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Sample records for cells mediates mechanical

  1. Mast Cell-Mediated Mechanisms of Nociception

    PubMed Central

    Aich, Anupam; Afrin, Lawrence B.; Gupta, Kalpna

    2015-01-01

    Mast cells are tissue-resident immune cells that release immuno-modulators, chemo-attractants, vasoactive compounds, neuropeptides and growth factors in response to allergens and pathogens constituting a first line of host defense. The neuroimmune interface of immune cells modulating synaptic responses has been of increasing interest, and mast cells have been proposed as key players in orchestrating inflammation-associated pain pathobiology due to their proximity to both vasculature and nerve fibers. Molecular underpinnings of mast cell-mediated pain can be disease-specific. Understanding such mechanisms is critical for developing disease-specific targeted therapeutics to improve analgesic outcomes. We review molecular mechanisms that may contribute to nociception in a disease-specific manner. PMID:26690128

  2. Fas and its ligand in a general mechanism of T-cell-mediated cytotoxicity.

    PubMed Central

    Hanabuchi, S; Koyanagi, M; Kawasaki, A; Shinohara, N; Matsuzawa, A; Nishimura, Y; Kobayashi, Y; Yonehara, S; Yagita, H; Okumura, K

    1994-01-01

    To investigate the mechanisms of T-cell-mediated cytotoxicity, we estimated the involvement of apoptosis-inducing Fas molecule on the target cells and its ligand on the effector cells. When redirected by ConA or anti-CD3 monoclonal antibody, a CD4+ T-cell clone, BK1, could lyse the target cells expressing wild-type Fas molecule but not those expressing death signaling-deficient mutants. This indicates the involvement of Fas-mediated signal transduction in the target cell lysis by BK1. Anti-CD3-activated but not resting BK1 expressed Fas ligand as detected by binding of a soluble Fas-Ig fusion protein, and the BK1-mediated cytotoxicity was blocked by the addition of Fas-Ig, implicating the inducible Fas ligand in the BK1 cytotoxicity. Ability to exert the Fas-mediated cytotoxicity was not confined to BK1, but splenic CD4+ T cells and, to a lesser extent, CD8+ T cells could also exert the Fas-dependent target cell lysis. This indicates that the Fas-mediated target cell lytic pathway can be generally involved in the T-cell-mediated cytotoxicity. Interestingly, CD4+ T cells prepared from gld/gld mice did not mediate the Fas-mediated cytotoxicity, indicating defective expression of functional Fas ligand in gld mice. PMID:7515183

  3. Ciprofloxacin mediates cancer stem cell phenotypes in lung cancer cells through caveolin-1-dependent mechanism.

    PubMed

    Phiboonchaiyanan, Preeyaporn Plaimee; Kiratipaiboon, Chayanin; Chanvorachote, Pithi

    2016-04-25

    Cancer stem cells (CSCs), a subpopulation of cancer cells with high aggressive behaviors, have been identified in many types of cancer including lung cancer as one of the key mediators driving cancer progression and metastasis. Here, we have reported for the first time that ciprofloxacin (CIP), a widely used anti-microbial drug, has a potentiating effect on CSC-like features in human non-small cell lung cancer (NSCLC) cells. CIP treatment promoted CSC-like phenotypes, including enhanced anchorage-independent growth and spheroid formation. The known lung CSC markers: CD133, CD44, ABCG2 and ALDH1A1 were found to be significantly increased, while the factors involving in epithelial to mesenchymal transition (EMT): Slug and Snail, were depleted. Also, self-renewal transcription factors Oct-4 and Nanog were found to be up-regulated in CIP-treated cells. The treatment of CIP on CSC-rich populations obtained from secondary spheroids resulted in the further increase of CSC markers. In addition, we have proven that the mechanistic insight of the CIP induced stemness is through Caveolin-1 (Cav-1)-dependent mechanism. The specific suppression of Cav-1 by stably transfected Cav-1 shRNA plasmid dramatically reduced the effect of CIP on CSC markers as well as the CIP-induced spheroid formation ability. Cav-1 was shown to activate protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) pathways in CSC-rich population; however, such an effect was rarely found in the main lung cancer cells population. These findings reveal a novel effect of CIP in positively regulating CSCs in lung cancer cells via the activation of Cav-1, Akt and ERK, and may provoke the awareness of appropriate therapeutic strategy in cancer patients.

  4. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  5. P-cadherin promotes collective cell migration via a Cdc42-mediated increase in mechanical forces

    PubMed Central

    Plutoni, Cédric; Bazellieres, Elsa; Le Borgne-Rochet, Maïlys; Comunale, Franck; Brugues, Agusti; Séveno, Martial; Planchon, Damien; Thuault, Sylvie; Morin, Nathalie; Bodin, Stéphane; Trepat, Xavier

    2016-01-01

    Collective cell migration (CCM) is essential for organism development, wound healing, and metastatic transition, the primary cause of cancer-related death, and it involves cell–cell adhesion molecules of the cadherin family. Increased P-cadherin expression levels are correlated with tumor aggressiveness in carcinoma and aggressive sarcoma; however, how P-cadherin promotes tumor malignancy remains unknown. Here, using integrated cell biology and biophysical approaches, we determined that P-cadherin specifically induces polarization and CCM through an increase in the strength and anisotropy of mechanical forces. We show that this mechanical regulation is mediated by the P-cadherin/β-PIX/Cdc42 axis; P-cadherin specifically activates Cdc42 through β-PIX, which is specifically recruited at cell–cell contacts upon CCM. This mechanism of cell polarization and migration is absent in cells expressing E- or R-cadherin. Thus, we identify a specific role of P-cadherin through β-PIX–mediated Cdc42 activation in the regulation of cell polarity and force anisotropy that drives CCM. PMID:26783302

  6. Autophagy as a Survival Mechanism for Squamous Cell Carcinoma Cells in Endonuclease G-Mediated Apoptosis

    PubMed Central

    Masui, Atsushi; Hamada, Masakazu; Kameyama, Hiroyasu; Wakabayashi, Ken; Takasu, Ayako; Imai, Tomoaki; Iwai, Soichi; Yura, Yoshiaki

    2016-01-01

    Safingol, L- threo-dihydrosphingosine, induces cell death in human oral squamous cell carcinoma (SCC) cells through an endonuclease G (endoG) -mediated pathway. We herein determined whether safingol induced apoptosis and autophagy in oral SCC cells. Safingol induced apoptotic cell death in oral SCC cells in a dose-dependent manner. In safingol-treated cells, microtubule-associated protein 1 light chain 3 (LC3)-I was changed to LC3-II and the cytoplasmic expression of LC3, amount of acidic vesicular organelles (AVOs) stained by acridine orange and autophagic vacuoles were increased, indicating the occurrence of autophagy. An inhibitor of autophagy, 3-methyladenine (3-MA), enhanced the suppressive effects of safingol on cell viability, and this was accompanied by an increase in the number of apoptotic cells and extent of nuclear fragmentation. The nuclear translocation of endoG was minimal at a low concentration of safingol, but markedly increased when combined with 3-MA. The suppressive effects of safingol and 3-MA on cell viability were reduced in endoG siRNA- transfected cells. The scavenging of reactive oxygen species (ROS) prevented cell death induced by the combinational treatment, whereas a pretreatment with a pan-caspase inhibitor z-VAD-fmk did not. These results indicated that safingol induced apoptosis and autophagy in SCC cells and that the suppression of autophagy by 3-MA enhanced apoptosis. Autophagy supports cell survival, but not cell death in the SCC cell system in which apoptosis occurs in an endoG-mediated manner. PMID:27658240

  7. T-cell-mediated drug hypersensitivity: immune mechanisms and their clinical relevance.

    PubMed

    Yun, James; Cai, Fenfen; Lee, Frederick J; Pichler, Werner J

    2016-04-01

    T-cell-mediated drug hypersensitivity represents a significant proportion of immune mediated drug hypersensitivity reactions. In the recent years, there has been an increase in understanding the immune mechanisms behind T-cell-mediated drug hypersensitivity. According to hapten mechanism, drug specific T-cell response is stimulated by drug-protein conjugate presented on major histocompatibility complex (MHC) as it is presented as a new antigenic determinant. On the other hand, p-i concept suggests that a drug can stimulate T cells via noncovalent direct interaction with T-cell receptor and/or peptide-MHC. The drug binding site is quite variable and this leads to several different mechanisms within p-i concept. Altered peptide repertoire can be regarded as an 'atypical' subset of p-i concept since the mode of the drug binding and the binding site are essentially identical to p-i concept. However, the intracellular binding of abacavir to HLA-B(*)57:01 additionally results in alteration in peptide repertoire. Furthermore the T-cell response to altered peptide repertoire model is only shown for abacavir and HLA-B(*)57:01 and therefore it may not be generalised to other drug hypersensitivity. Danger hypothesis has been postulated to play an important role in drug hypersensitivity by providing signal 2 but its experimental data is lacking at this point in time. Furthermore, the recently described allo-immune response suggests that danger signal may be unnecessary. Finally, in view of these new understanding, the classification and the definition of type B adverse drug reaction should be revised. PMID:27141480

  8. T-cell-mediated drug hypersensitivity: immune mechanisms and their clinical relevance

    PubMed Central

    Cai, Fenfen; Lee, Frederick J; Pichler, Werner J

    2016-01-01

    T-cell-mediated drug hypersensitivity represents a significant proportion of immune mediated drug hypersensitivity reactions. In the recent years, there has been an increase in understanding the immune mechanisms behind T-cell-mediated drug hypersensitivity. According to hapten mechanism, drug specific T-cell response is stimulated by drug-protein conjugate presented on major histocompatibility complex (MHC) as it is presented as a new antigenic determinant. On the other hand, p-i concept suggests that a drug can stimulate T cells via noncovalent direct interaction with T-cell receptor and/or peptide-MHC. The drug binding site is quite variable and this leads to several different mechanisms within p-i concept. Altered peptide repertoire can be regarded as an 'atypical' subset of p-i concept since the mode of the drug binding and the binding site are essentially identical to p-i concept. However, the intracellular binding of abacavir to HLA-B*57:01 additionally results in alteration in peptide repertoire. Furthermore the T-cell response to altered peptide repertoire model is only shown for abacavir and HLA-B*57:01 and therefore it may not be generalised to other drug hypersensitivity. Danger hypothesis has been postulated to play an important role in drug hypersensitivity by providing signal 2 but its experimental data is lacking at this point in time. Furthermore, the recently described allo-immune response suggests that danger signal may be unnecessary. Finally, in view of these new understanding, the classification and the definition of type B adverse drug reaction should be revised. PMID:27141480

  9. Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration

    PubMed Central

    Fang, Yuqiang; Iu, Catherine Y. Y.; Lui, Cathy N. P.; Zou, Yukai; Fung, Carmen K. M.; Li, Hung Wing; Xi, Ning; Yung, Ken K. L.; Lai, King W. C.

    2014-01-01

    Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases. PMID:25399549

  10. Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration

    NASA Astrophysics Data System (ADS)

    Fang, Yuqiang; Iu, Catherine Y. Y.; Lui, Cathy N. P.; Zou, Yukai; Fung, Carmen K. M.; Li, Hung Wing; Xi, Ning; Yung, Ken K. L.; Lai, King W. C.

    2014-11-01

    Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.

  11. Cell-mediated BMP-2 liberation promotes bone formation in a mechanically unstable implant environment.

    PubMed

    Hägi, Tobias T; Wu, Gang; Liu, Yuelian; Hunziker, Ernst B

    2010-05-01

    The flexible alloplastic materials that are used in bone-reconstruction surgery lack the mechanical stability that is necessary for sustained bone formation, even if this process is promoted by the application of an osteogenic agent, such as BMP-2. We hypothesize that if BMP-2 is delivered gradually, in a cell-mediated manner, to the surgical site, then the scaffolding material's lack of mechanical stability becomes a matter of indifference. Flexible discs of Ethisorb were functionalized with BMP-2, which was either adsorbed directly onto the material (rapid release kinetics) or incorporated into a calcium-phosphate coating (slow release kinetics). Unstabilized and titanium-plate-stabilized samples were implanted subcutaneously in rats and retrieved up to 14 days later for a histomorphometric analysis of bone and cartilage volumes. On day 14, the bone volume associated with titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2 was 10-fold higher than that associated with their mechanically unstabilized counterparts. The bone volume associated with discs bearing a coating-incorporated depot of BMP-2 was similar in the mechanically unstabilized and titanium-plate-stabilized groups, and comparable to that associated with the titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2. Hence, if an osteogenic agent is delivered in a cell-mediated manner (via coating degradation), ossification can be promoted even within a mechanically unstable environment.

  12. Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

    PubMed

    Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

    2013-12-15

    The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level.

  13. Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

    PubMed

    Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

    2013-12-15

    The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level. PMID:24021157

  14. Reduced expression IRF7 in nasal epithelial cells from smokers as a potential mechanism mediating enhanced susceptibility to influenza

    EPA Science Inventory

    Rationale: Smokers are more susceptible to viral infections, including influenza virus, yet the mechanisms mediating this effect are not known. Methods: We have established an in vitro model of differentiated nasal epithelial cells from smokers, which maintain enhanced levels...

  15. TRPM2 Channel-Mediated ROS-Sensitive Ca2+ Signaling Mechanisms in Immune Cells

    PubMed Central

    Syed Mortadza, Sharifah Alawieyah; Wang, Lu; Li, Dongliang; Jiang, Lin-Hua

    2015-01-01

    Transient receptor potential melastatin 2 (TRPM2) proteins form Ca2+-permeable cationic channels that are potently activated by reactive oxygen species (ROS). ROS are produced during immune responses as signaling molecules as well as anti-microbial agents. ROS-sensitive TRPM2 channels are widely expressed in cells of the immune system and located on the cell surface as a Ca2+ influx pathway in macrophages, monocytes, neutrophils, lymphocytes, and microglia but preferentially within the lysosomal membranes as a Ca2+ release mechanism in dendritic cells; ROS activation of the TRPM2 channels, regardless of the subcellular location, results in an increase in the intracellular Ca2+ concentrations. Recent studies have revealed that TRPM2-mediated ROS-sensitive Ca2+ signaling mechanisms play a crucial role in a number of processes and functions in immune cells. This mini-review discusses the recent advances in revelation of the various roles the TRPM2 channels have in immune cell functions and the implications in inflammatory diseases. PMID:26300888

  16. CDI Systems Are Stably Maintained by a Cell-Contact Mediated Surveillance Mechanism

    PubMed Central

    Chen, Annette J.; Leung, Nicole Y.; Hayes, Christopher S.; Low, David A.

    2016-01-01

    Contact-dependent growth inhibition (CDI) systems are widespread amongst Gram-negative bacteria where they play important roles in inter-cellular competition and biofilm formation. CDI+ bacteria use cell-surface CdiA proteins to bind neighboring bacteria and deliver C-terminal toxin domains. CDI+ cells also express CdiI immunity proteins that specifically neutralize toxins delivered from adjacent siblings. Genomic analyses indicate that cdi loci are commonly found on plasmids and genomic islands, suggesting that these Type 5 secretion systems are spread through horizontal gene transfer. Here, we examine whether CDI toxin and immunity activities serve to stabilize mobile genetic elements using a minimal F plasmid that fails to partition properly during cell division. This F plasmid is lost from Escherichia coli populations within 50 cell generations, but is maintained in ~60% of the cells after 100 generations when the plasmid carries the cdi gene cluster from E. coli strain EC93. By contrast, the ccdAB "plasmid addiction" module normally found on F exerts only a modest stabilizing effect. cdi-dependent plasmid stabilization requires the BamA receptor for CdiA, suggesting that plasmid-free daughter cells are inhibited by siblings that retain the CDI+ plasmid. In support of this model, the CDI+ F plasmid is lost rapidly from cells that carry an additional cdiI immunity gene on a separate plasmid. These results indicate that plasmid stabilization occurs through elimination of non-immune cells arising in the population via plasmid loss. Thus, genetic stabilization reflects a strong selection for immunity to CDI. After long-term passage for more than 300 generations, CDI+ plasmids acquire mutations that increase copy number and result in 100% carriage in the population. Together, these results show that CDI stabilizes genetic elements through a toxin-mediated surveillance mechanism in which cells that lose the CDI system are detected and eliminated by their siblings

  17. Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action.

    PubMed Central

    Nelson, R D; Shibata, N; Podzorski, R P; Herron, M J

    1991-01-01

    The ability of Candida albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan. Mannan and oligosaccharide fragments of mannan are potent inhibitors of cell-mediated immunity and appear to reproduce the immune deficit of patients with the mucocutaneous form of candidiasis. However, neither the exact structures of these inhibitory species nor their mechanisms of action have yet been clearly defined. Different investigators have proposed that mannan or mannan catabolites act upon monocytes or suppressor T lymphocytes, but research from unrelated areas has provided still other possibilities for consideration. These include interference with cytokine activities, lymphocyte-monocyte interactions, and leukocyte homing. To stimulate further research of the immunosuppressive property of C. albicans mannan, we have reviewed (i) the relationship of mannan to other antigens and virulence factors of the fungus; (ii) the chemistry of mannan, together with methods for preparation of mannan and mannan fragments; and (iii) the historical evidence for immunosuppression by Candida mannan and the mechanisms currently proposed for this property; and (iv) we have speculated upon still other mechanisms by which mannan might influence host defense functions. It is possible that understanding the immunosuppressive effects of mannan will provide clues to novel therapies for candidiasis that will enhance the efficacy of both available and future anti-Candida agents. PMID:2004345

  18. Ceruloplasmin enhances smooth muscle cell- and endothelial cell-mediated low density lipoprotein oxidation by a superoxide-dependent mechanism

    NASA Technical Reports Server (NTRS)

    Mukhopadhyay, C. K.; Ehrenwald, E.; Fox, P. L.

    1996-01-01

    Cultured vascular smooth muscle cells (SMC) and endothelial cells (EC) stimulate low density lipoprotein (LDL) oxidation by free radical-mediated, transition metal-dependent mechanisms. The physiological source(s) of metal ions is not known; however, purified ceruloplasmin, a plasma protein containing 7 coppers, oxidizes LDL in vitro. We now show that ceruloplasmin also increases LDL oxidation by vascular cells. In metal ion-free medium, human ceruloplasmin increased bovine aortic SMC- and EC-mediated LDL oxidation by up to 30- and 15-fold, respectively. The maximal response was at 100-300 microg ceruloplasmin/ml, a level at or below the unevoked physiological plasma concentration. Oxidant activity was dependent on protein structure as a specific proteolytic cleavage or removal of one of the seven ceruloplasmin copper atoms inhibited activity. Three lines of evidence indicated a critical role for cellular superoxide (O2.) in ceruloplasmin-stimulated oxidation. First, the rate of production of O2. by cells correlated with their rates of LDL oxidation. Second, superoxide dismutase effectively blocked ceruloplasmin-stimulated oxidation by both cell types. Finally, O2. production by SMC quantitatively accounted for the observed rate of LDL oxidation. To show this, the course of O2. production by SMC was simulated by repeated addition of xanthine and xanthine oxidase to culture medium under cell-free conditions. Neither ceruloplasmin nor O2. alone increased LDL oxidation, but together they completely reconstituted the oxidation rate of ceruloplasmin-stimulated SMC. These results are the first to show that ceruloplasmin stimulates EC- and SMC-mediated oxidation of LDL and that cell-derived O2. accounts quantitatively for metal-dependent, free radical-initiated oxidation of LDL by these cells.

  19. Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms.

    PubMed Central

    Rogatsky, I; Trowbridge, J M; Garabedian, M J

    1997-01-01

    Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor. PMID:9154817

  20. Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

    PubMed

    Zhou, Junmin; Donatelli, Sarah S; Gilvary, Danielle L; Tejera, Melba M; Eksioglu, Erika A; Chen, Xianghong; Coppola, Domenico; Wei, Sheng; Djeu, Julie Y

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b(+)Ly6G(+)Ly6C(low) granulocytic (G)-MDSCs, sparing CD11b(+)Ly6G(-)Ly6C(high) M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7(+) Dectin-1(-)M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68(+) macrophages but not in immature CD33(+) immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents. PMID:27405665

  1. Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin

    PubMed Central

    Zhou, Junmin; Donatelli, Sarah S.; Gilvary, Danielle L.; Tejera, Melba M.; Eksioglu, Erika A.; Chen, Xianghong; Coppola, Domenico; Wei, Sheng; Djeu, Julie Y.

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b+Ly6G+Ly6Clow granulocytic (G)-MDSCs, sparing CD11b+Ly6G−Ly6Chigh M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7+ Dectin-1−M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68+ macrophages but not in immature CD33+ immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents. PMID:27405665

  2. Mechanisms of PECAM-1-mediated cytoprotection and implications for cancer cell survival.

    PubMed

    Bergom, Carmen; Gao, Cunji; Newman, Peter J

    2005-10-01

    Defects in apoptotic pathways can promote cancer development and cause cancers to become resistant to chemotherapy. The cell adhesion and signaling molecule PECAM-1 has been shown to potently suppress apoptosis in a variety of cellular systems. PECAM-1 expression has been reported on a variety of human malignancies-especially hematopoietic and vascular cell cancers-but the significance of this expression has not been fully explored. The ability of PECAM-1 to inhibit apoptosis makes it an attractive candidate as a molecule that may promote cancer development and/or confer resistance to chemotherapeutic treatment. The exact mechanisms by which PECAM-1 mediates its cytoprotection have not been fully defined, but its anti-apoptotic effects have been shown to require both homophilic binding and intracellular signaling via its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. In this review, we will discuss the data regarding PECAM-1's anti-apoptotic effects and ways in which this cytoprotection may be clinically relevant to the development and/or treatment of hematologic malignancies that express this vascular cell-specific surface molecule. PMID:16194886

  3. Vimentin contributes to epithelial-mesenchymal transition cancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation

    PubMed Central

    Liu, Ching-Yi; Lin, Hsi-Hui; Tang, Ming-Jer; Wang, Yang-Kao

    2015-01-01

    Modulations of cytoskeletal organization and focal adhesion turnover correlate to tumorigenesis and epithelial-mesenchymal transition (EMT), the latter process accompanied by the loss of epithelial markers and the gain of mesenchymal markers (e.g., vimentin). Clinical microarray results demonstrated that increased levels of vimentin mRNA after chemotherapy correlated to a poor prognosis of breast cancer patients. We hypothesized that vimentin mediated the reorganization of cytoskeletons to maintain the mechanical integrity in EMT cancer cells. By using knockdown strategy, the results showed reduced cell proliferation, impaired wound healing, loss of directional migration, and increased large membrane extension in MDA-MB 231 cells. Vimentin depletion also induced reorganization of cytoskeletons and reduced focal adhesions, which resulted in impaired mechanical strength because of reduced cell stiffness and contractile force. In addition, overexpressing vimentin in MCF7 cells increased cell stiffness, elevated cell motility and directional migration, reoriented microtubule polarity, and increased EMT phenotypes due to the increased β1-integrin and the loss of junction protein E-cadherin. The EMT-related transcription factor slug was also mediated by vimentin. The current study demonstrated that vimentin serves as a regulator to maintain intracellular mechanical homeostasis by mediating cytoskeleton architecture and the balance of cell force generation in EMT cancer cells. PMID:25965826

  4. Neurodegenerative mechanisms in Alzheimer disease. A role for oxidative damage in amyloid beta protein precursor-mediated cell death.

    PubMed

    Sopher, B L; Fukuchi, K; Kavanagh, T J; Furlong, C E; Martin, G M

    1996-01-01

    We have established a stably transformed human neuroblastoma cell line (MC65) that conditionally expresses a C-terminal derivative of the amyloid beta protein precursor (beta PP) termed S beta C (a fusion protein composed of the amino-17 and carboxyl-99 residues of beta PP). Conditional expression of S beta C (mediated by the withdrawal of tetracycline from the culture medium) induces pronounced nuclear DNA fragmentation and cytotoxicity in this cell line. These effects are enhanced by hyperoxygen and suppressed by hypooxygen and antioxidants. This cell line is relatively insensitive to the extracellular application of amyloid beta 25-35, and coculture experiments suggest that this cytotoxicity is mediated by an intracellular process. These findings suggest that the overexpression of the C-terminal domain of beta PP can disrupt normal cellular processes in these cells in such a way as to induce a directed (deoxyribonuclease-mediated) mechanism of cell death. This process appears to be modulated and/or mediated by a reactive oxygen specie(s) (ROS). Consistent with a role for ROS in the process of S beta C-mediated toxicity, we have found that the MC65 cell line is hypersensitive to oxidative stress and that it is this sensitivity that appears (at least in part) to underlie its susceptibility to S beta C.

