP-1 cell lines exhibited biological functions such as endocytosis and respiratory burst activities. Collectively, we conclude that while IL-34 does not induce cell growth or proliferation, it is able to induce differentiation of leukemia cell lines from monoblastic precursor cells towards monocyte- and macrophage-like cells, mediated through the JAK/STAT and PI3K/Akt pathways. To our knowledge, this is the first report that IL-34 induces differentiation in human leukemic cells, let alone any cancer model. PMID:26045972

  19. SKW 6.4 cell differentiation induced by interleukin 6 is stimulated by butyrate.

    PubMed

    Kawamoto, T; Gohda, E; Iji, H; Fujiwara, M; Yamamoto, I

    1998-08-01

    We investigated if sodium butyrate (NaBu), an inhibitor of histone deacetylase, and its analogs modulate cytokine-induced differentiation of the human B cell line SKW 6.4 transformed by the Epstein-Barr virus. NaBu markedly enhanced interleukin (IL)-6-induced IgM production with an accompanying increase in the level of histone H4 acetylation and augmented IgM production induced by IL-4 and phorbol 12-myristate 13-acetate. From both the enhancing effect of cell differentiation and the effect of inducing histone hyperacetylation in SKW 6.4 cells, other histone deacetylase inhibitors and NaBu analogs were divided into three groups: those that increased both IL-6-induced antibody production and histone acetylation, those that caused histone hyperacetylation, but failed to induce the differentiation, and those that were ineffective at inducing either activity. No agent that enhanced IgM production without inducing histone hyperacetylation was found among the inhibitors and analogs we tested. These results suggest that the increase in the histone acetylation is necessary, but it is insufficient to augment differentiation of SKW 6.4 cells. Thus another activity of NaBu in addition to the inhibition of histone deacetylase may be involved in promoting IL-6-induced differentiation. Our results also suggest that fatty acids that have a straight chain of four carbon atoms or are branched with four and five carbon atoms, which contain no hydrophilic substituents, or those with similar structures, show this other activity. PMID:9826026

  20. [Transcriptomics and proteomics in studies of induced differentiation of leukemia cells].

    PubMed

    Novikova, S E; Zgoda, V G

    2015-01-01

    Induced differentiation of leukemia cells is in the focus of basic and applied biomedical studies medicine and biology for more than 30 years. During this period specific regulatory molecules involved in the maturation process have been identified by biochemical and molecular biological methods. Recent developments of high-throughput transcriptomic and proteomic techniques made it possible to analyze large sets of mRNA and proteins; this resulted in identification of functionally important signal transduction pathways and networks of molecular interactions, and thus extent existing knowledge on the molecular mechanisms of induced differentiation. Despite significant advances in mechanisms of induced differentiation, many problems related to the molecular mechanism of cell maturation, a phenomenon of therapeutic resistance of leukemic cells need better understanding and thus require further detailed study. Transcriptomics and proteomics methods provide a suitable methodological platform for the implementation of such studies. This review highlights the use of transcriptomic and proteomic methods in studies aimed at various aspects of the induced differentiation. Special attention is paid to the employment of the systems approach for investigation of various aspects of cell maturation. The use of the systems approach in studies of induced differentiation is an important step for the transition from the formal data accumulation on expression of mRNA and proteins towards creating models of biological processes in silico. PMID:26539862

  1. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    PubMed

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-01

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. PMID:27233607

  2. Inhibition of KDM6 activity during murine ESC differentiation induces DNA damage.

    PubMed

    Hofstetter, Christine; Kampka, Justyna M; Huppertz, Sascha; Weber, Heike; Schlosser, Andreas; Müller, Albrecht M; Becker, Matthias

    2016-02-15

    Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2- and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs. PMID:26759175

  3. The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

    PubMed

    Ibabao, Christopher N; Bunaciu, Rodica P; Schaefer, Deanna M W; Yen, Andrew

    2015-01-01

    In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr. PMID:25941627

  4. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    SciTech Connect

    Morizane, Ryuji; Monkawa, Toshiaki; Itoh, Hiroshi

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  5. SRY alone can induce normal male sexual differentiation

    SciTech Connect

    Lopez, M.; Torres, L.; Cervantes, A.