  5. Immune Cell Inhibition by SLAMF7 Is Mediated by a Mechanism Requiring Src Kinases, CD45, and SHIP-1 That Is Defective in Multiple Myeloma Cells

    PubMed Central

    Guo, Huaijian; Cruz-Munoz, Mario-Ernesto; Wu, Ning; Robbins, Michael

    2014-01-01

    Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells. PMID:25312647

  6. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  7. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    NASA Astrophysics Data System (ADS)

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-08-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells.

  8. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells.

    PubMed

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including "p38 MAPK Signaling", "p53 Signaling", "14-3-3-mediated Signaling", "p70S6K Signaling" and "Protein Ubiquitination Pathway". This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  9. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    PubMed Central

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  10. Mechanisms of tumor and viral immune escape from natural killer cell-mediated surveillance.

    PubMed

    Groth, Ariane; Klöss, Stephan; von Strandmann, Elke Pogge; Koehl, Ulrike; Koch, Joachim

    2011-01-01

    Human natural killer (NK) cells recognize and efficiently eliminate MHC class I low or negative malignant targets and virally infected host cells, without requirement for prior sensitization. However, viruses and various tumor cells display elaborate adaptations to evade and overcome immunosurveillance. The current review focuses on escape mechanisms of viruses and malignantly transformed 'stressed' cells to evade from NK cell cytotoxicity. A general overview of recent clinical studies using allogeneic donor NK cells is given, summarizing first data about a possible benefit for patients suffering from high-risk leukemia and solid tumors. Finally, the review discusses the future perspectives and hypotheses aiming to improve therapeutic NK cell strategies against tumor immune escape mechanisms.

  11. Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes.

    PubMed

    Liu, Betty Revon; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2015-04-01

    Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

  12. Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells – Role of Anchored Protein Kinase A Signaling Units

    PubMed Central

    Wehbi, Vanessa L.; Taskén, Kjetil

    2016-01-01

    The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A-kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system, cAMP is a potent negative regulator of T cell receptor-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50, and the anchoring protein phosphoprotein associated with glycosphingolipid-enriched microdomains holding Csk. As PKA activates Csk and Csk inhibits Lck, this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators, such as prostaglandins (PGs), adrenergic stimuli, adenosine, and a number of other ligands. A major inducer of T cell cAMP levels is PG E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced Tregs express cyclooxygenase-2, secrete PGE2, and elicit the immunosuppressive cAMP pathway in Teff as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms, such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review, we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical

  13. Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells - Role of Anchored Protein Kinase A Signaling Units.

    PubMed

    Wehbi, Vanessa L; Taskén, Kjetil

    2016-01-01

    The cyclic AMP/protein kinase A (cAMP/PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. A-kinase anchoring proteins (AKAPs) target PKA to specific substrates and distinct subcellular compartments providing spatial and temporal specificity for mediation of biological effects channeled through the cAMP/PKA pathway. In the immune system, cAMP is a potent negative regulator of T cell receptor-mediated activation of effector T cells (Teff) acting through a proximal PKA/Csk/Lck pathway anchored via a scaffold consisting of the AKAP Ezrin holding PKA, the linker protein EBP50, and the anchoring protein phosphoprotein associated with glycosphingolipid-enriched microdomains holding Csk. As PKA activates Csk and Csk inhibits Lck, this pathway in response to cAMP shuts down proximal T cell activation. This immunomodulating pathway in Teff mediates clinically important responses to regulatory T cell (Treg) suppression and inflammatory mediators, such as prostaglandins (PGs), adrenergic stimuli, adenosine, and a number of other ligands. A major inducer of T cell cAMP levels is PG E2 (PGE2) acting through EP2 and EP4 prostanoid receptors. PGE2 plays a crucial role in the normal physiological control of immune homeostasis as well as in inflammation and cancer immune evasion. Peripherally induced Tregs express cyclooxygenase-2, secrete PGE2, and elicit the immunosuppressive cAMP pathway in Teff as one tumor immune evasion mechanism. Moreover, a cAMP increase can also be induced by indirect mechanisms, such as intercellular transfer between T cells. Indeed, Treg, known to have elevated levels of intracellular cAMP, may mediate their suppressive function by transferring cAMP to Teff through gap junctions, which we speculate could also be regulated by PKA/AKAP complexes. In this review, we present an updated overview on the influence of cAMP-mediated immunoregulatory mechanisms acting through localized cAMP signaling and the therapeutical

  14. Flagella and pili-mediated near-surface single-cell motility mechanisms in P. aeruginosa.

    PubMed

    Conrad, Jacinta C; Gibiansky, Maxsim L; Jin, Fan; Gordon, Vernita D; Motto, Dominick A; Mathewson, Margie A; Stopka, Wiktor G; Zelasko, Daria C; Shrout, Joshua D; Wong, Gerard C L

    2011-04-01

    Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology. PMID:21463573

  15. Bypassing Mechanisms of Mitochondria-Mediated Cancer Stem Cells Resistance to Chemo- and Radiotherapy

    PubMed Central

    Lyakhovich, Alex; Lleonart, Matilde E.

    2016-01-01

    Cancer stem cells (CSCs) are highly resistant to conventional chemo- and radiotherapeutic regimes. Therefore, the multiple drug resistance (MDR) of cancer is most likely due to the resistance of CSCs. Such resistance can be attributed to some bypassing pathways including detoxification mechanisms of reactive oxygen and nitrogen species (RO/NS) formation or enhanced autophagy. Unlike in normal cells, where RO/NS concentration is maintained at certain threshold required for signal transduction or immune response mechanisms, CSCs may develop alternative pathways to diminish RO/NS levels leading to cancer survival. In this minireview, we will focus on elaborated mechanisms developed by CSCs to attenuate high RO/NS levels. Gaining a better insight into the mechanisms of stem cell resistance to chemo- or radiotherapy may lead to new therapeutic targets thus serving for better anticancer strategies. PMID:26697128

  16. The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease.

    PubMed

    Richards, Robert I; Robertson, Sarah A; O'Keefe, Louise V; Fornarino, Dani; Scott, Andrew; Lardelli, Michael; Baune, Bernhard T

    2016-01-01

    Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an "intelligent system," in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated damage

  17. The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease

    PubMed Central

    Richards, Robert I.; Robertson, Sarah A.; O'Keefe, Louise V.; Fornarino, Dani; Scott, Andrew; Lardelli, Michael; Baune, Bernhard T.

    2016-01-01

    Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an “intelligent system,” in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated

  18. A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway.

    PubMed

    Said, Hamid M; Ortiz, Alvaro; Ma, Thomas Y

    2003-11-01

    Vitamin B6 is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B6 and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B6 homeostasis. Due to the water-soluble nature of vitamin B6 and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B6 in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na+ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (Km) of 11.99 +/- 1.41 microM and a maximal velocity (Vmax) of 67.63 +/- 3.87 pmol. mg protein-1. 3 min-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (Ki) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca+2/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the Vmax, but not the

  19. Molecular Mechanisms Mediating Retinal Reactive Gliosis Following Bone Marrow Mesenchymal Stem Cell Transplantation

    PubMed Central

    Tassoni, Alessia; Gutteridge, Alex; Barber, Amanda C.; Osborne, Andrew

    2015-01-01

    abstract A variety of diseases lead to degeneration of retinal ganglion cells (RGCs) and their axons within the optic nerve resulting in loss of visual function. Although current therapies may delay RGC loss, they do not restore visual function or completely halt disease progression. Regenerative medicine has recently focused on stem cell therapy for both neuroprotective and regenerative purposes. However, significant problems remain to be addressed, such as the long‐term impact of reactive gliosis occurring in the host retina in response to transplanted stem cells. The aim of this work was to investigate retinal glial responses to intravitreally transplanted bone marrow mesenchymal stem cells (BM‐MSCs) to help identify factors able to modulate graft‐induced reactive gliosis. We found in vivo that intravitreal BM‐MSC transplantation is associated with gliosis‐mediated retinal folding, upregulation of intermediate filaments, and recruitment of macrophages. These responses were accompanied by significant JAK/STAT3 and MAPK (ERK1/2 and JNK) cascade activation in retinal Muller glia. Lipocalin‐2 (Lcn‐2) was identified as a potential new indicator of graft‐induced reactive gliosis. Pharmacological inhibition of STAT3 in BM‐MSC cocultured retinal explants successfully reduced glial fibrillary acidic protein expression in retinal Muller glia and increased BM‐MSC retinal engraftment. Inhibition of stem cell‐induced reactive gliosis is critical for successful transplantation‐based strategies for neuroprotection, replacement, and regeneration of the optic nerve. Stem Cells 2015;33:3006–3016 PMID:26175331

  20. Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism

    PubMed Central

    Donadelli, M; Dando, I; Zaniboni, T; Costanzo, C; Dalla Pozza, E; Scupoli, M T; Scarpa, A; Zappavigna, S; Marra, M; Abbruzzese, A; Bifulco, M; Caraglia, M; Palmieri, M

    2011-01-01

    Gemcitabine (GEM, 2′,2′-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl--cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells. PMID:21525939

  1. Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism.

    PubMed

    Donadelli, M; Dando, I; Zaniboni, T; Costanzo, C; Dalla Pozza, E; Scupoli, M T; Scarpa, A; Zappavigna, S; Marra, M; Abbruzzese, A; Bifulco, M; Caraglia, M; Palmieri, M

    2011-04-28

    Gemcitabine (GEM, 2',2'-difluorodeoxycytidine) is currently used in advanced pancreatic adenocarcinoma, with a response rate of < 20%. The purpose of our work was to improve GEM activity by addition of cannabinoids. Here, we show that GEM induces both cannabinoid receptor-1 (CB1) and cannabinoid receptor-2 (CB2) receptors by an NF-κB-dependent mechanism and that its association with cannabinoids synergistically inhibits pancreatic adenocarcinoma cell growth and increases reactive oxygen species (ROS) induced by single treatments. The antiproliferative synergism is prevented by the radical scavenger N-acetyl-L-cysteine and by the specific NF-κB inhibitor BAY 11-7085, demonstrating that the induction of ROS by GEM/cannabinoids and of NF-κB by GEM is required for this effect. In addition, we report that neither apoptotic nor cytostatic mechanisms are responsible for the synergistic cell growth inhibition, which is strictly associated with the enhancement of endoplasmic reticulum stress and autophagic cell death. Noteworthy, the antiproliferative synergism is stronger in GEM-resistant pancreatic cancer cell lines compared with GEM-sensitive pancreatic cancer cell lines. The combined treatment strongly inhibits growth of human pancreatic tumor cells xenografted in nude mice without apparent toxic effects. These findings support a key role of the ROS-dependent activation of an autophagic program in the synergistic growth inhibition induced by GEM/cannabinoid combination in human pancreatic cancer cells.

  2. Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms.

    PubMed

    Lecona, Emilio; Olmo, Nieves; Turnay, Javier; Santiago-Gómez, Angélica; López de Silanes, Isabel; Gorospe, Myriam; Lizarbe, M Antonia

    2008-01-01

    Butyrate has antitumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration, which is regulated by its transport. We have analysed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT-29), in butyrate-resistant cells (BCS-TC2.BR2) and in normal colonic cells (FHC). The properties of transport were analysed with structural analogues, specific inhibitors and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K(m)=109+/-16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K(m)=17.9+/-4.0 microM in BCS-TC2 cells) proton-monocarboxylate co-transporter that was energy-dependent and activated via PKCdelta (protein kinase Cdelta). All adenocarcinoma cells analysed express MCT (monocarboxylate transporter) 1, MCT4, ancillary protein CD147 and AE2 (anion exchanger 2). Silencing experiments show that MCT1, whose expression increases with butyrate treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate/bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumour regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes.

  3. Complement-mediated muscle cell lysis: a possible mechanism of myonecrosis in anti-SRP associated necrotizing myopathy (ASANM).

    PubMed

    Rojana-udomsart, Arada; Mitrpant, Chalermchai; Bundell, Christine; Price, Loren; Luo, Yue-Bei; Fabian, Victoria; Wilton, Steve D; Hollingsworth, Peter; Mastaglia, Frank L

    2013-11-15

    The mechanism of necrotizing myopathy associated with antibodies to signal recognition particle (SRP) remains unclear. We investigated the effect of anti-SRP+serum and complement on cell viability in myoblast cultures. Cell viability was only slightly reduced by incubation with anti-SRP+serum compared with control serum. However, the addition of fresh complement resulted in a marked reduction in cell survival. Surface immunostaining for SRP, C3c and C5b-9 was demonstrated in cultures pre-incubated with anti-SRP+serum and complement, and in muscle biopsies from patients with myopathy. These findings provide further support for a complement-dependent antibody-mediated mechanism in anti-SRP associated myopathy.

  4. An E-cadherin-mediated hitchhiking mechanism for C. elegans germ cell internalization during gastrulation

    PubMed Central

    Chihara, Daisuke; Nance, Jeremy

    2012-01-01

    Gastrulation movements place endodermal precursors, mesodermal precursors and primordial germ cells (PGCs) into the interior of the embryo. Somatic cell gastrulation movements are regulated by transcription factors that also control cell fate, coupling cell identity and position. By contrast, PGCs in many species are transcriptionally quiescent, suggesting that they might use alternative gastrulation strategies. Here, we show that C. elegans PGCs internalize by attaching to internal endodermal cells, which undergo morphogenetic movements that pull the PGCs into the embryo. We show that PGCs enrich HMR-1/E-cadherin at their surfaces to stick to endoderm. HMR-1 expression in PGCs is necessary and sufficient to ensure internalization, suggesting that HMR-1 can promote PGC-endoderm adhesion through a mechanism other than homotypic trans interactions between the two cell groups. Finally, we demonstrate that the hmr-1 3′ untranslated region promotes increased HMR-1 translation in PGCs. Our findings reveal that quiescent PGCs employ a post-transcriptionally regulated hitchhiking mechanism to internalize during gastrulation, and demonstrate a morphogenetic role for the conserved association of PGCs with the endoderm. PMID:22675206

  5. Zinc deficiency impairs neuronal precursor cell proliferation and induces apoptosis via p53-mediated mechanisms.

    PubMed

    Corniola, Rikki S; Tassabehji, Nadine M; Hare, Joan; Sharma, Girdhari; Levenson, Cathy W

    2008-10-27

    The potential importance of stem cells in the adult central nervous system (CNS) that cannot only divide, but also participate in neurogenesis, is now widely appreciated. While we know that the trace element zinc is needed for brain development, the role of this essential nutrient in adult stem cell proliferation and neurogenesis has not been investigated. Adult male rats fed a zinc-restricted diet had approximately 50% fewer Ki67-positive stem cells in the subgranular zone (SGZ) and granular cell layer of the dentate gyrus compared to both zinc-adequate and pair-fed controls (p<0.05). Zinc-deficient rats also had a significant increase the number of TUNEL-labeled cells in the SGZ compared to pair-fed rats (p<0.05). To explore the mechanisms responsible for the effects of zinc deficiency, cultured human Ntera-2 (NT2) neuronal precursor cells were deprived of zinc using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Consistent with the effects of deficiency in vivo, TPEN treatment resulted in a significant decrease in cellular proliferation, as measured by bromodeoxyuridine (BrdU) uptake, and an increase in caspase3/7-dependent apoptosis. These changes were accompanied by increases in nuclear p53. Oligonucleotide arrays, coupled with use of a dominant-negative p53 construct in NT2 cells, identified 14 differentially regulated p53 target genes. In the early phases zinc deficiency, p53 targets responsible for cell cycle arrest were induced. Continuation of deficiency resulted in the induction of a variety of pro-apoptotic genes such as transforming growth factor-beta (TGF-beta) and retinoblastoma-1 (Rb-1), as well as cellular protection genes such as glutathione peroxidase (GPx). These data suggest that zinc plays a role in neurogenesis by regulating p53-dependent molecular mechanisms that control neuronal precursor cell proliferation and survival.

  6. Mechanism of parathyroid hormone-mediated suppression of calcification markers in human intervertebral disc cells.

    PubMed

    Madiraju, P; Gawri, R; Wang, H; Antoniou, J; Mwale, F

    2013-01-01

    In degenerative intervertebral discs (IVD), type X collagen (COL X) expression (associated with hypertrophic differentiation) and calcification has been demonstrated. Suppression of COL X expression and calcification during disc degeneration can be therapeutic. In the present study we investigated the potential of human parathyroid hormone 1-34 (PTH) in suppressing indicators of calcification potential (alkaline phosphatase (ALP), Ca(2+), inorganic phosphate (Pi)), and COL X expression. Further, we sought to elucidate the mechanism of PTH action in annulus fibrosus (AF) and nucleus pulposus (NP) cells from human lumbar IVDs with moderate to advanced degeneration. Mitogen activated protein kinase (MAPK) signalling and alterations in the markers of calcification potential were analysed. PTH increased type II collagen (COL II) expression in AF (~200 %) and NP cells (~163 %) and decreased COL X levels both in AF and NP cells (~75 %). These changes in the expression of collagens were preceded by MAPK phosphorylation, which was increased in both AF and NP cells by PTH after 30 min. MAPK signalling inhibitor U0126 and protein kinase-A inhibitor H-89 DCH attenuated PTH stimulated COL II expression in both cell types. PTH decreased ALP activity and increased Ca(2+) release only in NP cells. The present study demonstrates that PTH can potentially retard IVD degeneration by stimulating matrix synthesis and suppressing markers of calcification potential in degenerated disc cells via both MAPK and PKA signalling pathways. Inhibition of further mineral deposition may therefore be a viable therapeutic option for improving the status of degenerating discs.

  7. IFNγR signaling in non-T cell targets regulates T cell-mediated intestinal inflammation through multiple mechanisms

    PubMed Central

    Do, Jeong-su; Asosingh, Kewal; Baldwin, William M.; Min, Booki

    2014-01-01

    Naïve CD4 T cells transferred into lymphopenic mice undergo spontaneous proliferation and induce chronic inflammation in the intestine. Cellular mechanisms regulating the proliferative and inflammatory processes are not fully understood. In this study, we report that IFNγ signaling in host cells plays a major role in limiting both T cell expansion and T cell-induced intestinal inflammation. However, the role for IFNγ appears to be distinct depending on the target cells. IFNγ signaling in DCs controls T cell expansion, while IFNγ signaling in neutrophils seems to regulate both T cell expansion and inflammation. IFNγ signaling in non-hematopoietic cells may control inflammation. Therefore, our results suggest novel immunoregulatory functions for IFNγ to orchestrate colitogenic T cell responses through its distinct action on different non-T cell target cells. PMID:24523506

  8. A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells.

    PubMed

    Nabokina, Svetlana M; Said, Hamid M

    2012-08-01

    All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [(3)H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na(+)-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent K(m) = 0.157 ± 0.028 μM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca(2+)/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

  9. A complex mechanism for HDGF-mediated cell growth, migration, invasion, and TMZ chemosensitivity in glioma.

    PubMed

    Song, Ye; Hu, Zheng; Long, Hao; Peng, Yuping; Zhang, Xi'an; Que, Tianshi; Zheng, Shihao; Li, Zhiyong; Wang, Gang; Yi, Liu; Liu, Zhen; Fang, Weiyi; Qi, Songtao

    2014-09-01

    HDGF is overexpressed in gliomas as compared to normal brain. We therefore analyzed the molecular mechanisms of HDGF action in gliomas. HDGF was downregulated in normal brain tissue as compared to glioma specimens at both the mRNA and the protein levels. In glioma samples, increased HDGF expression was associated with disease progression. Knocking down HDGF expression not only significantly decreased cellular proliferation, migration, invasion, and tumorigenesis, but also markedly enhanced TMZ-induced cytotoxicity and apoptosis in glioma cells. Mechanistic analyses revealed that CCND1, c-myc, and TGF-β were downregulated after stable HDGF knockdown in the U251 and U87 glioma cells. HDGF knockdown restored E-cadherin expression and suppressed mesenchymal cell markers such as vimentin, β-catenin, and N-cadherin. The expression of cleaved caspase-3 increased, while Bcl-2 decreased in each cell line following treatment with shHDGF and TMZ, as compared to TMZ alone. Furthermore, RNAi-based knockdown study revealed that HDGF is probably involved in the activation of both the PI3K/Akt and the TGF-β signaling pathways. Together, our data suggested that HDGF regulates glioma cell growth, apoptosis and epithelial-mesenchymal transition (EMT) probably through the Akt and the TGF-β signaling pathways. These results provide evidence that targeting HDGF or its downstream targets may lead to novel therapies for gliomas.

  10. Mechanisms which mediate the antiapoptotic effects of angiopoietin-1 on endothelial cells.

    PubMed

    Harfouche, Rania; Hasséssian, Haroutioun M; Guo, Yang; Faivre, Valérie; Srikant, Coimbatore B; Yancopoulos, George D; Hussain, Sabah N A

    2002-07-01

    The main objective of this study was to identify molecular mechanisms through which angiopoietin-1 (Ang-1), a ligand for Tie-2 receptors, influences endothelial cell apoptosis. Human umbilical vein endothelial cells were cultured in a medium enriched with 2% fetal bovine serum (FBS) and growth supplements. Apoptosis was induced over 24 h by reducing FBS to 0.1%. Activation of caspase-9, -8, -7, and -3 and the expression of Bcl-2 family proteins, inhibitors of apoptosis (IAPs), cytochrome c, as well as Smac proteins were evaluated with immunoblotting. Ang-1 clearly attenuated serum deprivation-evoked apoptosis, an effect which required Tie-2 receptor activation. Activation of caspase-9, -7, and -3, but not caspase-8, was inhibited by Ang-1. The inhibitory effects of Ang-1 on apoptosis and caspase activation were reversed by a PI-3 kinase inhibitor (wortmannin). Ang-1 exposure upregulated the expression of Survivin but not XIAP (members of IAPs), reduced the cystosolic levels of Smac, but not that of cytochrome c, and had no effect on the expression of Bcl-2 family proteins. This is the first study to report on the mitochondrial mechanisms through which Ang-1 inhibits apoptosis and to investigate the role of the newly discovered Smac. We conclude that Ang-1 inhibits endothelial cell apoptosis through several pathways, which include PI-3 kinase/AKT activation, inhibition of Smac release from the mitochondria, and upregulation of Survivin protein.

  11. Amiloride-Insensitive Salt Taste Is Mediated by Two Populations of Type III Taste Cells with Distinct Transduction Mechanisms

    PubMed Central

    Sukumaran, Sunil K.; Margolskee, Robert F.; Bachmanov, Alexander A.

    2016-01-01

    Responses in the amiloride-insensitive (AI) pathway, one of the two pathways mediating salty taste in mammals, are modulated by the size of the anion of a salt. This “anion effect” has been hypothesized to result from inhibitory transepithelial potentials (TPs) generated across the lingual epithelium as cations permeate through tight junctions and leave their larger and less permeable anions behind (Ye et al., 1991). We tested directly the necessity of TPs for the anion effect by measuring responses to NaCl and Na-gluconate (small and large anion sodium salts, respectively) in isolated taste cells from mouse circumvallate papillae. Using calcium imaging, we identified AI salt-responsive type III taste cells and demonstrated that they compose a subpopulation of acid-responsive taste cells. Even in the absence of TPs, many (66%) AI salt-responsive type III taste cells still exhibited the anion effect, demonstrating that some component of the transduction machinery for salty taste in type III cells is sensitive to anion size. We hypothesized that osmotic responses could explain why a minority of type III cells (34%) had AI salt responses but lacked anion sensitivity. All AI type III cells had osmotic responses to cellobiose, which were significantly modulated by extracellular sodium concentration, suggesting the presence of a sodium-conducting osmotically sensitive ion channel. However, these responses were significantly larger in AI type III cells that did not exhibit the anion effect. These findings indicate that multiple mechanisms could underlie AI salt responses in type III taste cells, one of which may contribute to the anion effect. SIGNIFICANCE STATEMENT Understanding the mechanisms underlying salty taste will help inform strategies to combat the health problems associated with NaCl overconsumption by humans. Of the two pathways underlying salty taste in mammals, the amiloride-insensitive (AI) pathway is the least understood. Using calcium imaging of

  12. Cell type-dependent mechanisms for formin-mediated assembly of filopodia.

    PubMed

    Young, Lorna E; Heimsath, Ernest G; Higgs, Henry N

    2015-12-15

    Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex-nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.

  13. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells.

    PubMed

    Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena; Ma, Jianpeng; Wang, Qinghua

    2015-10-13

    Leiomodin (Lmod) is a class of potent tandem-G-actin-binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin-Lmod2162-495 nucleus. The structure contains two actin subunits connected by one Lmod2162-495 molecule in a non-filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin-binding nucleators. PMID:26417072

  14. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

    PubMed Central

    Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena; Ma, Jianpeng; Wang, Qinghua

    2015-01-01

    Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2162–495 nucleus. The structure contains two actin subunits connected by one Lmod2162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators. PMID:26417072

  15. Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel

    PubMed Central

    Desarnaud, Frank; Geck, Peter; Parkin, Christopher; Carpinito, Gino

    2011-01-01

    The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation. PMID:21057205

  16. The biochemical pathway mediating the proliferative response of bone cells to a mechanical stimulus.

    PubMed

    Brighton, C T; Fisher, J R; Levine, S E; Corsetti, J R; Reilly, T; Landsman, A S; Williams, J L; Thibault, L E

    1996-09-01

    Calvarial bone cells of rats were subjected to either a cyclic biaxial strain of 0.17 per cent (1700 microstrain) or a hydrostatic pressure of 2.5, five, or ten pounds per square inch (17.2, 34.5, or sixty-nine kilopascals). The frequency was held constant at one hertz for both types of mechanical stimulation. When cultured bone cells that had been subjected to a cyclic biaxial strain for two hours were harvested twenty-two hours later, it was found that the level of prostaglandin E2 had increased significantly (p < 0.01) as had cellular proliferation (p < 0.01), as indicated by the incorporation of [3H]-thymidine. The addition to the medium of indomethacin, an inhibitor of prostaglandin synthesis, at a ten-micromolar concentration significantly inhibited (p < 0.01) the increase in prostaglandin E2 synthesis but had no effect on the strain-induced increase in cellular proliferation, as indicated by the incorporation of [3H]-thymidine. Twenty-four hours after exposure to the same cyclic biaxial strain for thirty seconds, other cultured bone cells showed a significant increase in the level of cytoskeletal calmodulin (p < 0.05) and in the DNA content (p < 0.05). N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist, was added to the medium at a one-micromolar concentration, which had been shown to have no effect on the increase in the DNA content of control cells; W-7 completely blocked the increase in the level of cytoskeletal calmodulin and in the DNA content in the cells that were subjected to a cyclic biaxial strain. The bone cells subjected to a hydrostatic pressure showed a dose-dependent increase in the concentration of cytosolic Ca2+, as measured with Fura 2-AM, a fluorescent indicator of intracellular calcium. With a pressure of ten pounds per square inch (sixty-nine kilopascals), the increase in the concentration of cytosolic Ca2+ was nearly eight times greater than that at 2.5 pounds per square inch (17.2 kilopascals) (126

  17. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications

    PubMed Central

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods. PMID:26157424

  18. Hydrogels with Differential and Patterned Mechanics to Study Stiffness-Mediated Myofibroblastic Differentiation of Hepatic Stellate Cells

    PubMed Central

    Guvendiren, Murat; Perepelyuk, Maryna; Wells, Rebecca G.; Burdick, Jason A.

    2014-01-01

    The differentiation of hepatic stellate cells (HSCs) into myofbroblasts is a key event in liver fibrosis. Due to the local stiffening of the extracellular matrix (ECM) during fibrosis, it is of great interest to develop mimics that can be used to investigate the cellular response to changes in mechanics. Here, we used a step-wise hydrogel crosslinking technique, where macromolecules are crosslinked using a sequence of addition then UV light-mediated radical crosslinking, to generate hydrogels with tunable stiffness. Freshly isolated HSCs remained rounded with lipid droplets and high levels of PPARγ expression on soft substrates (E~2 kPa); however, HSCs spread, lost their lipid droplets, and expressed high levels of α-smooth muscle actin (α-SMA) and type I collagen on stiff substrates (E~ 24 kPa). Similarly, fully differentiated cells reverted to a quiescent state when plated on soft substrates. Stiffness-induced differentiation of HSCs was enhanced in the presence of exogenous TGF-β1, a dominant signal in fibrosis. When the UV-induced secondary crosslinking was restricted with a photomask to spatially control mechanics, HSCs responded based on the local hydrogel stiffness, although they remained quiescent on stiff substrates if the stiff feature size was not sufficient to allow cell spreading. This hydrogel system permits the investigation of HSC response to materials with diverse levels and spatially heterogeneous mechanical properties. PMID:24361340

  19. Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

    PubMed

    Gänzle, Michael; Liu, Yang

    2015-01-01

    High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

  20. New animal model to study epigenetic mechanisms mediating altered gravity effects upon cell growth and morphogenesis

    NASA Astrophysics Data System (ADS)

    Grigoryan, Eleonora N.; Dvorochkin, Natasha; Radugina, Elena A.; Poplinskaya, Valentina; Novikova, Julia; Almeida, Eduardo

    The gravitational field and its variations act as a major environmental factor that can impact morphogenesis developing through epigenetic molecular mechanisms. The mechanisms can be thoroughly investigated by using adequate animal models that reveal changes in the morpho-genesis of a growing organ as a function of gravitational effects. Two cooperative US/Russian experiments on Foton-M2 (2005) and Foton-M3 (2007) were the first to demonstrate differences in the shape of regenerating tails of space-flown and ground control newts. The space-flown and aquarium (simulated microgravity) animals developed lancet-shaped tails whereas 1 g con-trols (kept in space-type habitats) showed hook-like regenerates. These visual observations were supported by computer-aided processing of the images and statistical analysis of the results. Morphological examinations and cell proliferation measurements using BrdU demon-strated dorsal-ventral asymmetry as well as enhanced epithelial growth on the dorsal area of regenerating tails in 1 g newts. These findings were reproduced in laboratory tests on newts kept at 1 g and in large water tanks at cut g. The 1 g animals showed statistically significant deviations of the lancet-like tail shape typically seen in aquarium animals. Such modifications were found as early as regeneration stages III-IV and proved irreversible. The authors believe that the above phenomenon detected in newts used in many space experiments can serve as an adequate model for studying molecular mechanisms underlying gravitational effects upon animal morphogenesis.

  1. The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells.

    PubMed

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

    Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.

  2. The molecular mechanism of sensitization to Fas-mediated apoptosis by 2-methoxyestradiol in PC3 prostate cancer cells.

    PubMed

    Shimada, Keiji; Nakamura, Mitsutoshi; Ishida, Eiwa; Kishi, Munehiro; Matsuyoshi, Syuchi; Konishi, Noboru

    2004-01-01

    It is widely known that death receptor Fas-dependent apoptotic signals are associated with development of prostate cancer, but the key pathways involved in sensitivity to the apoptosis remain unclear. Here we investigated the molecular mechanism by which 2-methoxyestradiol (2-ME) effectively sensitizes a human prostate cancer cell line, PC3, to Fas-mediated apoptosis. 2-ME significantly inhibited nuclear factor-kappaB (NF-kappaB) activation and downregulated Fas-associated death domain (FADD) protein interluekin-1beta-converting enzyme inhibitory protein (FLIP). Overexpression of the dominant negative mutant form of IkappaBalpha (d/n IkappaBalpha) or treatment with Ikappa kinase-specific inhibitor Bay117082 gave the same results, although the sensitizing effect was not as pronounced. A selective inhibitor of Akt phosphorylation, LY294002, accelerated formation of the death-inducing signaling complex (DISC) not only by FLIP reduction but also by enhancement of recruitment of the FADD to Fas, thereby sensitizing PC3 cells to apoptosis similar to the case with 2-ME stimulation. Moreover, we found that inhibition of 2-ME-induced extracellular signal-regulated kinase (ERK) activation by the upstream kinase inhibitor PD98059 significantly enhanced 2-ME-mediated suppression of Akt activation, resulting in much greater sensitization to apoptosis. Taken together, the present findings indicate that 2-ME suppresses NF-kappaB/FLIP signaling and enhances DISC formation through inhibition of Akt, and that PC3 cells thereby are being sensitized to Fas-mediated apoptosis and by a process closely associated with ERK.

  3. Inhibition of cell proliferation: a mechanism likely to mediate the prevention of neuronal cell death by melatonin.

    PubMed

    Mayo, J C; Sainz, R M; Uría, H; Antolín, I; Estéban, M M; Rodríguez

    1998-08-01

    In a previous work we demonstrated that melatonin is able to prevent apoptosis induced by low doses of 6-hydroxydopamine (6-OHDA) in undifferentiated and neuronal PC12 cells. We also reported how this neurohormone was able to prevent the decrease in the mRNA for antioxidant enzymes caused by 6-OHDA. Although the antioxidant capability of melatonin seems to be clearly implicated in its antiapoptotic activity, literature suggests that its antiproliferative property could also be involved in its prevention of apoptosis. In the present work we demonstrated that melatonin is able to inhibit cell proliferation in undifferentiated PC12 cells, decreasing cell number and the total amount of DNA, and the mRNA for the histone H4, which are known to increase during DNA synthesis. Melatonin does not decrease the number of cells in nonproliferating PC12 cells, indicating that it does not cause cell death. Additionally, we demonstrate that other inhibitors of cell proliferation, as well as other antioxidants, are able to mimic the antiapoptotic effect of melatonin. This is interpreted to mean that melatonin acts by both mechanisms to inhibit apoptosis caused by 6-OHDA and the findings support the hypothesis of a relationship between oxidative stress and regulation of the cell cycle.

  4. Cytokines induce protein kinase A-mediated signalling by a redox-dependent mechanism in rat renal mesangial cells.

    PubMed

    Beck, Karl-Friedrich; Euler, Johannes; Eisel, Florian; Beck, Martina; Köhler, Yvette; Sha, Lisa Katharina; von Knethen, Andreas; Longen, Sebastian; Pfeilschifter, Josef

    2015-02-01

    Glomerular mesangial cells are smooth muscle cell-like pericytes and are regarded as key players in kidney diseases. In an inflammatory setting, these cells produce high amounts of inflammatory cytokines, chemokines and redox mediators such as reactive oxygen species or nitric oxide (NO). The temporal production of ROS, NO and other redox mediators markedly contributes to the final outcome of inflammatory diseases. Recently, we reported that platelet-derived growth factor forced mesangial cells to activate the regulatory subunit of protein kinase A (PKA RI) by a redox-dependent mechanism but independent from changes in cyclic AMP. This prompted us to further analyze the dimerization of PKA RI and activation of PKA-driven signalling in an inflammatory context. Stimulation of rat mesangial cells with interleukin-1β and tumour necrosis factor-α [2 nM] induced the formation of PKA RI heterodimers in a time-dependent manner. PKA RI dimerization was accompanied with the formation of ROS, NO and peroxynitrite as well as a depletion of reduced glutathione. Furthermore, dimerization of PKA RI was paralleled by enhanced activity of PKA as shown by the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 157 that was independent of the formation of cyclic AMP. Remarkably, exogenously administered peroxynitrite potently induced dimerization of PKA RI, whereas pharmacologic inhibition of inducible NO synthase (iNOS) and scavenging of peroxynitrite reduced PKA RI dimerization and VASP phosphorylation to control levels thus clearly indicating a causal role for endogenously formed peroxynitrite on PKA signalling. Consequently, the treatment of inflammatory diseases with anti-oxidants or NOS inhibitors may alter PKA activity.

  5. Intracellular mechanisms of hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells.

    PubMed

    Okayama, N; Coe, L; Oshima, T; Itoh, M; Alexander, J S

    1999-03-01

    We examined which endothelial second messengers are involved in peroxide-mediated endothelial-neutrophil adhesion with respect to endothelial P-selectin expression and platelet-activating factor (PAF). Peroxide (0.5 mM)-mediated adhesion was blocked by a protein kinase C (PKC) inhibitor, Gö6976 (10 nM); an intracellular calcium chelator, TMB-8 (0.1 mM); and a protein kinase G (PKG) inhibitor, KT5823 (0.5 microM); but not by a tyrosine kinase inhibitor, genistein (1 microM), or a protein kinase A inhibitor, H-89 (0.1 microM). These data were consistent with the proadhesive effects of PMA (0.1 microM), a PKC activator; a calcium ionophore, A23187 (1 microM); and dibutyryl cGMP (0.5 and 1 mM); but not phenylarsine oxide (0.1 mM), a tyrosine phosphatase inhibitor, or dibutyryl cAMP (1 mM). Conversely, peroxide-mediated P-selectin expression was blocked by Gö6976 and KT5823, but not by TMB-8. These data are strengthened by the observation that PMA and dibutyryl cGMP, but not A23187, increased P-selectin expression. WEB 2086 (10 microM), a PAF-receptor antagonist, blocked peroxide-, PMA-, and A23187-mediated adhesion, but not peroxide-mediated P-selectin expression. PAF itself (10 nM) stimulated adhesion, but not P-selectin expression. These data indicate that PKC and PKG are involved in peroxide-mediated neutrophil adhesion via P-selectin mobilization and PAF synthesis; however, intracellular calcium appears to mediate adhesion only through PAF synthesis.

  6. Induction of osteogenic differentiation of adipose derived stem cells by microstructured nitinol actuator-mediated mechanical stress.

    PubMed

    Strauß, Sarah; Dudziak, Sonja; Hagemann, Ronny; Barcikowski, Stephan; Fliess, Malte; Israelowitz, Meir; Kracht, Dietmar; Kuhbier, Jörn W; Radtke, Christine; Reimers, Kerstin; Vogt, Peter M

    2012-01-01

    The development of large tissue engineered bone remains a challenge in vitro, therefore the use of hybrid-implants might offer a bridge between tissue engineering and dense metal or ceramic implants. Especially the combination of the pseudoelastic implant material Nitinol (NiTi) with adipose derived stem cells (ASCs) opens new opportunities, as ASCs are able to differentiate osteogenically and therefore enhance osseointegration of implants. Due to limited knowledge about the effects of NiTi-structures manufactured by selective laser melting (SLM) on ASCs the study started with an evaluation of cytocompatibility followed by the investigation of the use of SLM-generated 3-dimensional NiTi-structures preseeded with ASCs as osteoimplant model. In this study we could demonstrate for the first time that osteogenic differentiation of ASCs can be induced by implant-mediated mechanical stimulation without support of osteogenic cell culture media. By use of an innovative implant design and synthesis via SLM-technique we achieved high rates of vital cells, proper osteogenic differentiation and mechanically loadable NiTi-scaffolds could be achieved.

  7. Agent-based modeling traction force mediated compaction of cell-populated collagen gels using physically realistic fibril mechanics.

    PubMed

    Reinhardt, James W; Gooch, Keith J

    2014-02-01

    Agent-based modeling was used to model collagen fibrils, composed of a string of nodes serially connected by links that act as Hookean springs. Bending mechanics are implemented as torsional springs that act upon each set of three serially connected nodes as a linear function of angular deflection about the central node. These fibrils were evaluated under conditions that simulated axial extension, simple three-point bending and an end-loaded cantilever. The deformation of fibrils under axial loading varied <0.001% from the analytical solution for linearly elastic fibrils. For fibrils between 100 μm and 200 μm in length experiencing small deflections, differences between simulated deflections and their analytical solutions were <1% for fibrils experiencing three-point bending and <7% for fibrils experiencing cantilever bending. When these new rules for fibril mechanics were introduced into a model that allowed for cross-linking of fibrils to form a network and the application of cell traction force, the fibrous network underwent macroscopic compaction and aligned between cells. Further, fibril density increased between cells to a greater extent than that observed macroscopically and appeared similar to matrical tracks that have been observed experimentally in cell-populated collagen gels. This behavior is consistent with observations in previous versions of the model that did not allow for the physically realistic simulation of fibril mechanics. The significance of the torsional spring constant value was then explored to determine its impact on remodeling of the simulated fibrous network. Although a stronger torsional spring constant reduced the degree of quantitative remodeling that occurred, the inclusion of torsional springs in the model was not necessary for the model to reproduce key qualitative aspects of remodeling, indicating that the presence of Hookean springs is essential for this behavior. These results suggest that traction force mediated matrix

  8. In vivo testing confirms a blunting of the human cell-mediated immune mechanism during space flight

    NASA Technical Reports Server (NTRS)

    Taylor, G. R.; Janney, R. P.

    1992-01-01

    The cell-mediated immune (CMI) mechanism was evaluated in 10 space shuttle astronauts by measuring their delayed-type hypersensitivity response to seven common recall antigens. The Multitest CMI test system was used to administer antigens of tetanus, diphtheria, Streptococcus, Proteus, old tuberculin, Candida, and Trichophyton to the forearm 46 h before nominal mission termination; readings were conducted 2 h after landing. The mean number of reactions was reduced from 4.5 preflight to 3.0 inflight, and the mean reaction score was reduced from 21.4 to 13.7 mm inflight. The data presented suggest that the CMI system is still being degraded by space flight conditions on day 4 and that between day 5 and day 10, the depression maximizes and the system begins to adjust to the new conditions. The relation of these in vivo findings to previously reported in vitro results is discussed.

  9. A Bax-mediated Mechanism for Obatoclax-induced Apoptosis of Cholangiocarcinoma Cells

    PubMed Central

    Smoot, Rory L.; Blechacz, Boris R.A.; Werneburg, Nathan W.; Bronk, Steve F.; Sinicrope, Frank A.; Sirica, Alphonse E.; Gores, Gregory J.

    2010-01-01

    Apoptosis induction by BH3 mimetics is a therapeutic strategy for human cancer. These mimetics exert single-agent activity in cells “primed” for cell death. Primed cells are dependent upon antiapoptotic Bcl-2 proteins for survival, and are characterized by the ability of the BH3-mimetic to induce cytochrome c release from their isolated mitochondria. Our aim was to examine the single-agent activity of obatoclax, a BH3 mimetic in cholangiocarcinoma cell lines. In clonogenic assays, inhibition of colony formation was observed by obatoclax treatment. Despite single-agent activity by obatoclax, the mitochondria from these cells did not release cytochrome c following incubation with this BH3 mimetic. However, immunofluorescence and cell fractionation studies identified Bax activation and translocation to mitochondria following treatment with obatoclax. shRNA targeted knockdown of Bax doubled the IC50 for obatoclax, but did not abrogate its cytotoxicity, while knockdown of Bak did not alter the IC50. In a cell free system, obatoclax induced an activating conformational change of Bax which was attenuated by site-directed mutagenesis of a previously identified protein activation site. Finally, the drug also elicited a significant in vivo response in a rodent model of this disease. In conclusion, single agent obatoclax treatment results in Bax activation which contributes, in part, to cell death in cholangiocarcinoma cells. These data indicate that BH3 mimetics may also function as direct activators of Bax and induce cytotoxicity in cells not otherwise primed for cell death. PMID:20160031

  10. Mitochondrial pathogenic mechanism and degradation in optineurin E50K mutation-mediated retinal ganglion cell degeneration

    PubMed Central

    Shim, Myoung Sup; Takihara, Yuji; Kim, Keun-Young; Iwata, Takeshi; Yue, Beatrice Y. J. T.; Inatani, Masaru; Weinreb, Robert N.; Perkins, Guy A.; Ju, Won-Kyu

    2016-01-01

    Mutations in optineurin (OPTN) are linked to the pathology of primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis. Emerging evidence indicates that OPTN mutation is involved in accumulation of damaged mitochondria and defective mitophagy. Nevertheless, the role played by an OPTN E50K mutation in the pathogenic mitochondrial mechanism that underlies retinal ganglion cell (RGC) degeneration in POAG remains unknown. We show here that E50K expression induces mitochondrial fission-mediated mitochondrial degradation and mitophagy in the axons of the glial lamina of aged E50K−tg mice in vivo. While E50K activates the Bax pathway and oxidative stress, and triggers dynamics alteration-mediated mitochondrial degradation and mitophagy in RGC somas in vitro, it does not affect transport dynamics and fission of mitochondria in RGC axons in vitro. These results strongly suggest that E50K is associated with mitochondrial dysfunction in RGC degeneration in synergy with environmental factors such as aging and/or oxidative stress. PMID:27654856

  11. Concurrent Phosphorus Recovery and Energy Generation in Mediator-Less Dual Chamber Microbial Fuel Cells: Mechanisms and Influencing Factors.

    PubMed

    Almatouq, Abdullah; Babatunde, Akintunde O

    2016-03-29

    This study investigated the mechanism and key factors influencing concurrent phosphorus (P) recovery and energy generation in microbial fuel cells (MFC) during wastewater treatment. Using a mediator-less dual chamber microbial fuel cell operated for 120 days; P was shown to precipitate as struvite when ammonium and magnesium chloride solutions were added to the cathode chamber. Monitoring data for chemical oxygen demand (COD), pH, oxidation reduction potential (ORP) and aeration flow rate showed that a maximum 38% P recovery was achieved; and this corresponds to 1.5 g/L, pH > 8, -550 ± 10 mV and 50 mL/min respectively, for COD, pH(cathode), ORP and cathode aeration flow rate. More importantly, COD and aeration flow rate were shown to be the key influencing factors for the P recovery and energy generation. Results further show that the maximum P recovery corresponds to 72 mW/m² power density. However, the energy generated at maximum P recovery was not the optimum; this shows that whilst P recovery and energy generation can be concurrently achieved in a microbial fuel cell, neither can be at the optimal value.

  12. Concurrent Phosphorus Recovery and Energy Generation in Mediator-Less Dual Chamber Microbial Fuel Cells: Mechanisms and Influencing Factors

    PubMed Central

    Almatouq, Abdullah; Babatunde, Akintunde O.

    2016-01-01

    This study investigated the mechanism and key factors influencing concurrent phosphorus (P) recovery and energy generation in microbial fuel cells (MFC) during wastewater treatment. Using a mediator-less dual chamber microbial fuel cell operated for 120 days; P was shown to precipitate as struvite when ammonium and magnesium chloride solutions were added to the cathode chamber. Monitoring data for chemical oxygen demand (COD), pH, oxidation reduction potential (ORP) and aeration flow rate showed that a maximum 38% P recovery was achieved; and this corresponds to 1.5 g/L, pH > 8, −550 ± 10 mV and 50 mL/min respectively, for COD, pHcathode, ORP and cathode aeration flow rate. More importantly, COD and aeration flow rate were shown to be the key influencing factors for the P recovery and energy generation. Results further show that the maximum P recovery corresponds to 72 mW/m2 power density. However, the energy generated at maximum P recovery was not the optimum; this shows that whilst P recovery and energy generation can be concurrently achieved in a microbial fuel cell, neither can be at the optimal value. PMID:27043584

  13. Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

    PubMed

    Sartorius, C A; Hanna, C T; Gril, B; Cruz, H; Serkova, N J; Huber, K M; Kabos, P; Schedin, T B; Borges, V F; Steeg, P S; Cittelly, D M

    2016-06-01

    Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These

  14. Mechanism of T cell mediated protection in newborn mice against a Chlamydia infection

    PubMed Central

    Pal, Sukumar; de la Maza, Luis M.

    2013-01-01

    To determine the immune components needed for protection of newborn mice against Chlamydia muridarum, animals born to Chlamydia-immunized and to sham-immunized dams were infected intranasally with C. muridarum at 2 post-natal days. T-cells isolated from immunized or sham-immunized adult mice were adoptively transferred to newborn mice at the time of infection. Also, to establish what cytokines are involved in protection, IFN-γ TNF-α, IL-10, and IL-12 were passively transferred to newborn mice. To assess the Chlamydia burden in the lungs mice were euthanized at 12 post-natal days. When T-cells from immunized adult mice were transferred, mice born to and fed by immunized dams were significantly protected as evidenced by the reduced number of Chlamydia isolated from the lungs compared to mice born to and fed by sham-immunized dams. Transfer of IFN-γ and TNF-α also significantly reduced the number of Chlamydia in the lungs of mice born to immunized dams. Transfer of IL-10 or IL-12 did not result in a significant reduction of Chlamydia. In vitro T-cell proliferation data suggest that neonatal antigen presenting cells can present Chlamydia antigens to adult T-cells. In conclusion, maternal antibodies and Chlamydia specific T-cells or Th1 cytokines are required for protection of neonates against this pathogen. PMID:23644176

  15. Mechanism of T-cell mediated protection in newborn mice against a Chlamydia infection.

    PubMed

    Pal, Sukumar; de la Maza, Luis M

    2013-01-01

    To determine the immune components needed for protection of newborn mice against Chlamydia muridarum, animals born to Chlamydia-immunized and to sham-immunized dams were infected intranasally with C. muridarum at 2 post-natal days. T-cells isolated from immunized or sham-immunized adult mice were adoptively transferred to newborn mice at the time of infection. Also, to establish what cytokines are involved in protection, IFN-γ, TNF-α, IL-10, and IL-12 were passively transferred to newborn mice. To assess the Chlamydia burden in the lungs mice were euthanized at 12 post-natal days. When T-cells from immunized adult mice were transferred, mice born to and fed by immunized dams were significantly protected as evidenced by the reduced number of Chlamydia isolated from the lungs compared to mice born to and fed by sham-immunized dams. Transfer of IFN-γ and TNF-α also significantly reduced the number of Chlamydia in the lungs of mice born to immunized dams. Transfer of IL-10 or IL-12 did not result in a significant reduction of Chlamydia. In vitro T-cell proliferation data suggest that neonatal antigen presenting cells can present Chlamydia antigens to adult T-cells. In conclusion, maternal antibodies and Chlamydia specific T-cells or Th1 cytokines are required for protection of neonates against this pathogen.