    1995-01-30

    Most individuals with the rare 46,XX male {open_quotes}syndrome{close_quotes} arise due to an unequal interchange between Xp and Yp termini during paternal meiosis. The pattern of Y-sequences in these patients varies considerably, but very few cases have been reported showing only SRY. The phenotype in these patients is also variable ranging from severe impairment of the external genitalia through hypospadias and/or cryptorchidism to occasional normal male phenotype. We report a Mexican 46,XX male patient without genital ambiguities in whom DNA analysis showed the presence of SRY and the absence of ZFY. We conclude that in this case SRY alone was enough for complete male sexual differentiation. 25 refs., 1 fig.

  6. Differentiation of Isomeric Ginsenosides by Using Electron-Induced Dissociation Mass Spectrometry.

    PubMed

    Wong, Y-L Elaine; Chen, Xiangfeng; Li, Wan; Wang, Ze; Hung, Y-L Winnie; Wu, Ri; Chan, T-W Dominic

    2016-06-01

    Current phytochemical research on ginsengs focuses on the structural characterization and isomer differentiation of ginsenosides. In this Letter, electron-induced dissociation (EID) was initially investigated by analyzing isomeric ginsenosides. EID provided more structural information on their differentiation than collision-induced dissociation (CID) did. Glycosyl group migration previously observed in the CID of oligosaccharide ions could also be found in the EID of protonated Rg1. This rearrangement reaction would show substantial ambiguities in differentiating Rg1 from Rf. Although other charge carriers could alleviate this problem, the use of EID in dissociating deprotonated ginsenoside ions was superior to other techniques in terms of eliminating glycosyl group migration and generating diagnostic fragment ions for the differentiation of structural isomers. This study demonstrates a potential method to analyze natural products and thus help discover and evaluate novel compounds. PMID:27181402

  7. The Salmonella effector SopB prevents ROS-induced apoptosis of epithelial cells by retarding TRAF6 recruitment to mitochondria.

    PubMed

    Ruan, Haihua; Zhang, Zhen; Tian, Li; Wang, Suying; Hu, Shuangyan; Qiao, Jian-Jun

    2016-09-16

    Microbial pathogens enter host cells by injecting effector proteins of the Type III secretion system (T3SS), which facilitate pathogen translocation across the host cell membrane. These effector proteins exert their effects by modulating a variety of host innate immune responses, thereby facilitating bacterial replication and systemic infection. Salmonella enterica serovar typhimurium (S.typhimurium) is a clinically important pathogen that causes food poisoning and gastroenteritis. The SopB effector protein of S. typhimurium, encoded by Salmonella pathogenicity islands (SPI)-1 T3SS, protects host epithelial cells from infection-induced apoptosis. However, how SopB influences apoptosis induction remains unclear. Here, we investigated the mechanism of SopB action in host cells. We found that SopB inhibits infection-induced apoptosis by attenuating the production of reactive oxygen species (ROS) in mitochondria, the crucial organelles for apoptosis initiation. Further investigation revealed that SopB binds to cytosolic tumor necrosis factor receptor associated factor 6 (TRAF6) and forms a trap preventing the mitochondrial recruitment of TRAF6, an essential event for ROS generation within mitochondria. By studying the response of Traf6(+/+) and Traf6(-/-)mouse embryonic fibroblasts to S. typhimurium infection, we found that TRAF6 promoted apoptosis by increasing ROS accumulation, which led to increased Bax/Bcl-2 ratio, Bax recruitment to mitochondrial membrane, and release of Cyt c into the cytoplasm. These findings show that SopB suppresses host cell apoptosis by binding to TRAF6 and preventing mitochondrial ROS generation. PMID:27473656

  8. Hepatitis C Virus Sensing by Human Trophoblasts Induces Innate Immune Responses and Recruitment of Maternal NK Cells: Potential Implications for Limiting Vertical Transmission.