  16. Microglial VPAC1R mediates a novel mechanism of neuroimmune-modulation of hippocampal precursor cells via IL-4 release

    PubMed Central

    Nunan, Robert; Sivasathiaseelan, Harri; Khan, Damla; Zaben, Malik; Gray, William

    2014-01-01

    Neurogenesis, the production of new neurons from neural stem/progenitor cells (NSPCs), occurs throughout adulthood in the dentate gyrus of the hippocampus, where it supports learning and memory. The innate and adaptive immune systems are increasingly recognized as important modulators of hippocampal neurogenesis under both physiological and pathological conditions. However, the mechanisms by which the immune system regulates hippocampal neurogenesis are incompletely understood. In particular, the role of microglia, the brains resident immune cell is complex, as they have been reported to both positively and negatively regulate neurogenesis. Interestingly, neuronal activity can also regulate the function of the immune system. Here, we show that depleting microglia from hippocampal cultures reduces NSPC survival and proliferation. Furthermore, addition of purified hippocampal microglia, or their conditioned media, is trophic and proliferative to NSPCs. VIP, a neuropeptide released by dentate gyrus interneurons, enhances the proliferative and pro-neurogenic effect of microglia via the VPAC1 receptor. This VIP-induced enhancement is mediated by IL-4 release, which directly targets NSPCs. This demonstrates a potential neuro-immuno-neurogenic pathway, disruption of which may have significant implications in conditions where combined cognitive impairments, interneuron loss, and immune system activation occurs, such as temporal lobe epilepsy and Alzheimer's disease. PMID:24801739

  17. Microglial VPAC1R mediates a novel mechanism of neuroimmune-modulation of hippocampal precursor cells via IL-4 release.

    PubMed

    Nunan, Robert; Sivasathiaseelan, Harri; Khan, Damla; Zaben, Malik; Gray, William

    2014-08-01

    Neurogenesis, the production of new neurons from neural stem/progenitor cells (NSPCs), occurs throughout adulthood in the dentate gyrus of the hippocampus, where it supports learning and memory. The innate and adaptive immune systems are increasingly recognized as important modulators of hippocampal neurogenesis under both physiological and pathological conditions. However, the mechanisms by which the immune system regulates hippocampal neurogenesis are incompletely understood. In particular, the role of microglia, the brains resident immune cell is complex, as they have been reported to both positively and negatively regulate neurogenesis. Interestingly, neuronal activity can also regulate the function of the immune system. Here, we show that depleting microglia from hippocampal cultures reduces NSPC survival and proliferation. Furthermore, addition of purified hippocampal microglia, or their conditioned media, is trophic and proliferative to NSPCs. VIP, a neuropeptide released by dentate gyrus interneurons, enhances the proliferative and pro-neurogenic effect of microglia via the VPAC1 receptor. This VIP-induced enhancement is mediated by IL-4 release, which directly targets NSPCs. This demonstrates a potential neuro-immuno-neurogenic pathway, disruption of which may have significant implications in conditions where combined cognitive impairments, interneuron loss, and immune system activation occurs, such as temporal lobe epilepsy and Alzheimer's disease.

  18. Effective killing of Gleevec-resistant CML cells with T315I mutation by a natural compound PEITC through redox-mediated mechanism

    PubMed Central

    Zhang, H; Trachootham, D; Lu, W; Carew, J; Giles, FJ; Keating, MJ; Arlinghaus, RB; Huang, P

    2008-01-01

    Mutation of Bcr-Abl is an important mechanism by which chronic myelogenous leukemia (CML) cells become resistant to Gleevec. The T315I mutation is clinically significant since CML cells harboring this mutation are insensitive to Gleevec and other Bcr-Abl-targeted drugs. Identification of new agents capable of effectively killing CML cells with T315I mutation would have important therapeutic implications in Gleevec-resistant CML. Here, we showed that β-phenylethyl isothiocyanate (PEITC), a natural compound found in vegetables, is effective in killing CML cells expressing T315I BCR-ABL. Treatment of leukemia cell lines harboring wild-type or mutant Bcr-Abl with 10 μm PEITC resulted in an elevated ROS stress and a redox-mediated degradation of the BCR-ABL protein, leading to massive death of the leukemia cells. Antioxidant NAC attenuated the PEITC-induced oxidative stress in CML cells and prevented the degradation of BCR-ABL, caspase-3 activation and cell death. We further showed that the ROS-induced degradation of BCR-ABL was mediated partially by caspase-3 and the proteasome pathway. The ability of PEITC to effectively kill T315I-positive CML cells was further confirmed using primary leukemia cells isolated from CML patients. Our results suggest that PEITC is a promising compound capable of killing Gleevec-resistant CML cells through a ROS-mediated mechanism and warrants further investigations. PMID:18385754

  19. Diabetes reduces mesenchymal stem cells in fracture healing through a TNFα-mediated mechanism

    PubMed Central

    Tian, Chen; Alblowi, Jazia; Kayal, Rayyan A.; Einhorn, Thomas A.; Gerstenfeld, Louis C.; Pignolo, Robert J.; Graves, Dana T.

    2015-01-01

    Aims/hypothesis Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair. Methods Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNFα inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA. Results Diabetes significantly increased TNFα levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNFα significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNFα alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNFα-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes. Conclusions/interpretation Diabetes-enhanced TNFα significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNFα reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNFα in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients. PMID:25563724

  20. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis

    PubMed Central

    Sawcer, Stephen; Hellenthal, Garrett; Pirinen, Matti; Spencer, Chris C.A.; Patsopoulos, Nikolaos A.; Moutsianas, Loukas; Dilthey, Alexander; Su, Zhan; Freeman, Colin; Hunt, Sarah E.; Edkins, Sarah; Gray, Emma; Booth, David R.; Potter, Simon C.; Goris, An; Band, Gavin; Oturai, Annette Bang; Strange, Amy; Saarela, Janna; Bellenguez, Céline; Fontaine, Bertrand; Gillman, Matthew; Hemmer, Bernhard; Gwilliam, Rhian; Zipp, Frauke; Jayakumar, Alagurevathi; Martin, Roland; Leslie, Stephen; Hawkins, Stanley; Giannoulatou, Eleni; D’alfonso, Sandra; Blackburn, Hannah; Boneschi, Filippo Martinelli; Liddle, Jennifer; Harbo, Hanne F.; Perez, Marc L.; Spurkland, Anne; Waller, Matthew J; Mycko, Marcin P.; Ricketts, Michelle; Comabella, Manuel; Hammond, Naomi; Kockum, Ingrid; McCann, Owen T.; Ban, Maria; Whittaker, Pamela; Kemppinen, Anu; Weston, Paul; Hawkins, Clive; Widaa, Sara; Zajicek, John; Dronov, Serge; Robertson, Neil; Bumpstead, Suzannah J.; Barcellos, Lisa F.; Ravindrarajah, Rathi; Abraham, Roby; Alfredsson, Lars; Ardlie, Kristin; Aubin, Cristin; Baker, Amie; Baker, Katharine; Baranzini, Sergio E.; Bergamaschi, Laura; Bergamaschi, Roberto; Bernstein, Allan; Berthele, Achim; Boggild, Mike; Bradfield, Jonathan P.; Brassat, David; Broadley, Simon A.; Buck, Dorothea; Butzkueven, Helmut; Capra, Ruggero; Carroll, William M.; Cavalla, Paola; Celius, Elisabeth G.; Cepok, Sabine; Chiavacci, Rosetta; Clerget-Darpoux, Françoise; Clysters, Katleen; Comi, Giancarlo; Cossburn, Mark; Cournu-Rebeix, Isabelle; Cox, Mathew B.; Cozen, Wendy; Cree, Bruce A.C.; Cross, Anne H.; Cusi, Daniele; Daly, Mark J.; Davis, Emma; de Bakker, Paul I.W.; Debouverie, Marc; D’hooghe, Marie Beatrice; Dixon, Katherine; Dobosi, Rita; Dubois, Bénédicte; Ellinghaus, David; Elovaara, Irina; Esposito, Federica; Fontenille, Claire; Foote, Simon; Franke, Andre; Galimberti, Daniela; Ghezzi, Angelo; Glessner, Joseph; Gomez, Refujia; Gout, Olivier; Graham, Colin; Grant, Struan F.A.; Guerini, Franca Rosa; Hakonarson, Hakon; Hall, Per; Hamsten, Anders; Hartung, Hans-Peter; Heard, Rob N.; Heath, Simon; Hobart, Jeremy; Hoshi, Muna; Infante-Duarte, Carmen; Ingram, Gillian; Ingram, Wendy; Islam, Talat; Jagodic, Maja; Kabesch, Michael; Kermode, Allan G.; Kilpatrick, Trevor J.; Kim, Cecilia; Klopp, Norman; Koivisto, Keijo; Larsson, Malin; Lathrop, Mark; Lechner-Scott, Jeannette S.; Leone, Maurizio A.; Leppä, Virpi; Liljedahl, Ulrika; Bomfim, Izaura Lima; Lincoln, Robin R.; Link, Jenny; Liu, Jianjun; Lorentzen, Åslaug R.; Lupoli, Sara; Macciardi, Fabio; Mack, Thomas; Marriott, Mark; Martinelli, Vittorio; Mason, Deborah; McCauley, Jacob L.; Mentch, Frank; Mero, Inger-Lise; Mihalova, Tania; Montalban, Xavier; Mottershead, John; Myhr, Kjell-Morten; Naldi, Paola; Ollier, William; Page, Alison; Palotie, Aarno; Pelletier, Jean; Piccio, Laura; Pickersgill, Trevor; Piehl, Fredrik; Pobywajlo, Susan; Quach, Hong L.; Ramsay, Patricia P.; Reunanen, Mauri; Reynolds, Richard; Rioux, John D.; Rodegher, Mariaemma; Roesner, Sabine; Rubio, Justin P.; Rückert, Ina-Maria; Salvetti, Marco; Salvi, Erika; Santaniello, Adam; Schaefer, Catherine A.; Schreiber, Stefan; Schulze, Christian; Scott, Rodney J.; Sellebjerg, Finn; Selmaj, Krzysztof W.; Sexton, David; Shen, Ling; Simms-Acuna, Brigid; Skidmore, Sheila; Sleiman, Patrick M.A.; Smestad, Cathrine; Sørensen, Per Soelberg; Søndergaard, Helle Bach; Stankovich, Jim; Strange, Richard C.; Sulonen, Anna-Maija; Sundqvist, Emilie; Syvänen, Ann-Christine; Taddeo, Francesca; Taylor, Bruce; Blackwell, Jenefer M.; Tienari, Pentti; Bramon, Elvira; Tourbah, Ayman; Brown, Matthew A.; Tronczynska, Ewa; Casas, Juan P.; Tubridy, Niall; Corvin, Aiden; Vickery, Jane; Jankowski, Janusz; Villoslada, Pablo; Markus, Hugh S.; Wang, Kai; Mathew, Christopher G.; Wason, James; Palmer, Colin N.A.; Wichmann, H-Erich; Plomin, Robert; Willoughby, Ernest; Rautanen, Anna; Winkelmann, Juliane; Wittig, Michael; Trembath, Richard C.; Yaouanq, Jacqueline; Viswanathan, Ananth C.; Zhang, Haitao; Wood, Nicholas W.; Zuvich, Rebecca; Deloukas, Panos; Langford, Cordelia; Duncanson, Audrey; Oksenberg, Jorge R.; Pericak-Vance, Margaret A.; Haines, Jonathan L.; Olsson, Tomas; Hillert, Jan; Ivinson, Adrian J.; De Jager, Philip L.; Peltonen, Leena; Stewart, Graeme J.; Hafler, David A.; Hauser, Stephen L.; McVean, Gil; Donnelly, Peter; Compston, Alastair

    2011-01-01

    Multiple sclerosis (OMIM 126200) is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability.1 Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals;2,3 and systematic attempts to identify linkage in multiplex families have confirmed that variation within the Major Histocompatibility Complex (MHC) exerts the greatest individual effect on risk.4 Modestly powered Genome-Wide Association Studies (GWAS)5-10 have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects play a key role in disease susceptibility.11 Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the Class I region. Immunologically relevant genes are significantly over-represented amongst those mapping close to the identified loci and particularly implicate T helper cell differentiation in the pathogenesis of multiple sclerosis. PMID:21833088

  1. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

    PubMed

    Sawcer, Stephen; Hellenthal, Garrett; Pirinen, Matti; Spencer, Chris C A; Patsopoulos, Nikolaos A; Moutsianas, Loukas; Dilthey, Alexander; Su, Zhan; Freeman, Colin; Hunt, Sarah E; Edkins, Sarah; Gray, Emma; Booth, David R; Potter, Simon C; Goris, An; Band, Gavin; Oturai, Annette Bang; Strange, Amy; Saarela, Janna; Bellenguez, Céline; Fontaine, Bertrand; Gillman, Matthew; Hemmer, Bernhard; Gwilliam, Rhian; Zipp, Frauke; Jayakumar, Alagurevathi; Martin, Roland; Leslie, Stephen; Hawkins, Stanley; Giannoulatou, Eleni; D'alfonso, Sandra; Blackburn, Hannah; Martinelli Boneschi, Filippo; Liddle, Jennifer; Harbo, Hanne F; Perez, Marc L; Spurkland, Anne; Waller, Matthew J; Mycko, Marcin P; Ricketts, Michelle; Comabella, Manuel; Hammond, Naomi; Kockum, Ingrid; McCann, Owen T; Ban, Maria; Whittaker, Pamela; Kemppinen, Anu; Weston, Paul; Hawkins, Clive; Widaa, Sara; Zajicek, John; Dronov, Serge; Robertson, Neil; Bumpstead, Suzannah J; Barcellos, Lisa F; Ravindrarajah, Rathi; Abraham, Roby; Alfredsson, Lars; Ardlie, Kristin; Aubin, Cristin; Baker, Amie; Baker, Katharine; Baranzini, Sergio E; Bergamaschi, Laura; Bergamaschi, Roberto; Bernstein, Allan; Berthele, Achim; Boggild, Mike; Bradfield, Jonathan P; Brassat, David; Broadley, Simon A; Buck, Dorothea; Butzkueven, Helmut; Capra, Ruggero; Carroll, William M; Cavalla, Paola; Celius, Elisabeth G; Cepok, Sabine; Chiavacci, Rosetta; Clerget-Darpoux, Françoise; Clysters, Katleen; Comi, Giancarlo; Cossburn, Mark; Cournu-Rebeix, Isabelle; Cox, Mathew B; Cozen, Wendy; Cree, Bruce A C; Cross, Anne H; Cusi, Daniele; Daly, Mark J; Davis, Emma; de Bakker, Paul I W; Debouverie, Marc; D'hooghe, Marie Beatrice; Dixon, Katherine; Dobosi, Rita; Dubois, Bénédicte; Ellinghaus, David; Elovaara, Irina; Esposito, Federica; Fontenille, Claire; Foote, Simon; Franke, Andre; Galimberti, Daniela; Ghezzi, Angelo; Glessner, Joseph; Gomez, Refujia; Gout, Olivier; Graham, Colin; Grant, Struan F A; Guerini, Franca Rosa; Hakonarson, Hakon; Hall, Per; Hamsten, Anders; Hartung, Hans-Peter; Heard, Rob N; Heath, Simon; Hobart, Jeremy; Hoshi, Muna; Infante-Duarte, Carmen; Ingram, Gillian; Ingram, Wendy; Islam, Talat; Jagodic, Maja; Kabesch, Michael; Kermode, Allan G; Kilpatrick, Trevor J; Kim, Cecilia; Klopp, Norman; Koivisto, Keijo; Larsson, Malin; Lathrop, Mark; Lechner-Scott, Jeannette S; Leone, Maurizio A; Leppä, Virpi; Liljedahl, Ulrika; Bomfim, Izaura Lima; Lincoln, Robin R; Link, Jenny; Liu, Jianjun; Lorentzen, Aslaug R; Lupoli, Sara; Macciardi, Fabio; Mack, Thomas; Marriott, Mark; Martinelli, Vittorio; Mason, Deborah; McCauley, Jacob L; Mentch, Frank; Mero, Inger-Lise; Mihalova, Tania; Montalban, Xavier; Mottershead, John; Myhr, Kjell-Morten; Naldi, Paola; Ollier, William; Page, Alison; Palotie, Aarno; Pelletier, Jean; Piccio, Laura; Pickersgill, Trevor; Piehl, Fredrik; Pobywajlo, Susan; Quach, Hong L; Ramsay, Patricia P; Reunanen, Mauri; Reynolds, Richard; Rioux, John D; Rodegher, Mariaemma; Roesner, Sabine; Rubio, Justin P; Rückert, Ina-Maria; Salvetti, Marco; Salvi, Erika; Santaniello, Adam; Schaefer, Catherine A; Schreiber, Stefan; Schulze, Christian; Scott, Rodney J; Sellebjerg, Finn; Selmaj, Krzysztof W; Sexton, David; Shen, Ling; Simms-Acuna, Brigid; Skidmore, Sheila; Sleiman, Patrick M A; Smestad, Cathrine; Sørensen, Per Soelberg; Søndergaard, Helle Bach; Stankovich, Jim; Strange, Richard C; Sulonen, Anna-Maija; Sundqvist, Emilie; Syvänen, Ann-Christine; Taddeo, Francesca; Taylor, Bruce; Blackwell, Jenefer M; Tienari, Pentti; Bramon, Elvira; Tourbah, Ayman; Brown, Matthew A; Tronczynska, Ewa; Casas, Juan P; Tubridy, Niall; Corvin, Aiden; Vickery, Jane; Jankowski, Janusz; Villoslada, Pablo; Markus, Hugh S; Wang, Kai; Mathew, Christopher G; Wason, James; Palmer, Colin N A; Wichmann, H-Erich; Plomin, Robert; Willoughby, Ernest; Rautanen, Anna; Winkelmann, Juliane; Wittig, Michael; Trembath, Richard C; Yaouanq, Jacqueline; Viswanathan, Ananth C; Zhang, Haitao; Wood, Nicholas W; Zuvich, Rebecca; Deloukas, Panos; Langford, Cordelia; Duncanson, Audrey; Oksenberg, Jorge R; Pericak-Vance, Margaret A; Haines, Jonathan L; Olsson, Tomas; Hillert, Jan; Ivinson, Adrian J; De Jager, Philip L; Peltonen, Leena; Stewart, Graeme J; Hafler, David A; Hauser, Stephen L; McVean, Gil; Donnelly, Peter; Compston, Alastair

    2011-08-11

    Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

  2. Nonsense-mediated mRNA decay - mechanisms of substrate mRNA recognition and degradation in mammalian cells.

    PubMed

    Schweingruber, Christoph; Rufener, Simone C; Zünd, David; Yamashita, Akio; Mühlemann, Oliver

    2013-01-01

    The nonsense-mediated mRNA decay (NMD) pathway is well known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with truncated open reading frames (ORF) due to the presence of a premature termination codon (PTC). However, a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD targets. In this review, we focus on mechanistic aspects of target mRNA identification and degradation in mammalian cells, based on the available biochemical and genetic data, and point out knowledge gaps. Translation termination in a messenger ribonucleoprotein particle (mRNP) environment lacking necessary factors for proper translation termination emerges as a key determinant for subjecting an mRNA to NMD, and we therefore review recent structural and mechanistic insight into translation termination. In addition, the central role of UPF1, its crucial phosphorylation/dephosphorylation cycle and dynamic interactions with other NMD factors are discussed. Moreover, we address the role of exon junction complexes (EJCs) in NMD and summarize the functions of SMG5, SMG6 and SMG7 in promoting mRNA decay through different routes. This article is part of a Special Issue entitled: RNA Decay mechanisms.

  3. A novel profibrotic mechanism mediated by TGF-β-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells

    PubMed Central

    Luo, Yongfeng; Xu, Wei; Chen, Hui; Warburton, David; Dong, Rachel; Qian, Bangping; Selman, Moisés; Gauldie, Jack; Kolb, Martin; Shi, Wei

    2015-01-01

    Idiopathic pulmonary fibrosis is a severe chronic lung disease with a high mortality rate. Excessive TGF-β signaling is recognized as a central player in lung fibrosis. However, the related mechanisms remain unclear. Herein we used a novel Tbx4 lung enhancer-driven Tet-On transgenic system to inhibit TGF-β signaling in mouse lung resident mesenchymal cells at different stages of bleomycin-induced fibrosis by conditionally knocking out TGF-β receptor II or expressing a dominant-negative TGF-β receptor II. Abrogation of mesenchymal TGF-β signaling markedly attenuated bleomycin-induced fibrotic pathology, which was independent of altered early inflammation. Furthermore, a novel TGF-β downstream target gene P4HA3 (an α-subunit of collagen prolyl hydroxylase) was identified, and its expression was significantly increased in fibroblastic foci of both bleomycin-induced fibrotic mouse lungs and idiopathic pulmonary fibrosis patients’ lungs. The relationship between activated TGF-β signaling, upregulation of P4HA3, as well as increased hydroxyproline/collagen production was further verified in cultured lung fibroblasts. Moreover, inhibition of collagen prolyl hydroxylase by pyridine-2,5-dicarboxylate attenuated both TGF-β-stimulated collagen production in cultured fibroblasts and bleomycin-induced mouse lung fibrosis. These data indicate that increased expression and activity of collagen prolyl hydroxylase is one of the important mechanisms underlying TGF-β-mediated profibrotic effects. Inhibition of collagen prolyl hydroxylase may be a new promising approach for preventing and treating pulmonary fibrosis. PMID:25779936

  4. Oxidative stress and mitochondria-mediated cell death mechanisms triggered by the familial Danish dementia ADan amyloid.

    PubMed

    Todd, Krysti; Ghiso, Jorge; Rostagno, Agueda

    2016-01-01

    Familial Danish Dementia (FDD), an early-onset non-amyloid-β (Aβ) cerebral amyloidosis, is neuropathologically characterized by widespread cerebral amyloid angiopathy, parenchymal amyloid and preamyloid deposits, as well as neurofibrillary degeneration indistinguishable to that seen in Alzheimer's disease (AD). The main amyloid subunit composing FDD lesions, a 34-amino acid de-novo generated peptide ADan, is the direct result of a genetic defect at the 3'-end of the BRI2 gene and the physiologic action of furin-like proteolytic processing at the C-terminal region of the ADan precursor protein. We aimed to study the impact of the FDD mutation, the additional formation of the pyroglutamate (pE) posttranslational modification as well as the relevance of C-terminal truncations -all major components of the heterogeneous FDD deposits- on the structural and neurotoxic properties of the molecule. Our data indicates that whereas the mutation generated a β-sheet-rich hydrophobic ADan subunit of high oligomerization/fibrillization propensity and the pE modification further enhanced these properties, C-terminal truncations had the opposite effect mostly abolishing these features. The potentiation of pro-amyloidogenic properties correlated with the initiation of neuronal cell death mechanisms involving oxidative stress, perturbation of mitochondrial membrane potential, release of mitochondrial cytochrome c, and downstream activation of caspase-mediated apoptotic pathways. The amyloid-induced toxicity was inhibited by targeting specific components of these detrimental cellular pathways, using reactive oxygen scavengers and monoclonal antibodies recognizing the pathological amyloid subunit. Taken together, the data indicate that the FDD mutation and the pE posttranslational modification are both primary elements driving intact ADan into an amyloidogenic/neurotoxic pathway while truncations at the C-terminus eliminate the pro-amyloidogenic characteristics of the molecule

  5. Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

    PubMed Central

    Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth AS; Nallasamy, Palanisamy; Zhu, Hong

    2015-01-01

    Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity. PMID:25504014

  6. Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

    PubMed

    Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth A S; Nallasamy, Palanisamy; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

    2015-10-01

    Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.

  7. Distinct kinetic and mechanical properties govern mucin 16- and podocalyxin-mediated tumor cell adhesion to E- and L-selectin in shear flow

    PubMed Central

    Shea, Daniel J.; Wirtz, Denis; Stebe, Kathleen J.; Konstantopoulos, Konstantinos

    2015-01-01

    Selectin-mediated tumor cell tethering to host cells, such as vascular endothelial cells, is a critical step in the process of cancer metastasis. We recently identified sialofucosylated mucin16 (MUC16) and podocalyxin (PODXL) as the major functional E- and L-selectin ligands expressed on the surface of metastatic pancreatic cancer cells. While the biophysics of leukocyte binding to selectins has been well studied, little is known about the mechanics of selectin-mediated adhesion pertinent to cancer metastasis. We thus sought to evaluate the critical parameters of selectin-mediated pancreatic tumor cell tethering and rolling. Using force spectroscopy, we characterized the binding interactions of MUC16 and PODXL to E- and L-selectin at the single-molecule level. To further analyze the response of these molecular interactions under physiologically relevant regimes, we used a microfluidic assay in conjunction with a mathematical model to study the biophysics of selectin-ligand binding as a function of fluid shear stress. We demonstrate that both MUC16 and PODXL-E-selectin-mediated interactions are mechanically stronger than like L-selectin interactions at the single-molecule level, and display a higher binding frequency at all contact times. The single-molecule kinetic and micromechanical properties of selectin-ligand bonds, along with the number of receptor-ligand bonds needed to initiate tethering, regulate the average velocity of ligand-coated microspheres rolling on selectin-coated surfaces in shear flow. Understanding the biophysics of selectin-ligand bonds and their responses to physiologically relevant shear stresses is vital for developing diagnostic assays and/or preventing the metastatic spread of tumor cells by interfering with selectin-mediated adhesion. PMID:26329844

  8. Cell-Mediated Drugs Delivery

    PubMed Central

    Batrakova, Elena V.; Gendelman, Howard E.; Kabanov, Alexander V.

    2011-01-01

    INTRODUCTION Drug targeting to sites of tissue injury, tumor or infection with limited toxicity is the goal for successful pharmaceutics. Immunocytes (including mononuclear phagocytes (dendritic cells, monocytes and macrophages), neutrophils, and lymphocytes) are highly mobile; they can migrate across impermeable barriers and release their drug cargo at sites of infection or tissue injury. Thus immune cells can be exploited as trojan horses for drug delivery. AREAS COVERED IN THIS REVIEW This paper reviews how immunocytes laden with drugs can cross the blood brain or blood tumor barriers, to facilitate treatments for infectious diseases, injury, cancer, or inflammatory diseases. The promises and perils of cell-mediated drug delivery are reviewed, with examples of how immunocytes can be harnessed to improve therapeutic end points. EXPERT OPINION Using cells as delivery vehicles enables targeted drug transport, and prolonged circulation times, along with reductions in cell and tissue toxicities. Such systems for drug carriage and targeted release represent a novel disease combating strategy being applied to a spectrum of human disorders. The design of nanocarriers for cell-mediated drug delivery may differ from those used for conventional drug delivery systems; nevertheless, engaging different defense mechanisms into drug delivery may open new perspectives for the active delivery of drugs. PMID:21348773

  9. Molecular mechanisms involved in TFF3 peptide-mediated modulation of the E-cadherin/catenin cell adhesion complex.

    PubMed

    Meyer zum Büschenfelde, Dirk; Hoschützky, Heinz; Tauber, Rudolf; Huber, Otmar

    2004-05-01

    TFF3 is a member of the TFF-domain peptide family which is constitutively expressed in mucous epithelial tissues where it acts as a motogenic factor and plays an important role during epithelial restitution after wounding and during inflammation. In contrast to these beneficial functions, TFFs were also reported to be involved in cell scattering and tumor invasion. These changes in epithelial cell morphology and motility are associated with a modulation of cell contacts. In this respect, we here investigated the E-cadherin/catenin cell adhesion complex in FLAG-hTFF3-transfected HT29/B6 and MDCK cells. In hTFF3-transfected cells the amount of E-cadherin is reduced with a concomitant reduction of alpha- and beta-catenin levels. On one hand, E-cadherin expression is lowered at the transcriptional level as shown by multiplex RT-PCR analysis. This decrease does not depend on differences in the promoter methylation status as shown by methylation-specific PCR. On the other hand, pulse-chase experiments showed a reduction in the E-cadherin half-life in hTFF3-transfected cells reflecting increased E-cadherin degradation. In summary, hTFF3 induces transcriptional and posttranslational processes resulting in a modulation of E-cadherin-mediated cell-cell contacts that may play an important role in the paradoxical benefical and pathogenic function of TFF peptides.

  10. Molecular mechanisms regulating CD13-mediated adhesion

    PubMed Central

    Ghosh, Mallika; Gerber, Claire; Rahman, M Mamunur; Vernier, Kaitlyn M; Pereira, Flavia E; Subramani, Jaganathan; Caromile, Leslie A; Shapiro, Linda H

    2014-01-01

    CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is expressed on myeloid cells, is up-regulated on endothelial cells at sites of inflammation and mediates monocyte/endothelial adhesion by homotypic interactions. In animal models the lack of CD13 alters the profiles of infiltrating inflammatory cells at sites of ischaemic injury. Here, we found that CD13 expression is enriched specifically on the pro-inflammatory subset of monocytes, suggesting that CD13 may regulate trafficking and function of specific subsets of immune cells. To further dissect the mechanisms regulating CD13-dependent trafficking we used the murine model of thioglycollate-induced sterile peritonitis. Peritoneal monocytes, macrophages and dendritic cells were significantly decreased in inflammatory exudates from global CD13KO animals when compared with wild-type controls. Furthermore, adoptive transfer of wild-type and CD13KO primary myeloid cells, or wild-type myeloid cells pre-treated with CD13-blocking antibodies into thioglycollate-challenged wild-type recipients demonstrated fewer CD13KO or treated cells in the lavage, suggesting that CD13 expression confers a competitive advantage in trafficking. Similarly, both wild-type and CD13KO cells were reduced in infiltrates in CD13KO recipients, confirming that both monocytic and endothelial CD13 contribute to trafficking. Finally, murine monocyte cell lines expressing mouse/human chimeric CD13 molecules demonstrated that the C-terminal domain of the protein mediates CD13 adhesion. Therefore, this work verifies that the altered inflammatory trafficking in CD13KO mice is the result of aberrant myeloid cell subset trafficking and further defines the molecular mechanisms underlying this regulation. PMID:24627994

  11. Calcium-mediated mechanisms of cystic expansion

    PubMed Central

    Abdul-Majeed, Shakila; Nauli, Surya M.

    2010-01-01

    In this review, we will discuss several well-accepted signaling pathways toward calcium-mediated mechanisms of cystic expansion. The second messenger calcium ion has contributed to a vast diversity of signal transduction pathways. We will dissect calcium signaling as a possible mechanism that contributes to renal cyst formation. Because cytosolic calcium also regulates an array of signaling pathways, we will first discuss cilia-induced calcium fluxes, followed by Wnt signaling that has attributed to much-discussed planar cell polarity. We will then look at the relationship between cytosolic calcium and cAMP as one of the most important aspects of cyst progression. The signaling of cAMP on MAPK and mTOR will also be discussed. We infer that while cilia-induced calcium fluxes may be the initial signaling messenger for various cellular pathways, no single signaling mediator or pathway is implicated exclusively in the progression of the cystic expansion. PMID:20932898

  12. Control of cell cycle by metabolites of prostaglandin D2 through a non-cAMP mediated mechanism

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Fukushima, M.

    1993-01-01

    The dehydration products of PGD2, 9-deoxy-9 prostaglandin D2(PGJ2), 9-deoxy-delta 9, delta 12, delta 13 dehydroprostaglandin D2 (delta 12 PGJ2), and PGA2 all contain an unsaturated cyclopentenone structure which is characteristic of prostaglandins which effectively inhibit cell growth. It has been suggested that the action of the inhibitory prostaglandins may be through a cAMP mechanism. In this study, we use S49 wild type (WT) and adenylate cyclase variant (cyc-) cells to show that PGD2 and PGJ2 are not acting via a cyclic AMP mechanism. First, the increase in cyclic AMP in wild type S-49 cells is not proportional to its effects on DNA synthesis. More importantly, when S-49 cyc- cells were exposed to PGJ2, the adenylate cyclase (cyc-) mutant had decreased DNA synthesis with no change in its nominal cAMP content. Short-term (2 hours or less) exposure of the cyc- cells to prostaglandin J2 caused an inhibition of DNA synthesis. PGJ2 caused cytolysis at high concentrations. Long-term exposure (>14 hrs) of the cells to PGJ2, delta 12PGJ2 or delta 12, delta 14PGJ2 caused a cell cycle arrest in G1 demonstrating a cell cycle specific mechanism of action for growth inhibition by naturally occurring biological products independent of cAMP.

  13. Mechanisms of CDDO-imidazolide-mediated cytoprotection against acrolein-induced neurocytotoxicity in SH-SY5Y cells and primary human astrocytes.

    PubMed

    Speen, Adam; Jones, Colton; Patel, Ruby; Shah, Halley; Nallasamy, Palanisamy; Brooke, Elizabeth A S; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

    2015-10-01

    Acrolein is a ubiquitous unsaturated aldehyde has been implicated in the pathogenesis of various neurological disorders. However, limited study has been conducted into potential therapeutic protection and underlying mechanism against acrolein-induced cytotoxicity via upregulation of cellular aldehyde-detoxification defenses. In this study we have utilized RA-differentiated human SH-SY5Y cells and primary human astrocytes to investigate the induction of glutathione (GSH) by the synthetic triterpenoid 2-cyano-3,12-dixooleana-1,9-dien-28-imidazolide (CDDO-Im) and the protective effects CDDO-Im-mediated antioxidant defenses on acrolein toxicity. Acrolein exposure to RA-differentiated SH-SY5Y cells resulted in a significant time dependent depletion of cellular GSH preceding a reduction in cell viability and LDH release. Further, we demonstrated the predominance of cellular GSH in protection against acrolein-induced cytotoxicity. Buthionine sulfoximine (BSO) at 25μM dramatically depleted GSH and significantly potentiated acrolein-induced cytotoxicity. Pretreatment of the cells with 100nM CDDO-Im afforded a dramatic protection against acrolein-induced cytotoxicity. Pretreatment of BSO and CDDO was found to prevent the CDDO-Im-mediated GSH induction and partially reversed the cytoprotective effects of CDDO-Im against acrolein cytotoxicity. Overall, this study represents for the first time the CDDO-Im mediated upregulation of GSH is a predominant mechanism against acrolein-induced neurotoxicity.

  14. NADPH oxidase-mediated generation of reactive oxygen species: A new mechanism for X-ray-induced HeLa cell death

    SciTech Connect

    Liu Qing; He Xiaoqing; Liu Yongsheng; Du Bingbing; Wang Xiaoyan; Zhang Weisheng; Jia Pengfei; Dong Jingmei; Ma Jianxiu; Wang Xiaohu; Li Sha; Zhang Hong

    2008-12-19

    Oxidative damage is an important mechanism in X-ray-induced cell death. Radiolysis of water molecules is a source of reactive oxygen species (ROS) that contribute to X-ray-induced cell death. In this study, we showed by ROS detection and a cell survival assay that NADPH oxidase has a very important role in X-ray-induced cell death. Under X-ray irradiation, the upregulation of the expression of NADPH oxidase membrane subunit gp91{sup phox} was dose-dependent. Meanwhile, the cytoplasmic subunit p47{sup phox} was translocated to the cell membrane and localized with p22{sup phox} and gp91{sup phox} to form reactive NADPH oxidase. Our data suggest, for the first time, that NADPH oxidase-mediated generation of ROS is an important contributor to X-ray-induced cell death. This suggests a new target for combined gene transfer and radiotherapy.

  15. Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism

    PubMed Central

    Zubair, Haseeb; Azim, Shafquat; Khan, Husain Yar; Ullah, Mohammad Fahad; Wu, Daocheng; Singh, Ajay Pratap; Hadi, Sheikh Mumtaz; Ahmad, Aamir

    2016-01-01

    There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach. PMID:27331811

  16. Bladder cancer cells acquire competent mechanisms to escape Fas-mediated apoptosis and immune surveillance in the course of malignant transformation

    PubMed Central

    Perabo, F G E; Kamp, S; Schmidt, D; Lindner, H; Steiner, G; Mattes, R H; Wirger, A; Pegelow, K; Albers, P; Kohn, E C; Ruecker, A von; Mueller, S C

    2001-01-01

    Mechanisms of resistance against Fas-mediated cell killing have been reported in different malignancies. However, the biological response of immune escape mechanisms might depend on malignant transformation of cancer cells. In this study we investigated different mechanisms of immune escape in 2 well-differentiated low-grade (RT4 and RT112) and 2 poorly differentiated high-grade (T24 and TCCSUP) bladder cancer cell lines. Fas, the receptor of Fas-ligand, is expressed and shedded by human transitional bladder carcinoma cell lines RT4, RT112, T24 and TCCSUP. Cytotoxicity and apoptosis assays demonstrate that in spite of the Fas expression, poorly differentiated T24 and TCCSUP cells are insensitive towards either recombinant Fas-ligand or agonistic apoptosis-inducing monoclonal antibody against Fas. In poorly differentiated T24 and TCCSUP cell lines we were able to detect marked Fas-ligand protein by flow cytometry and Western blot analysis. In grade 1 RT4 and RT112 cells only minor expression of Fas-ligand possibly because of proteinase action. Fas-ligand mRNA translation or post-translational processing seems to be regulated differentially in the cancer cell lines depending on malignant transformation. In co-culture experiments we show that poorly differentiated cells can induce apoptosis and cell death in Jurkat cells and activated peripheral blood mononuclear cells. This in vitro study suggests that bladder cancer cells can take advantage of different mechanisms of immune evasion and become more competent in avoiding immune surveillance during transformation to higher-grade malignant disease. © 2001 Cancer Research Campaign www.bjcancer.com PMID:11355943

  17. Analyses of CD20 Monoclonal Antibody–Mediated Tumor Cell Killing Mechanisms: Rational Design of Dosing Strategies

    PubMed Central

    Lindorfer, Margaret A.

    2014-01-01

    Since approval of rituximab for treatment of B cell non-Hodgkin lymphoma, development of monoclonal antibodies (mAbs) for cancer treatment and elucidation of their cytotoxic mechanisms have been subject to intense investigations. Compelling evidence indicates that rituximab and another CD20 mAb, ofatumumab, must use the body’s cellular and humoral immune effector functions to kill malignant cells. Other U.S. Food and Drug Administration–approved mAbs, including obinutuzumab, cetuximab, and trastuzumab, require, in part, these effector mechanisms to eliminate tumor cells. Although gram quantities of mAbs can be administered to patients, our investigations of CD20 mAb-based therapies for chronic lymphocytic leukemia (CLL), including correlative measurements in clinical trials and studies with primary cells and cell lines, indicate that effector mechanisms necessary for mAb activity can be saturated or exhausted if tumor burdens are high, thus substantially compromising the efficacy of high-dose mAb therapy. Under these conditions, another reaction (trogocytosis) predominates in which bound CD20 mAb and CD20 are removed from targeted cells by effector cells that express Fcγ receptors, thereby allowing malignant cells to escape unharmed and continue to promote disease pathology. To address this problem, we propose that a low-dose strategy, based on administering 30–50 mg of CD20 mAb three times per week, may be far more effective for CLL than standard dosing because it will minimize effector function saturation and reduce trogocytosis. This approach may have general applicability to other mAbs that use immune effector functions, and could be formulated into a subcutaneous treatment strategy that would be more accessible and possibly more efficacious for patients. PMID:24944188

  18. Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

    PubMed

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

  19. Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

    PubMed Central

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y.; Meininger, Gerald A.; Lee, James C-M.

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewisx (sLex) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

  20. Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

    PubMed

    Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

    2013-01-01

    Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

  1. Acetylcholine mediates the release of IL-8 in human bronchial epithelial cells by a NFkB/ERK-dependent mechanism.

    PubMed

    Profita, Mirella; Bonanno, Anna; Siena, Liboria; Ferraro, Maria; Montalbano, Angela M; Pompeo, Flora; Riccobono, Loredana; Pieper, Michael P; Gjomarkaj, Mark

    2008-03-17

    Acetylcholine may play a role in cell activation and airway inflammation. We evaluated the levels of both mRNA and protein of muscarinic M(1), M(2), M(3) receptors in human bronchial epithelial cell line (16HBE). 16HBE cells were also stimulated with acetylcholine and extracellular signal-regulated kinase1/2 (ERK1/2) and NFkB pathway activation as well as the IL-8 release was assessed in the presence or absence of the inhibitor of Protein-kinase (PKC) (GF109203X), of the inhibitor of mitogenic activated protein-kinase kinase (MAPKK) (PDO9805), of the inhibitor of kinaseB-alpha phosphorilation (pIkBalpha) (BAY11-7082), and of muscarinic receptor antagonists tiotropium bromide, 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), telenzepine, gallamine. Additionally, we tested the IL-8-mediated neutrophil chemotactic activity of 16HBE supernatants stimulated with acetylcholine in the presence or absence of tiotropium. 16HBE cells expressed both protein and mRNA for muscarinic M(3), M(2) and M(1) receptors with levels of muscarinic M(3) receptor>muscarinic M(1) receptor>muscarinic M(2) receptor. Acetylcholine (10 microM) significantly stimulated ERK1/2 and NFkB activation as well as IL-8 release in 16HBE cells when compared to basal values. Furthermore, while the use of tiotropium, 4-DAMP, GF109203X, PDO98059, BAY11-7082 completely abolished these events, the use of telenzepine and gallamine were only partially able to downregulate these effects. Additionally, acetylcholine-mediated IL-8 release from 16HBE cells significantly increased chemotaxis toward neutrophils and this effect was blocked by tiotropium. In conclusion, acetylcholine activates the release of IL-8 from 16HBE involving PKC, ERK1/2 and NFkB pathways via muscarinic receptors, suggesting that it is likely to contribute to IL-8 related neutrophilic inflammatory disorders in the airway. Thus, muscarinic antagonists may contribute to control inflammatory processes in airway diseases.

  2. An autologous T cell clone overcomes intra-melanoma heterogeneity for susceptibility to cell-mediated lysis by using multiple lytic mechanisms: in vitro and in vivo analysis.

    PubMed

    Mazzocchi, A; Rodolfo, M; Parmiani, G; Anichini, A

    1991-01-01

    An HLA-A2-specific cytotoxic T lymphocyte (CTL) clone (CTL49), capable of killing the HLA-A2-negative autologous melanoma (Me665/2) in a T cell receptor and MHC-independent fashion, lysed six of 16 Me665/2 tumour clones in short-term (4 and 18 hour) 51Cr-release assays. In long-term (96 hour) lytic assays, CTL49 could lyse all the 16 tumour clones. The lysis observed in the 96 hour assay could be enhanced by stimulating CTL49 with anti-CD3 monoclonal antibodies (MAb) and interleukin-2 (IL-2). Supernatants of anti-CD3- or antigen-stimulated CTL49, known to contain tumour necrosis factor (TNF) alpha and interferon (IFN)gamma, were also able to lyse all but one (665/2/51) of the tumour clones in 96 hour assays. Absence of lysis of tumour clone 2/51 by supernatants correlated with resistance of the same tumour clone to lysis by recombinant IFN-gamma plus TNF-alpha. Antibodies to TNF-alpha and, to a lesser extent, to IFN-gamma, reduced significantly the 96 hour lysis of Me2/9 and Me2/10, two of the tumour clones killed in long term but not in short term assays. Winn assays in nude mice revealed that CTL49, stimulated with anti-CD3-MAb plus IL-2, could abolish tumour cell growth when injected together with clones 2/9 or 2/10. These results indicate that intra-tumour heterogeneity for susceptibility to lysis can be overcome even by a single CTL clone providing that appropriate signals (i.e. anti-CD3-MAb and IL-2) are supplied to an effector able to mediate tumour cell lysis by multiple mechanisms. PMID:1841713

  3. Interleukin 1 and tumour necrosis factor alpha may be responsible for the lytic mechanism during anti-tumour antibody-dependent cell-mediated cytotoxicity.

    PubMed Central

    Pullyblank, A. M.; Guillou, P. J.; Monson, J. R.

    1995-01-01

    Antibodies are thought to bring about tumour cell lysis by antibody-dependent cell-mediated cytotoxicity (ADCC), but the exact mechanism is not well elucidated. Monocytes are known to be important mediators of anti-tumour ADCC and are also known to secrete the cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), both of which have been shown to bring about tumour cell lysis. We examined the release of these cytokines during ADCC and attempted to elucidate which components of the ADCC reaction were necessary for cytokine production. We measured TNF-alpha and IL-1 beta in supernatants collected from a standard ADCC assay using each of the anti-colorectal antibodies m17-1A, c17-1A and cSF25. We found that there was significant TNF-alpha and IL-1 beta release during ADCC mediated by each of these three antibodies and that the magnitude of cytokine release seemed to reflect the degree of tumour cell lysis produced by each antibody. Furthermore, we found that effector cells, target cells and a specific anti-tumour antibody were necessary for this to occur. The presence of only some of the components of the reaction or of an irrelevant antibody produced little or no TNF-alpha or IL-1 beta. We conclude that TNF-alpha and IL-1 beta are released when an effector and tumour target cell are united by a specific tumour antibody and that these cytokines may be important in bringing about tumour cell lysis during the ADCC reaction. PMID:7669568

  4. Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators

    PubMed Central

    Shpak, Guy; Zylbertal, Asaph; Wagner, Shlomo

    2015-01-01

    Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB) and accessory (AOB) olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican). In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs) following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states. PMID:25642164

  5. Androgen receptor–negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms

    PubMed Central

    Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W.; Zurita, Amado J.; Liu, Jie; Sikes, Charles; Multani, Asha S.; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V.; Prieto, Victor G.; Kundra, Vikas; Vazquez, Elba S.; Troncoso, Patricia; Raymond, Austin K.; Logothetis, Christopher J.; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M.

    2008-01-01

    In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor–negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer–induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor–null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. PMID:18618013

  6. Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms.

    PubMed

    Li, Zhi Gang; Mathew, Paul; Yang, Jun; Starbuck, Michael W; Zurita, Amado J; Liu, Jie; Sikes, Charles; Multani, Asha S; Efstathiou, Eleni; Lopez, Adriana; Wang, Jing; Fanning, Tina V; Prieto, Victor G; Kundra, Vikas; Vazquez, Elba S; Troncoso, Patricia; Raymond, Austin K; Logothetis, Christopher J; Lin, Sue-Hwa; Maity, Sankar; Navone, Nora M

    2008-08-01

    In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells. PMID:18618013

  7. Galectin-9-Mediated Protection from Allo-Specific T Cells as a Mechanism of Immune Privilege of Corneal Allografts

    PubMed Central

    Shimmura-Tomita, Machiko; Wang, Mingcong; Taniguchi, Hiroko; Akiba, Hisaya; Yagita, Hideo; Hori, Junko

    2013-01-01

    The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation. The immunosuppressive intraocular microenvironment is known as one of the mechanisms underlying immune privilege in the eye. T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand. We investigated the role of this pathway in establishing the immune-privileged status of corneal allografts in mice. Gal-9 is constitutively expressed on the corneal epithelium, endothelium and iris-ciliary body in normal mouse eyes and eyes bearing surviving allografts, and Tim-3 was expressed on CD8 T cells infiltrating the allografts. Allograft survival in recipients treated with anti-Tim-3 monoclonal antibody (mAb) or anti-Gal-9 mAb was significantly shorter than that in control recipients. In vitro, destruction of corneal endothelial cells by allo-reactive T cells was enhanced when the cornea was pretreated with anti-Gal-9 mAb. Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation. We propose that constitutive expression of Gal-9 plays an immunosuppressive role in corneal allografts. Gal-9 expressed on corneal endothelial cells protects them from destruction by allo-reactive T cells within the cornea. PMID:23667648

  8. Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

    PubMed

    Cardoso, Nancy; Franco-Mahecha, Olga Lucía; Czepluch, Wenzel; Quintana, María Eugenia; Malacari, Darío Amílcar; Trotta, Myrian Vanesa; Mansilla, Florencia Celeste; Capozzo, Alejandra Victoria

    2016-09-01

    Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis.

  9. Bovine Viral Diarrhea Virus Infects Monocyte-Derived Bovine Dendritic Cells by an E2-Glycoprotein-Mediated Mechanism and Transiently Impairs Antigen Presentation.