    PubMed

    Giugliano, Silvia; Petroff, Margaret G; Warren, Bryce D; Jasti, Susmita; Linscheid, Caitlin; Ward, Ashley; Kramer, Anita; Dobrinskikh, Evgenia; Sheiko, Melissa A; Gale, Michael; Golden-Mason, Lucy; Winn, Virginia D; Rosen, Hugo R

    2015-10-15

    Hepatitis C virus (HCV) is the world's most common blood-borne viral infection for which there is no vaccine. The rates of vertical transmission range between 3 and 6% with odds 90% higher in the presence of HIV coinfection. Prevention of vertical transmission is not possible because of lack of an approved therapy for use in pregnancy or an effective vaccine. Recently, HCV has been identified as an independent risk factor for preterm delivery, perinatal mortality, and other complications. In this study, we characterized the immune responses that contribute to the control of viral infection at the maternal-fetal interface (MFI) in the early gestational stages. In this study, we show that primary human trophoblast cells and an extravillous trophoblast cell line (HTR8), from first and second trimester of pregnancy, express receptors relevant for HCV binding/entry and are permissive for HCV uptake. We found that HCV-RNA sensing by human trophoblast cells induces robust upregulation of type I/III IFNs and secretion of multiple chemokines that elicit recruitment and activation of decidual NK cells. Furthermore, we observed that HCV-RNA transfection induces a proapoptotic response within HTR8 that could affect the morphology of the placenta. To our knowledge, for the first time, we demonstrate that HCV-RNA sensing by human trophoblast cells elicits a strong antiviral response that alters the recruitment and activation of innate immune cells at the MFI. This work provides a paradigm shift in our understanding of HCV-specific immunity at the MFI as well as novel insights into mechanisms that limit vertical transmission but may paradoxically lead to virus-related pregnancy complications. PMID:26342030

  9. Recombinant human bone morphogenetic protein-9 potently induces osteogenic differentiation of human periodontal ligament fibroblasts.

    PubMed

    Fuchigami, Sawako; Nakamura, Toshiaki; Furue, Kirara; Sena, Kotaro; Shinohara, Yukiya; Noguchi, Kazuyuki

    2016-04-01

    To accomplish effective periodontal regeneration for periodontal defects, several regenerative methods using growth and differentiation factors, including bone morphogenetic proteins (BMPs), have been developed. Bone morphogenetic protein-9 exhibits the most potent osteogenic activity of this growth factor family. However, it is unclear whether exogenous BMP-9 can induce osteogenic differentiation in human periodontal ligament (PDL) fibroblasts. Here, we examined the effects of recombinant human (rh) BMP-9 on osteoblastic differentiation in human PDL fibroblasts in vitro, compared with rhBMP-2. Recombinant human BMP-9 potently induced alkaline phosphatase (ALP) activity, mineralization, and increased expression of runt-related transcription factor-2/core binding factor alpha 1 (RUNX2/CBFA1), osterix, inhibitor of DNA binding/differentiation-1 (ID1), osteopontin, and bone sialoprotein genes, compared with rhBMP-2. The levels of rhBMP-9-induced osterix and ALP mRNA were significantly reduced in activin receptor-like kinase-1 and -2 small interfering RNA (siRNA)-transfected human PDL fibroblasts. Recombinant human BMP-9-induced ALP activity was not inhibited by noggin, in contrast to rhBMP-2 induced ALP activity, which was. Phosphorylation of SMAD1/5/8 in human PDL fibroblasts was induced by addition of rhBMP-9. Recombinant human BMP-9-induced ALP activity was suppressed by SB203580, SP600125, and U0126, which are inhibitors of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), respectively. Our data suggest that rhBMP-9 is a potent inducer of the differentiation of human PDL fibroblasts into osteoblast-like cells and that this may be mediated by the SMAD and mitogen-activated protein kinase (p38, ERK1/2, and JNK) pathways. PMID:26879145

  10. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    SciTech Connect

    Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min; Wang, Jianxiang

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.