    PubMed

    Cardoso, Nancy; Franco-Mahecha, Olga Lucía; Czepluch, Wenzel; Quintana, María Eugenia; Malacari, Darío Amílcar; Trotta, Myrian Vanesa; Mansilla, Florencia Celeste; Capozzo, Alejandra Victoria

    2016-09-01

    Infection of professional antigen presenting cells by viruses can have a marked effect on these cells and important consequences for the generation of subsequent immune responses. In this study, we demonstrate that different strains of bovine viral diarrhea virus (BVDV) infect bovine dendritic cells differentiated from nonadherent peripheral monocytes (moDCs). BVDV did not cause apoptosis in these cells. Infection of moDC was prevented by incubating the virus with anti-E2 antibodies or by pretreating the cells with recombinant E2 protein before BVDV contact, suggesting that BVDV infects moDC through an E2-mediated mechanism. Virus entry was not reduced by incubating moDC with Mannan or ethylenediaminetetraacetic acid (EDTA) before infection, suggesting that Ca(2+) and mannose receptor-dependent pathways are not mediating BVDV entry to moDC. Infected moDC did not completely upregulate maturation surface markers. Infection, but not treatment with inactivated virus, prevented moDC to present a third-party antigen to primed CD4(+) T cells within the first 24 hours postinfection (hpi). Antigen-presenting capacity was recovered when viral replication diminished at 48 hpi, suggesting that active infection may interfere with moDC maturation. Altogether, our results suggest an important role of infected DCs in BVDV-induced immunopathogenesis. PMID:27529119

  10. Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties.

    PubMed

    Meloni, Bruno P; Milani, Diego; Edwards, Adam B; Anderton, Ryan S; O'Hare Doig, Ryan L; Fitzgerald, Melinda; Palmer, T Norman; Knuckey, Neville W

    2015-09-01

    Several recent studies have demonstrated that TAT and other arginine-rich cell penetrating peptides (CPPs) have intrinsic neuroprotective properties in their own right. Examples, we have demonstrated that in addition to TAT, poly-arginine peptides (R8 to R18; containing 8-18 arginine residues) as well as some other arginine-rich peptides are neuroprotective in vitro (in neurons exposed to glutamic acid excitotoxicity and oxygen glucose deprivation) and in the case of R9 in vivo (after permanent middle cerebral artery occlusion in the rat). Based on several lines of evidence, we propose that this neuroprotection is related to the peptide's endocytosis-inducing properties, with peptide charge and arginine residues being critical factors. Specifically, we propose that during peptide endocytosis neuronal cell surface structures such as ion channels and transporters are internalised, thereby reducing calcium influx associated with excitotoxicity and other receptor-mediated neurodamaging signalling pathways. We also hypothesise that a peptide cargo can act synergistically with TAT and other arginine-rich CPPs due to potentiation of the CPPs endocytic traits rather than by the cargo-peptide acting directly on its supposedly intended intracellular target. In this review, we systematically consider a number of studies that have used CPPs to deliver neuroprotective peptides to the central nervous system (CNS) following stroke and other neurological disorders. Consequently, we critically review evidence that supports our hypothesis that neuroprotection is mediated by carrier peptide endocytosis. In conclusion, we believe that there are strong grounds to regard arginine-rich peptides as a new class of neuroprotective molecules for the treatment of a range of neurological disorders.

  11. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

    PubMed

    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  12. A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines.

    PubMed

    Sciamanna, Ilaria; Gualtieri, Alberto; Cossetti, Cristina; Osimo, Emanuele Felice; Ferracin, Manuela; Macchia, Gianfranco; Aricò, Eleonora; Prosseda, Gianni; Vitullo, Patrizia; Misteli, Tom; Spadafora, Corrado

    2013-12-01

    LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the

  13. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance

    PubMed Central

    2013-01-01

    Background The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Methods Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. Results PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. Conclusion The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells. PMID:23663432

  14. IL-10 Production in Macrophages Is Regulated by a TLR-Driven CREB-Mediated Mechanism That Is Linked to Genes Involved in Cell Metabolism

    PubMed Central

    Sanin, David E.; Prendergast, Catriona T.

    2015-01-01

    IL-10 is produced by macrophages in diverse immune settings and is critical in limiting immune-mediated pathology. In helminth infections, macrophages are an important source of IL-10; however, the molecular mechanism underpinning production of IL-10 by these cells is poorly characterized. In this study, bone marrow–derived macrophages exposed to excretory/secretory products released by Schistosoma mansoni cercariae rapidly produce IL-10 as a result of MyD88-mediated activation of MEK/ERK/RSK and p38. The phosphorylation of these kinases was triggered by TLR2 and TLR4 and converged on activation of the transcription factor CREB. Following phosphorylation, CREB is recruited to a novel regulatory element in the Il10 promoter and is also responsible for regulating a network of genes involved in metabolic processes, such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. Moreover, skin-resident tissue macrophages, which encounter S. mansoni excretory/secretory products during infection, are the first monocytes to produce IL-10 in vivo early postinfection with S. mansoni cercariae. The early and rapid release of IL-10 by these cells has the potential to condition the dermal microenvironment encountered by immune cells recruited to this infection site, and we propose a mechanism by which CREB regulates the production of IL-10 by macrophages in the skin, but also has a major effect on their metabolic state. PMID:26116503

  15. Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms.

    PubMed

    Meschiari, Cesar A; Izidoro-Toledo, Tatiane; Gerlach, Raquel F; Tanus-Santos, Jose E

    2013-06-01

    Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 μM) or SNAP (50-400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.

  16. Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms.

    PubMed

    Meschiari, Cesar A; Izidoro-Toledo, Tatiane; Gerlach, Raquel F; Tanus-Santos, Jose E

    2013-06-01

    Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 μM) or SNAP (50-400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases. PMID:23456480

  17. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2

    PubMed Central

    Neumann, Alexander J.; Gardner, Oliver F. W.; Williams, Rebecca; Alini, Mauro; Archer, Charles W.; Stoddart, Martin J.

    2015-01-01

    Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a

  18. Does a nonclassical signaling mechanism underlie an increase of estradiol-mediated gonadotropin-releasing hormone receptor binding in ovine pituitary cells?

    PubMed

    Davis, Tracy L; Whitesell, Jennifer D; Cantlon, Jeremy D; Clay, Colin M; Nett, Terry M

    2011-10-01

    Estradiol-17beta (E2) is the major regulator of GnRH receptor (GnRHR) gene expression and number during the periovulatory period; however, the mechanisms underlying E2 regulation of the GNRHR gene remain undefined. Herein, we find that E2 conjugated to BSA (E2-BSA) mimics the stimulatory effect of E2 on GnRH binding in primary cultures of ovine pituitary cells. The time course for maximal GnRH analog binding was similar for both E2 and E2-BSA. The ability of E2 and E2-BSA to increase GnRH analog binding was blocked by the estrogen receptor (ER) antagonist ICI 182,780. Also, increased GnRH analog binding in response to E2 and the selective ESR1 agonist propylpyrazole triol was blocked by expression of a dominant-negative form of ESR1 (L540Q). Thus, membrane-associated ESR1 is the likely candidate for mediating E2 activation of the GNRHR gene. As cAMP response element binding protein (CREB) is an established target for E2 activation in gonadotrophs, we next explored a potential role for this protein as an intracellular mediator of the E2 signal. Consistent with this possibility, adenoviral-mediated expression of a dominant-negative form of CREB (A-CREB) completely abolished the ability of E2 to increase GnRH analog binding in primary cultures of ovine pituitary cells. Finally, the presence of membrane-associated E2 binding sites on ovine pituitary cells was demonstrated using a fluorescein isothiocyanate conjugate of E2-BSA. We suggest that E2 regulation of GnRHR number during the preovulatory period reflects a membrane site of action and may proceed through a nonclassical signaling mechanism, specifically a CREB-dependent pathway.

  19. IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells

    PubMed Central

    2016-01-01

    Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed “myokines,” has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARα and/or PPARδ as targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARα inhibitor (GW-6471), a PPARδ inhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1β, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1β and Nrf1. However, with PPARδ inhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1β, UCP2, and Nrf1. Further, inhibition of PPARδ abolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδ activity is required for the beneficial metabolic effects of IL-15 signaling in SKM. PMID:27738421

  20. Activation of Proinflammatory Responses in Cells of the Airway Mucosa by Particulate Matter: Oxidant- and Non-Oxidant-Mediated Triggering Mechanisms

    PubMed Central

    Øvrevik, Johan; Refsnes, Magne; Låg, Marit; Holme, Jørn A.; Schwarze, Per E.

    2015-01-01

    Inflammation is considered to play a central role in a diverse range of disease outcomes associated with exposure to various types of inhalable particulates. The initial mechanisms through which particles trigger cellular responses leading to activation of inflammatory responses are crucial to clarify in order to understand what physico-chemical characteristics govern the inflammogenic activity of particulate matter and why some particles are more harmful than others. Recent research suggests that molecular triggering mechanisms involved in activation of proinflammatory genes and onset of inflammatory reactions by particles or soluble particle components can be categorized into direct formation of reactive oxygen species (ROS) with subsequent oxidative stress, interaction with the lipid layer of cellular membranes, activation of cell surface receptors, and direct interactions with intracellular molecular targets. The present review focuses on the immediate effects and responses in cells exposed to particles and central down-stream signaling mechanisms involved in regulation of proinflammatory genes, with special emphasis on the role of oxidant and non-oxidant triggering mechanisms. Importantly, ROS act as a central second-messenger in a variety of signaling pathways. Even non-oxidant mediated triggering mechanisms are therefore also likely to activate downstream redox-regulated events. PMID:26147224

  1. An Antisense RNA-mediated Mechanism Eliminates a Meiosis-specific Copper-regulated Transcript in Mitotic Cells*

    PubMed Central

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-01-01

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1+ gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1+ sense transcript, inhibiting cum1+ expression in vegetative cells. Inhibition of cum1+ transcription was not at the level of its initiation because fusion of the cum1+ promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1+-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1+ led to the exclusive production of a shorter iss1+ mRNA isoform, thereby allowing accumulation of cum1+ sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1+ antisense mRNA could pair with cum1+ sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1+ sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells. PMID:26229103

  2. An antisense RNA-mediated mechanism eliminates a meiosis-specific copper-regulated transcript in mitotic cells.

    PubMed

    Normant, Vincent; Beaudoin, Jude; Labbé, Simon

    2015-09-11

    Sense and antisense transcripts produced from convergent gene pairs could interfere with the expression of either partner gene. In Schizosaccharomyces pombe, we found that the iss1(+) gene produces two transcript isoforms, including a long antisense mRNA that is complementary to the meiotic cum1(+) sense transcript, inhibiting cum1(+) expression in vegetative cells. Inhibition of cum1(+) transcription was not at the level of its initiation because fusion of the cum1(+) promoter to the lacZ gene showed that activation of the reporter gene occurs in response to low copper conditions. Further analysis showed that the transcription factor Cuf1 and conserved copper-signaling elements (CuSEs) are required for induction of cum1(+)-lacZ transcription under copper deficiency. Insertion of a multipartite polyadenylation signal immediately downstream of iss1(+) led to the exclusive production of a shorter iss1(+) mRNA isoform, thereby allowing accumulation of cum1(+) sense mRNA in copper-limited vegetative cells. This finding suggested that the long iss1(+) antisense mRNA could pair with cum1(+) sense mRNA, thereby producing double-stranded RNA molecules that could induce RNAi. We consistently found that mutant strains for RNAi (dcr1Δ, ago1Δ, rdp1Δ, and clr4Δ) are defective in selectively eliminating cum1(+) sense transcript in the G1 phase of the cell cycle. Taken together, these results describe the first example of a copper-regulated meiotic gene repressed by an antisense transcription mechanism in vegetative cells.

  3. Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells

    PubMed Central

    Mehta, Anuradha; Trotta, Christopher R.; Peltz, Stuart W.

    2006-01-01

    Transcripts harboring 5′ upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5′ untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3′ UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3′ UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5′ uORF can be modulated by elements in their 3′ UTRs. PMID:16598037

  4. Notch-Mediated Cell Adhesion

    PubMed Central

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  5. Inactivation of Src-to-ezrin pathway: a possible mechanism in the ouabain-mediated inhibition of A549 cell migration.

    PubMed

    Shin, Hye Kyoung; Ryu, Byung Jun; Choi, Sik-Won; Kim, Seong Hwan; Lee, Kyunglim

    2015-01-01

    Ouabain, a cardiac glycoside found in plants, is primarily used in the treatment of congestive heart failure and arrhythmia because of its ability to inhibit Na(+)/K(+)-ATPase pump. Recently ouabain has been shown to exert anticancer effects but the underlying mechanism is not clear. Here, we explored the molecular mechanism by which ouabain exerts anticancer effects in human lung adenocarcinoma. Employing proteomic techniques, we found 7 proteins downregulated by ouabain in A549 including p-ezrin, a protein associated with pulmonary cancer metastasis in a dose-dependent manner. In addition, when the relative phosphorylation levels of 39 intracellular proteins were compared between control and ouabain-treated A549 cells, p-Src (Y416) was also found to be downregulated by ouabain. Furthermore, western blot revealed the ouabain-mediated downregulation of p-FAK (Y925), p-paxillin (Y118), p130CAS, and Na(+)/K(+)-ATPase subunits that have been shown to be involved in the migration of cancer cells. The inhibitory effect of ouabain and Src inhibitor PP2 on the migration of A549 cells was confirmed by Boyden chamber assay. Anticancer effects of ouabain in A549 cells appear to be related to its ability to regulate and inactivate Src-to-ezrin signaling, and proteins involved in focal adhesion such as Src, FAK, and p130CAS axis are proposed here. PMID:25866790

  6. Control of the Inflammatory Response Mechanisms Mediated by Natural and Induced Regulatory T-Cells in HCV-, HTLV-1-, and EBV-Associated Cancers

    PubMed Central

    Moralès, Olivier; Delhem, Nadira

    2014-01-01

    Virus infections are involved in chronic inflammation and, in some cases, cancer development. Although a viral infection activates the immune system's response that eradicates the pathogen mainly through inflammatory mechanisms, it is now recognized that this inflammatory condition is also favorable to the development of tumors. Indeed, it is well described that viruses, such as hepatitis C virus (HCV), Epstein Barr virus (EBV), human papillomavirus (HPV) or human T-cell lymphotropic virus type-1 (HTLV-1), are important risk factors for tumor malignancies. The inflammatory response is a fundamental immune mechanism which involves several molecular and cellular components consisting of cytokines and chemokines that are released by various proinflammatory cells. In parallel to this process, some endogenous recruited components release anti-inflammatory mediators to restore homeostasis. The development of tools and strategies using viruses to hijack the immune response is mostly linked to the presence of regulatory T-cells (Treg) that can inhibit inflammation and antiviral responses of other effector cells. In this review, we will focus on current understanding of the role of natural and induced Treg in the control and the resolution of inflammatory response in HCV-, HTLV-1-, and EBV-associated cancers. PMID:25525301

  7. Fibronectin mediates mesendodermal cell fate decisions

    PubMed Central

    Cheng, Paul; Andersen, Peter; Hassel, David; Kaynak, Bogac L.; Limphong, Pattraranee; Juergensen, Lonny; Kwon, Chulan; Srivastava, Deepak

    2013-01-01

    Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-β1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm. PMID:23715551

  8. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  9. The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism.

    PubMed

    Freeman, E J

    2000-02-15

    Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.

  10. Analysis of Host-Mediated Repair Mechanisms after Human CNS-Stem Cell Transplantation for Spinal Cord Injury: Correlation of Engraftment with Recovery

    PubMed Central

    Hooshmand, Mitra J.; Sontag, Christopher J.; Uchida, Nobuko; Tamaki, Stan

    2009-01-01

    Background Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study. Methods and Findings Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = −0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery. Conclusions Altogether

  11. Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-X(L)/p53-mediated amplification mechanisms.

    PubMed

    Shi, Y L; Feng, S; Chen, W; Hua, Z C; Bian, J J; Yin, W

    2014-12-18

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X(L) overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X(L) downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X(L) and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X(L) and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.

  12. Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism

    PubMed Central

    Luanpitpong, Sudjit; Chanvorachote, Pithi; Leonard, Stephen S.; Pongrakhananon, Varisa; Wang, Liying

    2016-01-01

    Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy. PMID:21573972

  13. Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism.

    PubMed

    Luanpitpong, Sudjit; Nimmannit, Ubonthip; Chanvorachote, Pithi; Leonard, Stephen S; Pongrakhananon, Varisa; Wang, Liying; Rojanasakul, Yon

    2011-08-01

    Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.

  14. The study of the mechanism of arsenite toxicity in respiration-deficient cells reveals that NADPH oxidase-derived superoxide promotes the same downstream events mediated by mitochondrial superoxide in respiration-proficient cells.

    PubMed

    Guidarelli, Andrea; Fiorani, Mara; Carloni, Silvia; Cerioni, Liana; Balduini, Walter; Cantoni, Orazio

    2016-09-15

    We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5μM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis. PMID:27450018

  15. Mechanics of Protein-Mediated DNA Looping

    NASA Astrophysics Data System (ADS)

    Meiners, Jens-Christian

    2009-03-01

    The formation of looped DNA-protein complexes in which a protein or protein assembly binds to multiple distant operator sites on the DNA is a common feature for many regulatory schemes on the transcriptional level. In a living cell, a multitude of mechanical forces and constraints act on these complexes, and it is imperative to understand their effects on biological function. For this aim, we study the lactose repressor as a model system for protein-mediated DNA looping in single-molecule experiments. Using a novel axial constant-force optical trapping scheme that allows us to manipulate sub-micron DNA fragments with well-controlled forces down to the 10 fN range, we show that mechanical tension in the substrate DNA of hundred femtonewton is sufficient to disrupt the loop formation process, which suggests that such mechanical tension may provide a mechanical pathway to controlling gene expression in vivo. From the force sensitivity of the loop formation process, we can also infer the topology of the looped complex; in our case an antiparallel conformation. In addition, we will present new tethered-particle microscopy data that shows lifetimes of the looped complexes that are two to three orders of magnitude shorter than those measured in biochemical competition assays and discuss possible interpretations, including the suggestion that operator binding of the lactose repressor tetramer leads to a destabilization of the dimer-dimer interface and that thus the loop breakdown process is mostly a dissociation of the tetramer into two dimers, instead, as widely assumed, an unbinding of the tetramer from the DNA.

  16. The apoptotic effect of nanosilver is mediated by a ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells.

    PubMed

    Hsin, Yi-Hong; Chen, Chun-Feng; Huang, Shing; Shih, Tung-Sheng; Lai, Ping-Shan; Chueh, Pin Ju

    2008-07-10

    Nanomaterials and nanoparticles have received considerable attention recently because of their unique properties and diverse biotechnology and life sciences applications. Nanosilver products, which have well-known antimicrobial properties, have been used extensively in a range of medical settings. Despite the widespread use of nanosilver products, relatively few studies have been undertaken to determine the biological effects of nanosilver exposure. The purpose of this study was to evaluate the toxicity of nanosilver and to elucidate possible molecular mechanisms underlying the biological effects of nanosilver. Here, we show that nanosilver is cytotoxic, inducing apoptosis in NIH3T3 fibroblast cells. Treatment with nanosilver induced the release of cytochrome c into the cytosol and translocation of Bax to mitochondria, indicating that nanosilver-mediated apoptosis is mitochondria-dependent. Nanosilver-induced apoptosis was associated with the generation of reactive oxygen species (ROS) and JNK activation, and inhibition of either ROS or JNK attenuated nanosilver-induced apoptosis. In nanosilver-resistant HCT116 cells, up-regulation of the anti-apoptotic proteins, Bcl-2 appeared to be associated with a diminished apoptotic response. Taken together, our results provide the first evidence for a molecular mechanism of nanosilver cytotoxicity, showing that nanosilver acts through ROS and JNK to induce apoptosis via the mitochondrial pathway.

  17. Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells

    PubMed Central

    Cai, Jin; Han, Yu; Ren, Hongmei; Chen, Caiyu; He, Duofen; Zhou, Lin; Eisner, Gilbert M.; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

    2013-01-01

    Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells. PMID:23580760

  18. Induction of the Lytic Cycle Sensitizes Epstein-Barr Virus-Infected B Cells to NK Cell Killing That Is Counteracted by Virus-Mediated NK Cell Evasion Mechanisms in the Late Lytic Cycle

    PubMed Central

    Williams, Luke R.; Quinn, Laura L.; Rowe, Martin

    2015-01-01

    ABSTRACT Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2

  19. Nuclear Mechanics and Stem Cell Differentiation.

    PubMed

    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  20. Hyaluronic acid-paclitaxel conjugate inhibits growth of human squamous cell carcinomas of the head and neck via a hyaluronic acid-mediated mechanism.

    PubMed

    Galer, Chad E; Sano, Daisuke; Ghosh, Sukhen C; Hah, Jeong H; Auzenne, Edmund; Hamir, Amirali N; Myers, Jeffrey N; Klostergaard, Jim

    2011-11-01

    Chemotherapeutic regimens incorporating taxanes significantly improve outcomes for patients with squamous cell carcinomas of the head and neck (SCCHN). However, treatment with taxanes is limited by toxicities, including bone marrow suppression and peripheral neuropathies. We proposed that conjugating taxanes to targeting carrier molecules would increase antitumor efficacy and decrease toxicity. The cell surface proteoglycan, CD44, is expressed on most SCCHNs, and we hypothesized that it is an attractive candidate for targeted therapy via its natural ligand, hyaluronic acid (HA). We determined whether HA-paclitaxel conjugates were able to decrease tumor growth and improve survival in orthotopic nude mouse human SCCHN xenograft models. HA-paclitaxel concentration-dependent growth inhibition of human SCCHN cell lines OSC-19 and HN5 in vitro, very similarly to free paclitaxel treatment. Tumor cell uptake of FITC-labeled HA-paclitaxel was significantly blocked with free HA, indicating the dependence of uptake on CD44. HA-paclitaxel administered intravenously once per week for three weeks at 120 mg/kg paclitaxel equivalents, far above the paclitaxel maximum tolerated dose, exerted superior tumor growth control to that of paclitaxel in both orthotopic OSC-19-luciferase and HN5 xenograft models in vivo. Mouse survival following HA-paclitaxel administration was prolonged compared with that of controls in mice implanted with either of these xenografts. Mice treated with HA-paclitaxel displayed increased TUNEL(+) cells in tumor tissue, as well as markedly reduced microvessel density compared to those treated with free paclitaxel. No acute histopathological changes were observed in mice treated with HA-paclitaxel. Thus, we conclude that HA-paclitaxel effectively inhibits tumor growth in human SCCHN xenografts via an HA-mediated mechanism and this conjugate should be considered for further preclinical development for this disease.

  1. Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties

    PubMed Central

    Thompson, Peter M.; Tolbert, Caitlin E.; Shen, Kai; Kota, Pradeep; Palmer, Sean M.; Plevock, Karen M.; Orlova, Albina; Galkin, Vitold E.; Burridge, Keith; Egelman, Edward H.; Dokholyan, Nikolay V.; Superfine, Richard; Campbell, Sharon L.

    2014-01-01

    SUMMARY Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling. PMID:24685146

  2. Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system

    PubMed Central

    Keshwani, Malik M.; Kanter, Joan R.; Ma, Yuliang; Wilderman, Andrea; Darshi, Manjula; Insel, Paul A.; Taylor, Susan S.

    2015-01-01

    Cyclic AMP/protein kinase A (cAMP/PKA) and glucocorticoids promote the death of many cell types, including cells of hematopoietic origin. In wild-type (WT) S49 T-lymphoma cells, signaling by cAMP and glucocorticoids converges on the induction of the proapoptotic B-cell lymphoma-family protein Bim to produce mitochondria-dependent apoptosis. Kin–, a clonal variant of WT S49 cells, lacks PKA catalytic (PKA-Cα) activity and is resistant to cAMP-mediated apoptosis. Using sorbitol density gradient fractionation, we show here that in kin– S49 cells PKA-Cα is not only depleted but the residual PKA-Cα mislocalizes to heavier cell fractions and is not phosphorylated at two conserved residues (Ser338 or Thr197). In WT S49 cells, PKA-regulatory subunit I (RI) and Bim coimmunoprecipitate upon treatment with cAMP analogs and forskolin (which increases endogenous cAMP concentrations). By contrast, in kin– cells, expression of PKA-RIα and Bim is prominently decreased, and increases in cAMP do not increase Bim expression. Even so, kin– cells undergo apoptosis in response to treatment with the glucocorticoid dexamethasone (Dex). In WT cells, glucorticoid-mediated apoptosis involves an increase in Bim, but in kin– cells, Dex-promoted cell death appears to occur by a caspase 3-independent apoptosis-inducing factor pathway. Thus, although cAMP/PKA-Cα and PKA-R1α/Bim mediate apoptotic cell death in WT S49 cells, kin– cells resist this response because of lower levels of PKA-Cα and PKA-RIα subunits as well as Bim. The findings for Dex-promoted apoptosis imply that these lymphoma cells have adapted to selective pressure that promotes cell death by altering canonical signaling pathways. PMID:26417071

  3. Endoglin involvement in integrin-mediated cell adhesion as a putative pathogenic mechanism in hereditary hemorrhagic telangiectasia type 1 (HHT1)

    PubMed Central

    Rossi, Elisa; Lopez-Novoa, José M.; Bernabeu, Carmelo

    2015-01-01

    Mutations in the endoglin gene (ENG) are responsible for ∼50% of all cases with hereditary hemorrhagic telangiectasia (HHT). Because of the absence of effective treatments for HHT symptoms, studies aimed at identifying novel biological functions of endoglin which could serve as therapeutic targets of the disease are needed. Endoglin is an endothelial membrane protein, whose most studied function has been its role as an auxiliary receptor in the TGF-β receptor complex. However, several lines of evidence suggest the involvement of endoglin in TGF-β-independent functions. Endoglin displays, within its zona pellucida domain, an RGD motif, which is a prototypic sequence involved in integrin-based interactions with other proteins. Indeed, we have recently described a novel role for endothelial endoglin in leukocyte trafficking and extravasation via its interaction with leukocyte integrins. In addition, functional, as well as protein and gene expression analysis have shown that ectopic endoglin represses the synthesis of several members of the integrin family and modulates integrin-mediated cell adhesions. This review focuses on the tight link between endoglin and integrins and how the role of endothelial endoglin in integrin-dependent cell adhesion processes can provide a better understanding of the pathogenic mechanisms leading to vascular lesions in endoglin haploinsufficient HHT1 patients. PMID:25709613

  4. Regulation on RhoA in vascular smooth muscle cells under inflammatory stimulation proposes a novel mechanism mediating the multiple-beneficial action of acetylsalicylic acid.

    PubMed

    Li, Dong-Bo; Yang, Guo-Jie; Xu, Hong-Wei; Fu, Zhi-Xuan; Wang, Shan-Wei; Hu, Shen-Jiang

    2013-12-01

    Recent studies have revealed the additional beneficial effects of acetylsalicylic acid (aspirin) in the medication of cardiovascular diseases. The small GTPase RhoA as an important signaling factor is implicated in a wide range of cell functions. This study aimed to investigate the regulatory effect of acetylsalicylic acid on RhoA in vascular smooth muscle cells (VSMCs). We found that aspirin at 300 μM suppressed VSMCs proliferation stimulated by LPS, and this inhibitory effect was partially mediated by inhibiting the iNOS/NO pathway. RhoA overexpression was downregulated by aspirin (both 30 and 300 μM) because of enhanced degradation of RhoA protein. The effect of LPS on increasing active RhoA level was significantly attenuated by aspirin (300 μM), which exerted no effect on RhoA translocation. The promoted RhoA phosphorylation under LPS stimulation, coupled with RhoA protein expression, was greatly decreased by aspirin treatment. No effect of aspirin was found on the expression, activation, and phosphorylation of RhoA in VSMCs devoid of inflammatory stimulation. Our investigation indicates that the regulation of RhoA by aspirin in VSMCs under inflammatory stimulus could be a novel mechanism via which aspirin, apart from the COX-dependent action, exerted the multiple beneficial effects.

  5. Infiltrating Macrophages Induce ERα Expression through an IL17A-mediated Epigenetic Mechanism to Sensitize Endometrial Cancer Cells to Estrogen.

    PubMed

    Ning, Chengcheng; Xie, Bingying; Zhang, Lin; Li, Chunsheng; Shan, Weiwei; Yang, Bingyi; Luo, Xuezhen; Gu, Chao; He, Qizhi; Jin, Hongyan; Chen, Xiaojun; Zhang, Zhenbo; Feng, Youji

    2016-03-15

    Persistent unopposed estrogen stimulation is a central oncogenic mechanism driving the formation of type I endometrial cancer. Recent epidemiologic and clinical studies of endometrial cancer have also revealed a role for insulin resistance, clinically manifested by chronic inflammation. However, the role of inflammation in estrogen-driven endometrial cancer is not well characterized. In this study, we investigated the association between infiltrating macrophages and estrogen sensitivity in endometrial cancer. Evaluating tissue samples and serum from patients with precancerous lesions or endometrial cancer, we found that tissue macrophage infiltration, but not serum estradiol levels, correlated positively with endometrial cancer development. Furthermore, IL4/IL13-induced CD68(+)CD163(+) macrophages enhanced the proliferative effects of estradiol in endometrial cancer cells by upregulating estrogen receptor alpha (ERα), but not ERβ. Mechanistic investigations revealed that CD68(+)CD163(+) macrophages secreted cytokines, such as IL17A, that upregulated ERα expression through TET1-mediated epigenetic modulation of the ERα gene. Overall, our findings show how cytokines produced by infiltrating macrophages in the endometrial microenvironment can induce epigenetic upregulation of ERα expression, which in turn sensitizes endometrial cells to estrogen stimulation. The concept that inflammation-induced estrogen sensitivity in the endometrium acts as a driver of type I endometrial cancer has implications for infiltrating macrophages as a prognostic biomarker of progression in this disease setting. PMID:26744532

  6. Suicide Gene-Mediated Sequencing Ablation Revealed the Potential Therapeutic Mechanism of Induced Pluripotent Stem Cell-Derived Cardiovascular Cell Patch Post-Myocardial Infarction

    PubMed Central

    Wang, Yuhua; Huang, Wei; Liang, Jialiang; Wen, Zhili; Chang, Dehua; Kang, Kai; Wang, Jiapeng; Xu, Meifeng; Millard, Ronald W.

    2014-01-01

    Abstract Aims: This study is designed to assess the protective cardiac effects after myocardial infarction (MI) of (i) cardiovascular progenitor cells (PC) differentiated directly into cardiomyocytes (CM) and endothelial cells (ECs) at the injury site, as separable from the effects of (ii) paracrine factors released from PC. Results: In vivo: bi-cell patch containing induced pluripotent stem cell (iPSC)-derived CM and EC (BIC) was transplanted onto the infarcted heart. BIC were transduced with herpes simplex virus thymidine kinase “suicide” gene driven by cardiac NCX1 or endothelial vascular endothelium-cadherin promoter. IGF-1α and VEGF levels released from ischemic tissues were significantly enhanced in the BIC patch treatment group. Heart function, infarction size, and vessel density were significantly improved after BIC patch treatment. These effects were completely abolished in the group given ganciclovir (GCV) at week 1 as a suicide gene activator, and partially abolished in the group given GCV at week 3 as compared with the untreated cell patch group. Innovation: This study was designed to distinguish between cell-based and noncell-based therapeutic effects of PC lineages after MI. PCs derived from iPSC were genetically modified to express “suicide” gene. iPSC-derived CM and EC were then ablated in situ at week 1 and 3 by intraperitoneal administration of GCV. This enabled direct assessment of the effects of iPSC transplantation on myocardial function and tissue regeneration potential. Conclusions: Data support a mechanism in which iPSC-derived cardiovascular lineages contribute directly to improved cardiac performance and attenuated remodeling. Paracrine factors provide additional support to the restoration of heart function. Antioxid. Redox Signal. 21, 2177–2191. PMID:24787391

  7. Mechanisms of corticosteroid action on lymphocyte subpopulations. III. Differential effects of dexamethasone administration on subpopulations of effector cells mediating cellular cytotoxicity in man

    PubMed Central

    Parrillo, J. E.; Fauci, A. S.

    1978-01-01

    The present study investigated the effect of dexamethasone (DEX) administration on different populations of mononuclear cells and neutrophils mediating antibody-dependent cellular cytotoxicity (ADCC) against different target cells. Mononuclear cells (lymphocytes and monocytes) and neutrophils were obtained from twenty-seven normal volunteers at 0, 4, 24 and 48 hr after oral administration of 21 mg of DEX. ADCC was determined utilizing the following targets: human red blood cells (HRBC), Chang liver cells (Ch) and human heart cells (HHC). The predominant mononuclear effector in HRBC killing was shown to be a monocyte and in Ch and HHC killing, a K cell. As previously shown, DEX produced a profound monocytopenia and lymphocytopenia at 4 hr with a return of lymphocyte counts to normal and monocyte counts to supra-normal at 24 hr. At the point of maximal monocytopenia, monocyte-mediated HRBC killing decreased from a geometric mean of 14 to 4 lytic units per 108 effector cells (P<0·05) and rebounded at 24 hr to a mean of 39 lytic units (P<0·02) with the rebound monocytosis. At the point of absolute lymphopenia (4 hr), there was a relative enrichment in the proportion of lymphocytes bearing an Fc receptor (K cells, P<0·01). Concomitant with this was an increase in ADCC against Ch and HHC from geometric means of 1121 to 7172 lytic units and 939 to 7354 lytic units (P<0·001) respectively. Thus, a major action of DEX administration on mononuclear ADCC was to differentially enrich or deplete different effector cells to and from the circulation, causing changes in cytotoxicity. Since the cytotoxicity paralleled the proportion of effector cells, the cells remaining in the circulation following DEX administration retained normal antibody-dependent cytotoxic capabilities. Neutrophil-mediated ADCC against HRBC significantly increased at 4 hr from a geometric mean of 3785 to 20142 lytic units (P<0·02) concomitant with the blood neutrophilia and remained elevated for 72 hr

  8. Estrogen-mediated mechanisms to control the growth and apoptosis of breast cancer cells: a translational research success story.

    PubMed

    McDaniel, Russell E; Maximov, Philipp Y; Jordan, V Craig

    2013-01-01

    The treatment and prevention of solid tumors have proved to be a major challenge for medical science. The paradigms for success in the treatment of childhood leukemia, Hodgkin's disease, Burkett's lymphoma, and testicular carcinoma with cytotoxic chemotherapy did not translate to success in solid tumors--the majority of cancers that kill. In contrast, significant success has accrued for patients with breast cancer with antihormone treatments (tamoxifen or aromatase inhibitors) that are proved to enhance survivorship, and remarkably, there are now two approved prevention strategies using either tamoxifen or raloxifene. This was considered impossible 40 years ago. We describe the major clinical advances with nonsteroidal antiestrogens that evolved into selective estrogen receptor modulators (SERMs) which successfully exploited the ER target selectively inside a woman's body. The standard paradigm that estrogen stimulates breast cancer growth has been successfully exploited for over 4 decades with therapeutic strategies that block (tamoxifen, raloxifene) or reduce (aromatase inhibitors) circulating estrogens in patients to stop breast tumor growth. But this did not explain why high-dose estrogen treatment that was the standard of care to treat postmenopausal breast cancer for 3 decades before tamoxifen caused tumor regression. This paradox was resolved with the discovery that breast cancer resistance to long-term estrogen deprivation causes tumor regression with physiologic estrogen through apoptosis. The new biology of estrogen action has been utilized to explain the findings in the Women's Health Initiative that conjugated equine estrogen alone given to postmenopausal women, average age 68, will produce a reduction of breast cancer incidence and mortality compared to no treatment. Estrogen is killing nascent breast cancer cells in the ducts of healthy postmenopausal women. The modulation of the ER using multifunctional medicines called SERMs has provided not only

  9. T-cell-mediated ganglionitis associated with acute sensory neuronopathy.

    PubMed

    Hainfellner, J A; Kristoferitsch, W; Lassmann, H; Bernheimer, H; Neisser, A; Drlicek, M; Beer, F; Budka, H

    1996-04-01

    A 67-year-old man presented with acute painful sensory loss, areflexia, ataxia, urinary retention, and severe constipation and became unable to walk within 2 weeks. He died suddenly 5 weeks after the onset of symptoms. Autopsy revealed widespread inflammation of sensory and autonomic ganglia with immunocytochemical evidence of a CD8+ T cell-mediated cytotoxic attack against ganglion neurons. This observation suggests a novel pathogenetic mechanism of immune-mediated human ganglion cell damage comparable to mechanisms operating in polymyositis.

  10. MicroRNA-mediated somatic cell reprogramming.

    PubMed

    Kuo, Chih-Hao; Ying, Shao-Yao

    2013-02-01

    Since the first report of induced pluripotent stem cells (iPSCs) using somatic cell nuclear transfer (SCNT), much focus has been placed on iPSCs due to their great therapeutic potential for diseases such as abnormal development, degenerative disorders, and even cancers. Subsequently, Takahashi and Yamanaka took a novel approach by using four defined transcription factors to generate iPSCs in mice and human fibroblast cells. Scientists have since been trying to refine or develop better approaches to reprogramming, either by using different combinations of transcription factors or delivery methods. However, recent reports showed that the microRNA expression pattern plays a crucial role in somatic cell reprogramming and ectopic introduction of embryonic stem cell-specific microRNAs revert cells back to an ESC-like state, although, the exact mechanism underlying this effect remains unclear. This review describes recent work that has focused on microRNA-mediated approaches to somatic cell reprogramming as well as some of the pros and cons to these approaches and a possible mechanism of action. Based on the pivotal role of microRNAs in embryogenesis and somatic cell reprogramming, studies in this area must continue in order to gain a better understanding of the role of microRNAs in stem cells regulation and activity. PMID:22961769

  11. Mechanical plasticity of cells

    NASA Astrophysics Data System (ADS)

    Bonakdar, Navid; Gerum, Richard; Kuhn, Michael; Spörrer, Marina; Lippert, Anna; Schneider, Werner; Aifantis, Katerina E.; Fabry, Ben

    2016-10-01

    Under mechanical loading, most living cells show a viscoelastic deformation that follows a power law in time. After removal of the mechanical load, the cell shape recovers only incompletely to its original undeformed configuration. Here, we show that incomplete shape recovery is due to an additive plastic deformation that displays the same power-law dynamics as the fully reversible viscoelastic deformation response. Moreover, the plastic deformation is a constant fraction of the total cell deformation and originates from bond ruptures within the cytoskeleton. A simple extension of the prevailing viscoelastic power-law response theory with a plastic element correctly predicts the cell behaviour under cyclic loading. Our findings show that plastic energy dissipation during cell deformation is tightly linked to elastic cytoskeletal stresses, which suggests the existence of an adaptive mechanism that protects the cell against mechanical damage.

  12. Surface mechanics mediate pattern formation in the developing retina.

    PubMed

    Hayashi, Takashi; Carthew, Richard W

    2004-10-01

    Pattern formation of biological structures involves organizing different types of cells into a spatial configuration. In this study, we investigate the physical basis of biological patterning of the Drosophila retina in vivo. We demonstrate that E- and N-cadherins mediate apical adhesion between retina epithelial cells. Differential expression of N-cadherin within a sub-group of retinal cells (cone cells) causes them to form an overall shape that minimizes their surface contact with surrounding cells. The cells within this group, in both normal and experimentally manipulated conditions, pack together in the same way as soap bubbles do. The shaping of the cone cell group and packing of its components precisely imitate the physical tendency for surfaces to be minimized. Thus, simple patterned expression of N-cadherin results in a complex spatial pattern of cells owing to cellular surface mechanics. PMID:15470418

  13. Chronic infection by Leishmania amazonensis mediated through MAPK ERK mechanisms

    PubMed Central

    Martinez, Pedro A.; Petersen, Christine A.

    2014-01-01

    Leishmania amazonensis is an intracellular protozoan parasite responsible for chronic cutaneous leishmaniasis (CL). CL is a neglected tropical disease responsible for infecting millions of people worldwide. L. amazonensis promotes alteration of various signaling pathways that are essential for host cell survival. Specifically, through parasite-mediated phosphorylation of extracellular signal regulated kinase (ERK), L. amazonensis inhibits cell-mediated parasite killing and promotes its own survival by co-opting multiple host cell functions. In this review we highlight Leishmania-host cell signaling alterations focusing on those specific to 1) motor proteins, 2) prevention of NADPH subunit phosphorylation impairing reactive oxygen species production (ROS), and 3) localized endosomal signaling to up-regulate ERK phosphorylation. This review will focus upon mechanisms and possible explanations as to how Leishmania spp. evades the various layers of defense employed by the host immune response. PMID:24838145

  14. Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

    2013-03-01

    Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

  15. Lifeguard inhibition of Fas-mediated apoptosis: A possible mechanism for explaining the cisplatin resistance of triple-negative breast cancer cells.

    PubMed

    Radin, Daniel; Lippa, Arnold; Patel, Parth; Leonardi, Donna

    2016-02-01

    Triple-negative breast cancer does not express estrogen receptor-α, progesterone or the HER2 receptor making hormone or antibody therapy ineffective. Cisplatin may initiate p73-dependent apoptosis in p53 mutant cell lines through Fas trimerization and Caspase-8 activation and Bax up regulation and subsequent Caspase-9 activation. The triple-negative breast cancer, MDA-MB-231, overexpresses the protein Lifeguard, which inhibits Fas-mediated apoptosis by inhibiting Caspase-8 activation after Fas trimerization. The relationship between Fas, Lifeguard and cisplatin is investigated by down regulating Lifeguard via shRNA. Results demonstrate that cisplatin's efficacy increases when Lifeguard is down regulated. Lifeguard Knockdown MDA-MB-231 continue to decrease in cell viability from 24 to 48h after cisplatin treatment while no additional decrease in viability is observed in the Wild-Type MDA over the same period. Higher Caspase-8 activity in the Lifeguard knockdown MDA after cisplatin administration could explain the significant decrease in cell viability from 24 to 48h. This cell type is also more sensitive to Fas ligand-mediated reductions in cell viability, confirming Lifeguard's anti-apoptotic function through the Fas receptor. This research suggests that the efficacy of chemotherapy acting through the Fas pathway would increase if Lifeguard were not overexpressed to inhibit Fas-mediated apoptosis. PMID:26796280

  16. Perspective and prejudice: antecedents and mediating mechanisms.

    PubMed

    Dovidio, John F; ten Vergert, Marleen; Stewart, Tracie L; Gaertner, Samuel L; Johnson, James D; Esses, Victoria M; Riek, Blake M; Pearson, Adam R

    2004-12-01

    The present work investigated mechanisms by which Whites' prejudice toward Blacks can be reduced (Study 1) and explored how creating a common ingroup identity can reduce prejudice by promoting these processes (Study 2). In Study 1, White participants who viewed a videotape depicting examples of racial discrimination and who imagined the victim's feelings showed greater decreases in prejudice toward Blacks than did those in the objective and no instruction conditions. Among the potential mediating affective and cognitive variables examined, reductions in prejudice were mediated primarily by feelings associated with perceived injustice. In Study 2, an intervention designed to increase perceptions of a common group identity before viewing the videotape, reading that a terrorist threat was directed at all Americans versus directed just at White Americans, also reduced prejudice toward Blacks through increases in feelings of injustice. PMID:15536238

  17. Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing.

    PubMed Central

    Merino, S; Camprubí, S; Albertí, S; Benedí, V J; Tomás, J M

    1992-01-01

    The different mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes K1, K10, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed. Images PMID:1587619

  18. Neuropeptide-mediated regulation of hapten-specific IgE responses in mice. II. Mechanisms of substance P-mediated isotype-specific suppression of BPO-specific IgE antibody-forming cell responses induced in vitro.

    PubMed

    Carucci, J A; Herrick, C A; Durkin, H G

    1994-01-01

    Previous studies in our laboratory have shown that substance P (SP), injected into benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) sensitized mice at the peak of the benzylpenicilloyl (BPO)-specific IgE response, suppressed these responses in isotype-specific fashion within 48 h. These studies also showed that SP, but not neurotensin (NT), serotonin (5-HT), somatostatin (SOM) or gastrin, suppressed BPO-specific memory IgE antibody-forming cell (AFC) responses induced in vitro, also in isotype-specific fashion. To investigate the mechanisms by which SP suppressed BPO-specific IgE AFC responses were induced in vitro, these responses were induced by culturing spleen cells from BPO-KLH sensitized mice for 5 days with BPO-KLH with or without whole SP, amino terminal SP (SP 1-4: Arg-Lys-Pro-Lys), or carboxy terminal SP (SP 8-11: Phe-Gly-Leu-Met). In some experiments, the SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP (D-SP) was included in culture. In other experiments anti-interferon monoclonal antibody (anti-IFN gamma mAb) was in culture. Whole SP and SP 8-11, but not SP 1-4, suppressed BPO-specific IgE AFC responses induced in vitro. The suppression obtained was IgE isotype-specific and dose-dependent. Inclusion of SP receptor antagonist (D-Pro2, D-Phe7, D-Trp9)-SP inhibited suppression of BPO-specific memory IgE AFC responses by SP or SP 8-11. The SP-mediated suppression of BPO-specific memory IgE responses appeared to involve interferon gamma (IFN gamma).

  19. Photodynamic Action Mechanism Mediated by Zinc(II) 2,9,16,23-Tetrakis[4-(N-methylpyridyloxy)]phthalocyanine in Candida albicans Cells.

    PubMed

    Di Palma, María Albana; Alvarez, María Gabriela; Durantini, Edgardo N

    2015-01-01

    The photoreaction type I/type II pathways mediated by zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc(4+) ) was studied in Candida albicans cells. This photosensitizer was strongly bound to C. albicans cells at short times. After 30 min irradiation, 5 μM ZnPPc(4+) produced ~5 log decrease in cell viability. Different probes were used to detect reactive oxygen species (ROS) in cell suspensions (~10(6) CFU mL(-1) ). Singlet molecular oxygen, O2 ((1) Δg ), was observed by the reaction with 9,10-dimethylanthracene (DMA) and tetrasodium 2,2-(anthracene-9,10-diyl)bis(methylmalonate) (ABMM), whereas the nitro blue tetrazolium (NBT) method was used to sense superoxide anion radical (O2·-). Moreover, the effects produced by an anoxic atmosphere and cell suspensions in D2 O, as well as the addition of sodium azide and mannitol as ROS trapping were evaluated in the PDI of C. albicans. These investigation indicates that O2 ((1) Δg ) is generated in the cells, although a minor extension other radical species can also be involved in the PDI of C. albicans mediated by ZnPPc(4+) . PMID:26108811

  20. Caffeine inhibits UV-mediated NF-kappaB activation in A2058 melanoma cells: an ATM-PKCdelta-p38 MAPK-dependent mechanism.

    PubMed

    Ravi, Dashnamoorthy; Muniyappa, Harish; Das, Kumuda C

    2008-01-01

    Mammalian ultraviolet (UV) radiation response is a gene induction cascade activated by several transcription factors, including NF-kappaB. Although NF-kappaB is induced by UV radiation, the signal transduction mechanism remains relatively unclear. In the present study, we show that UV-induced NF-kappaB activation is mediated by the activation of Ataxia telangiecia mutated (ATM) and protein kinase C (PKC). We also show that caffeine specifically inhibits UV-mediated NF-kappaB activation, but not TNFalpha-mediated NF-kappaB activation. In addition, our study shows that ATM, but not ATM-Rad3-related (ATR) or DNA-dependent protein kinase (DNA-PK) is involved in UV-induced NF-kappaB activation. Because SB203580 (a p38 MAPK inhibitor), or Calphostin C or rottlerin (PKC inhibitors) was able to inhibit UV-mediated NF-kappaB activation, we evaluated whether caffeine could inhibit p38 MAPK or PKC activity. Caffeine or rottlerin inhibited UV-induced phosphorylation of p38 MAPK, but not anisomycin-induced phosphorylation of p38 MAPK, suggesting that p38 MAPK is downstream of PKC. Additionally, caffeine could effectively inhibit UV-induced increases in PKC activity. Taken together, our study demonstrates that caffeine is a potent inhibitor of UV-induced NF-kappaB activation. Additionally, this inhibition occurs due to the inhibitory action of caffeine on ATM and PKC, resulting in the inhibition of p38 MAPK activation.

  1. Cell-mediated immunity in nutritional deficiency.

    PubMed

    McMurray, D N

    1984-01-01

    Dietary deficiencies of specific nutrients profoundly alter cell-mediated immune responses in man and experimental animals. Both moderate and severe deficiencies are associated with significant changes in immunocompetence. Diets with inadequate levels of protein, calories, vitamin A, pyridoxine, biotin and zinc result in loss of thymic cellularity. Secondary to thymic atrophy, the production of thymic hormones critical for the differentiation of T lymphocytes is reduced, especially in protein-calorie malnutrition and zinc deficiency. Confirmation of a T cell maturational defect in nutritional deprivation comes from the observations of decreased total (T3 and rosette-forming) T cells in the peripheral blood of children with kwashiorkor and marasmus, with preferential loss of helper/inducer (T4) T cell subsets. Reduced number and in vitro function of T cells have also been reported in experimental deficiencies of iron, zinc, copper, and vitamins A and E. Loss of cutaneous hypersensitivity to mitogens and antigens is a consistent sequela of dietary deficiencies of protein, vitamins A and C, pyridoxine, iron and zinc. Cell-mediated immunity directed against allogeneic histocompatibility antigens (e.g. mixed leukocyte cultures, graft versus host, skin graft rejection) may actually be enhanced by experimental protein and polyunsaturated fat deficiencies. Alternatively, pyridoxine, ascorbate and biotin deficiencies resulted in delayed rejection of skin allografts. Cytotoxic T lymphocyte (CTL) activity is impaired in zinc-, iron- and copper-deficient mice, as well as in scorbutic guinea pigs. Natural killer (NK) cell function may be either enhanced or depressed, depending upon the nutrient and its effects on interferon production. Several authors have demonstrated normal or enhanced macrophage activity in a variety of experimental deficiencies. The extrapolation of these observations to infectious disease resistance is not straightforward, and depends upon the nature of

  2. Mechanisms of interleukin-10-mediated immune suppression

    PubMed Central

    Akdis, Cezmi A; Blaser, Kurt

    2001-01-01

    Specific immune suppression and induction of anergy are essential processes in the regulation and circumvention of immune defence. Interleukin-10 (IL-10), a suppressor cytokine of T-cell proliferative and cytokine responses, plays a key regulatory role in tolerizing exogenous antigens during specific immunotherapy (SIT) of allergy and natural exposure to antigens. Specific T-cell tolerance is directed against the T-cell epitopes of an antigen and characterized by suppressed proliferative and T helper type 1 (Th1) and type 2 (Th2) cytokine responses. IL-10 elicits tolerance in T cells by selective inhibition of the CD28 co-stimulatory pathway and thereby controls suppression and development of antigen-specific immunity. IL-10 only inhibits T cells stimulated by low numbers of triggered T-cell receptors and which therefore depend on CD28 co-stimulation. T cells receiving a strong signal from the T-cell receptor alone, and thus not requiring CD28 co-stimulation, are not affected by IL-10. IL-10 inhibits CD28 tyrosine phosphorylation, the initial step of the CD28 signalling pathway, and consequently the phosphatidylinositol 3-kinase p85 binding to CD28. Together these results demonstrate that IL-10-induced selective inhibition of the CD28 co-stimulatory pathway acts as a decisive mechanism in determining whether a T cell will contribute to an immune response or become anergic. PMID:11412299

  3. Mechanisms for virus-induced liver disease: tumor necrosis factor-mediated pathology independent of natural killer and T cells during murine cytomegalovirus infection.

    PubMed Central

    Orange, J S; Salazar-Mather, T P; Opal, S M; Biron, C A

    1997-01-01

    The contribution of endogenous NK cells and cytokines to virus-induced liver pathology was evaluated during murine cytomegalovirus infections of mice. In immunocompetent C57BL/6 mice, the virus induced a self-limited liver disease characterized by hepatitis, with focal inflammation, and large grossly visible subcapsular necrotic foci. The inflammatory foci were most numerous and contained the greatest number of cells 3 days after infection; they colocalized with areas of viral antigen expression. The largest number of necrotic foci was found 2 days after infection. Overall hepatic damage, assessed as increased expression of liver enzymes in serum, accompanied the development of inflammatory and necrotic foci. Experiments with neutralizing antibodies demonstrated that although virus-induced tumor necrosis factor (TNF) can have antiviral effects, it also mediated significant liver pathology. TNF was required for development of hepatic necrotic foci and increased levels of liver enzymes in serum but not for increased numbers of inflammatory foci. The necrotic foci and liver enzyme indications of pathology occurred independently of NK and T cells, because mice rendered NK-cell deficient by treatment with antibodies, T- and B-cell-deficient Rag-/- mice, and NK- and T-cell-deficient E26 mice all manifested both parameters of disease. Development of necrotic foci and maximally increased levels of liver enzymes in serum also were TNF dependent in NK-cell-deficient mice. Moreover, in the immunodeficient E26 mice, virus-induced liver disease was progressive, with eventual death of the host, and neutralization of TNF significantly increased longevity. These results establish conditions separating hepatitis from significant liver damage and demonstrate a cytokine-mediated component to viral pathogenesis. PMID:9371583

  4. The spliceosomal phosphopeptide P140 controls the lupus disease by interacting with the HSC70 protein and via a mechanism mediated by gammadelta T cells.

    PubMed

    Page, Nicolas; Schall, Nicolas; Strub, Jean-Marc; Quinternet, Marc; Chaloin, Olivier; Décossas, Marion; Cung, Manh Thong; Van Dorsselaer, Alain; Briand, Jean-Paul; Muller, Sylviane

    2009-01-01

    The phosphopeptide P140 issued from the spliceosomal U1-70K snRNP protein is recognized by lupus CD4(+) T cells, transiently abolishes T cell reactivity to other spliceosomal peptides in P140-treated MRL/lpr mice, and ameliorates their clinical features. P140 modulates lupus patients' T cell response ex vivo and is currently included in phase IIb clinical trials. Its underlying mechanism of action remains elusive. Here we show that P140 peptide binds a unique cell-surface receptor, the constitutively-expressed chaperone HSC70 protein, known as a presenting-protein. P140 induces apoptosis of activated MRL/lpr CD4(+) T cells. In P140-treated mice, it increases peripheral blood lymphocyte apoptosis and decreases B cell, activated T cell, and CD4(-)CD8(-)B220(+) T cell counts via a specific mechanism strictly depending on gammadelta T cells. Expression of inflammation-linked genes is rapidly regulated in CD4(+) T cells. This work led us to identify a powerful pathway taken by a newly-designed therapeutic peptide to immunomodulate lupus autoimmunity.

  5. Regulation of the pentose phosphate pathway by an androgen receptor-mTOR-mediated mechanism and its role in prostate cancer cell growth.

    PubMed

    Tsouko, E; Khan, A S; White, M A; Han, J J; Shi, Y; Merchant, F A; Sharpe, M A; Xin, L; Frigo, D E

    2014-05-26

    Cancer cells display an increased demand for glucose. Therefore, identifying the specific aspects of glucose metabolism that are involved in the pathogenesis of cancer may uncover novel therapeutic nodes. Recently, there has been a renewed interest in the role of the pentose phosphate pathway in cancer. This metabolic pathway is advantageous for rapidly growing cells because it provides nucleotide precursors and helps regenerate the reducing agent NADPH, which can contribute to reactive oxygen species (ROS) scavenging. Correspondingly, clinical data suggest glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, is upregulated in prostate cancer. We hypothesized that androgen receptor (AR) signaling, which plays an essential role in the disease, mediated prostate cancer cell growth in part by increasing flux through the pentose phosphate pathway. Here, we determined that G6PD, NADPH and ribose synthesis were all increased by AR signaling. Further, this process was necessary to modulate ROS levels. Pharmacological or molecular inhibition of G6PD abolished these effects and blocked androgen-mediated cell growth. Mechanistically, regulation of G6PD via AR in both hormone-sensitive and castration-resistant models of prostate cancer was abolished following rapamycin treatment, indicating that AR increased flux through the pentose phosphate pathway by the mammalian target of rapamycin (mTOR)-mediated upregulation of G6PD. Accordingly, in two separate mouse models of Pten deletion/elevated mTOR signaling, Pb-Cre;Pten(f/f) and K8-CreER(T2);Pten(f/f), G6PD levels correlated with prostate cancer progression in vivo. Importantly, G6PD levels remained high during progression to castration-resistant prostate cancer. Taken together, our data suggest that AR signaling can promote prostate cancer through the upregulation of G6PD and therefore, the flux of sugars through the pentose phosphate pathway. Hence, these findings support a

  6. Force nanoscopy of cell mechanics and cell adhesion

    NASA Astrophysics Data System (ADS)

    Dufrêne, Yves F.; Pelling, Andrew E.

    2013-05-01

    Cells are constantly exposed to mechanical stimuli in their environment and have several evolved mechanisms to sense and respond to these cues. It is becoming increasingly recognized that many cell types, from bacteria to mammalian cells, possess a diverse set of proteins to translate mechanical cues into biochemical signalling and to mediate cell surface interactions such as cell adhesion. Moreover, the mechanical properties of cells are involved in regulating cell function as well as serving as indicators of disease states. Importantly, the recent development of biophysical tools and nanoscale methods has facilitated a deeper understanding of the role that physical forces play in modulating cell mechanics and cell adhesion. Here, we discuss how atomic force microscopy (AFM) has recently been used to investigate cell mechanics and cell adhesion at the single-cell and single-molecule levels. This knowledge is critical to our understanding of the molecular mechanisms that govern mechanosensing, mechanotransduction, and mechanoresponse in living cells. While pushing living cells with the AFM tip provides a means to quantify their mechanical properties and examine their response to nanoscale forces, pulling single surface proteins with a functionalized tip allows one to understand their role in sensing and adhesion. The combination of these nanoscale techniques with modern molecular biology approaches, genetic engineering and optical microscopies provides a powerful platform for understanding the sophisticated functions of the cell surface machinery, and its role in the onset and progression of complex diseases.

  7. Mechanisms for macrophage-mediated HIV-1 induction.

    PubMed

    Devadas, Krishnakumar; Hardegen, Neil J; Wahl, Larry M; Hewlett, Indira K; Clouse, Kathleen A; Yamada, Kenneth M; Dhawan, Subhash

    2004-12-01

    Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.

  8. Mechanism of Resistance and Novel Targets Mediating Resistance to EGFR and c-Met Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer

    PubMed Central

    Chhabra, Gagan; Nlend, Marie

    2015-01-01

    Tyrosine kinase inhibitors (TKIs) against EGFR and c-Met are initially effective when administered individually or in combination to non-small cell lung cancer (NSCLC) patients. However, the overall efficacies of TKIs are limited due to the development of drug resistance. Therefore, it is important to elucidate mechanisms of EGFR and c-Met TKI resistance in order to develop more effective therapies. Model NSCLC cell lines H1975 and H2170 were used to study the similarities and differences in mechanisms of EGFR/c-Met TKI resistance. H1975 cells are positive for the T790M EGFR mutation, which confers resistance to current EGFR TKI therapies, while H2170 cells are EGFR wild-type. Previously, H2170 cells were made resistant to the EGFR TKI erlotinib and the c-Met TKI SU11274 by exposure to progressively increasing concentrations of TKIs. In H2170 and H1975 TKI-resistant cells, key Wnt and mTOR proteins were found to be differentially modulated. Wnt signaling transducer, active β-catenin was upregulated in TKI-resistant H2170 cells when compared to parental cells. GATA-6, a transcriptional activator of Wnt, was also found to be upregulated in resistant H2170 cells. In H2170 erlotinib resistant cells, upregulation of inactive GSK3β (p-GSK3β) was observed, indicating activation of Wnt and mTOR pathways which are otherwise inhibited by its active form. However, in H1975 cells, Wnt modulators such as active β-catenin, GATA-6 and p-GSK3β were downregulated. Additional results from MTT cell viability assays demonstrated that H1975 cell proliferation was not significantly decreased after Wnt inhibition by XAV939, but combination treatment with everolimus (mTOR inhibitor) and erlotinib resulted in synergistic cell growth inhibition. Thus, in H2170 cells and H1975 cells, simultaneous inhibition of key Wnt or mTOR pathway proteins in addition to EGFR and c-Met may be a promising strategy for overcoming EGFR and c-Met TKI resistance in NSCLC patients. PMID:26301867

  9. Anoikis Mediators in Oral Squamous Cell Carcinoma

    PubMed Central

    Bunek, Julius; Kamarajan, Pachiyappan; Kapila, Yvonne L.

    2010-01-01

    Anoikis—apoptotic cell death triggered by loss of extracellular matrix (ECM) contacts— is dysregulated in many chronic debilitating and fatal diseases. Mechanisms rendering tumor cells resistant to anoikis, although not completely understood, possess significant therapeutic promise. In death receptor-mediated anoikis mechanisms, FAK and RIP dissociate, leading to association of RIP with Fas, formation of the death-inducing signaling complex (DISC), activation of caspase-3, and propagation of anoikis. In contrast, anoikis resistance is accomplished through constitutive activation of survival pathways that include integrin-dependent activation of focal adhesion kinase (FAK) and extracellular-signal-regulated kinase (ERK). In addition, FAK and receptor-interacting protein (RIP) association confers anoikis resistance by inhibiting the association of RIP with Fas and formation of the death signaling complex, which allows cells to escape anoikis. Up-regulation of CD44 also contributes to survival signals and promotes anoikis resistance. This review will focus on the roles of death receptors, pro-survival pathways, and the molecular players involved in anoikis escalation and resistance in OSCC. PMID:21114588

  10. Molecular Mechanisms of HTLV-1 Cell-to-Cell Transmission

    PubMed Central

    Gross, Christine; Thoma-Kress, Andrea K.

    2016-01-01

    The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation. PMID:27005656

  11. PERK-eIF2α-ATF4 pathway mediated by endoplasmic reticulum stress response is involved in osteodifferentiation of human periodontal ligament cells under cyclic mechanical force.

    PubMed

    Yang, Shuang-Yan; Wei, Fu-Lan; Hu, Li-Hua; Wang, Chun-Ling

    2016-08-01

    To prevent excess accumulation of unfolded proteins in endoplasmic reticulum (ER), eukaryotic cells have signaling pathways from the ER to the cytosol or nucleus. These processes are known as the endoplasmic reticulum stress (ERS) response. Protein kinase R like endoplasmic reticulum kinase (PERK) is a major transducer of the ERS response and it directly phosphorylate α-subunit of eukaryotic initiation factor 2 (eIF2α), resulting in translational attenuation. Phosphorylated eIF2α specifically promoted the translation of the activating transcription factor 4 (ATF4). ATF4 is a known important transcription factor which plays a pivotal role in osteoblast differentiation and bone formation. Furthermore, ATF4 is a downstream target of PERK. Studies have shown that PERK-eIF2α-ATF4 signal pathway mediated by ERS was involved in osteoblastic differentiation of osteoblasts. We have known that orthodontic tooth movement is a process of periodontal ligament cells (PDLCs) osteodifferentiation and alveolar bone remodeling under mechanical force. However, the involvement of PERK-eIF2α-ATF4 signal pathway mediated by ERS in osteogenic differentiation of PDLCs under mechanical force has not been unclear. In our study, we applied the cyclic mechanical force at 10% elongation with 0.5Hz to mimic occlusal force, and explored whether PERK-eIF2α-ATF4 signaling pathway mediated by ERS involved in osteogenic differentiation of PDLCs under mechanical force. Firstly, cyclic mechanical force will induce ERS and intensify several osteoblast marker genes (ATF4, OCN, and BSP). Next, we found that PERK overexpression increased eIF2α phosphorylation and expression of ATF4, furthermore induced BSP, OCN expression, thus it will promote osteodifferentiation of hPDLCs; mechanical force could promote this effect. However, PERK(-/-) cells showed the opposite changes, which will inhibit osteodifferentiation of hPDLCs. Taken together, our study proved that PERK-eIF2α-ATF4 signaling pathway

  12. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

    PubMed Central

    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  13. Mechanics of Cell Growth

    PubMed Central

    Ateshian, Gerard A.; Morrison, Barclay; Holmes, Jeffrey W.; Hung, Clark T.

    2012-01-01

    Cell growth describes an essential feature of biological tissues. This growth process may be modeled by using a set of relatively simple governing equations based on the axioms of mass and momentum balance, and using a continuum framework that describes cells and tissues as mixtures of a solid matrix, a solvent and multiple solutes. In this model the mechanics of cell growth is driven by osmotic effects, regulated by the cells’ active uptake of solutes and passive uptake of solvent. By accounting for the anisotropy of the cells’ cytoskeletal structures or extracellular matrix, as well as external constraints, a wide variety of growing shapes may be produced as illustrated in various examples. PMID:22904576

  14. Anti-apoptotic mechanism of Bacoside rich extract against reactive nitrogen species induced activation of iNOS/Bax/caspase 3 mediated apoptosis in L132 cell line.

    PubMed

    Anand, T; Pandareesh, M D; Bhat, Pratiksha V; Venkataramana, M

    2014-10-01

    Nitric oxide is a highly reactive free radical gas that reacts with a wide range of bio-molecules to produce reactive nitrogen species and exerts nitrative stress. Bacopa monniera is a traditional folk and ayurvedic medicine known to alleviate a variety of disorders. Aim of the present study is to evaluate the protective propensity of Bacopa monniera extract (BME) through its oxido-nitrosative and anti-apoptotic mechanism to attenuate sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132). Our results elucidate that pre-treatment of L132 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP as evidenced by MTT and LDH leakage assays. BME pre-treatment inhibited NO generation by down-regulating inducible nitric oxide synthase expression. BME exhibited potent antioxidant activity by up-regulating the antioxidant enzymes. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic biomarkers such as Bax, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. By considering all these findings, we report that BME protects L132 cells against SNP-induced toxicity via its free radical scavenging and anti-apoptotic mechanism.

  15. hCG-induced Sprouty2 mediates amphiregulin-stimulated COX-2/PGE2 up-regulation in human granulosa cells: a potential mechanism for the OHSS

    PubMed Central

    Cheng, Jung-Chien; Fang, Lanlan; Chang, Hsun-Ming; Sun, Ying-Pu; Leung, Peter C. K.

    2016-01-01

    Sprouty2 (SPRY2) is an important intracellular regulator for epidermal growth factor receptor (EGFR)-mediated ERK1/2 signaling. In human granulosa cells, although SPRY2 is expressed, its regulation and function remains complete unknown and must be defined. Our previous study has shown that human chorionic gonadotropin (hCG)/luteinizing hormone (LH) up-regulates the expression levels of EGF-like growth factor, amphiregulin (AREG), which subsequently contributes to the hCG/LH-induced COX-2 expression and PGE2 production. The aim of the present study was to investigate the effect of hCG on SPRY2 expression and the role of hCG-induced SPRY2 in AREG-stimulated COX-2 expression and PGE2 production in human granulosa cells. Our results demonstrated that the expression of SPRY2 was up-regulated by hCG treatment. Using pharmacological inhibitors and siRNA knockdown, we showed that activation of ERK1/2 signaling was required for hCG-induced up-regulation of SPRY2 expression. Further, SPRY2 knockdown attenuated the AREG-induced COX-2 expression and PGE2 production by inhibiting AREG-activated ERK1/2 signaling. Interestingly, we showed that SPRY2 expression levels were significantly increased in granulosa cells of ovarian hyperstimulation syndrome (OHSS) patients. These results for the first time elucidate the physiological roles of SPRY2 in human granulosa cells and suggest that aberrant expression of SPRY2 may contribute to the pathogenesis of OHSS. PMID:27539669

  16. Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

    PubMed

    Zafar, Atif; Singh, Swarnendra; Naseem, Imrana

    2016-07-01

    Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells. PMID:27260464

  17. Autocrine control of MIP-2 secretion from metastatic breast cancer cells is mediated by CXCR2: a mechanism for possible resistance to CXCR2 antagonists.

    PubMed

    Erin, Nuray; Nizam, Esra; Tanrıöver, Gamze; Köksoy, Sadi

    2015-02-01

    CXCR2 interacts with a wide range of chemokines and CXCR2 antagonists may have therapeutic value for treatment-resistant metastatic carcinomas. We aimed to explore regulation of activity of CXCR2 and its ligand, MIP-2, in metastatic breast carcinoma. We used mouse breast carcinoma cells metastasize to brain (4TBM), liver (4TLM), and heart (4THM) and explored the extra- and intracellular mechanisms effecting MIP-2 secretion using CXCR2 antagonist and inhibitors of downstream signaling molecules. 4TBM, 4TLM, and 4THM cells include cancer stem cell features and metastasize extensively. We also determined kinetics of MIP-2 secretion in 4T1 and non-metastatic 67NR mouse breast carcinoma cells. We found that there is an autocrine-inhibition of MIP-2 secretion. Specifically, metastatic cells selectively express CXCR2 only, and not CXCR1 and attenuating CXCR2 activity with SB225002 increased MIP-2 secretion. This may be due to the inhibition of protein kinase C (PKC) activity since RO318220; a specific inhibitor of PKC also increased MIP-2 secretion. Attenuating CXCR2 activity with SB225002, otherwise suppressed proliferation of 4THM and 4TBM cells. Tumor explants and cancer-associated fibroblasts obtained from 4TLM, 4THM, and 4TBM primary tumors secreted high levels of MIP-2. Surprisingly, CXCR2 expression was low in 4TLM cells demonstrating that liver metastatic cells might be resistant to the anti-tumoral effects of CXCR2 antagonists. Our results demonstrated that resistance to anti-proliferative effects of CXCR2 may also arise from feedback increases in MIP-2 secretion. Activation of PI3 K pathway augments MIP-2 secretion, hence possible resistance to the antitumor effects of CXCR2 antagonists might be prevented with inhibitors of PI3 K.

  18. Mechanical Properties of Cells

    NASA Technical Reports Server (NTRS)

    Bradley, Robert; Becerril, Joseph; Jeevarajan, Anthony

    2007-01-01

    Many physiologic and pathologic processes alter the biomechanical properties of the tissue they affect, and these changes may be manifest at the single cell level. The normal and abnormal mechanical properties of a given cell type can be established with the aid of an atomic force microscope (AFM), nonetheless, consistency in the area of the tip has been a mayor limitation of using the AFM for quantitative measurements of mechanical properties. This project attempts to overcome this limitation by using materials with a known elastic modulus, which resembles the one of the cell, to create force-deformation curves to calculate the area of indentation by means of Hooke s Law (sigma = E(epsilon)), which states that stress (sigma) is proportional to the strain (epsilon) where the constant of proportionality, E, is called the Young s modulus, also referred as the elastic modulus. Hook s Law can be rearranged to find the area of indentation (Area= Force/ E(epsilon)), where the indentation force is defined by the means of the added mass spring calibration method.

  19. Adipokines and the cardiovascular system: mechanisms mediating health and disease.

    PubMed

    Northcott, Josette M; Yeganeh, Azadeh; Taylor, Carla G; Zahradka, Peter; Wigle, Jeffrey T

    2012-08-01

    This review focuses on the role of adipokines in the maintenance of a healthy cardiovascular system, and the mechanisms by which these factors mediate the development of cardiovascular disease in obesity. Adipocytes are the major cell type comprising the adipose tissue. These cells secrete numerous factors, termed adipokines, into the blood, including adiponectin, leptin, resistin, chemerin, omentin, vaspin, and visfatin. Adipose tissue is a highly vascularised endocrine organ, and different adipose depots have distinct adipokine secretion profiles, which are altered with obesity. The ability of many adipokines to stimulate angiogenesis is crucial for adipose tissue expansion; however, excessive blood vessel growth is deleterious. As well, some adipokines induce inflammation, which promotes cardiovascular disease progression. We discuss how these 7 aforementioned adipokines act upon the various cardiovascular cell types (endothelial progenitor cells, endothelial cells, vascular smooth muscle cells, pericytes, cardiomyocytes, and cardiac fibroblasts), the direct effects of these actions, and their overall impact on the cardiovascular system. These were chosen, as these adipokines are secreted predominantly from adipocytes and have known effects on cardiovascular cells.

  20. Physiological mechanisms mediating aspartame-induced satiety.

    PubMed

    Hall, W L; Millward, D J; Rogers, P J; Morgan, L M

    2003-04-01

    Aspartame has been previously shown to increase satiety. This study aimed to investigate a possible role for the satiety hormones cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) in this effect. The effects of the constituents of aspartame, phenylalanine and aspartic acid, were also examined. Six subjects consumed an encapsulated preload consisting of either 400 mg aspartame, 176 mg aspartic acid+224 mg phenylalanine, or 400 mg corn flour (control), with 1.5 g paracetamol dissolved in 450 ml water to measure gastric emptying. A 1983-kJ liquid meal was consumed 60 min later. Plasma CCK, GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucose, and insulin were measured over 0-120 min. Gastric emptying was measured from 0 to 60 min. Plasma GLP-1 concentrations decreased following the liquid meal (60-120 min) after both the aspartame and amino acids preloads (control, 2096.9 pmol/l min; aspartame, 536.6 pmol/l min; amino acids, 861.8 pmol/l min; incremental area under the curve [AUC] 60-120 min, P<.05). Desire to eat was reduced from 60 to 120 min following the amino acids preload (control, -337.1 mm min; aspartame, -505.4 mm min; amino acids, -1497.1 mm min; incremental AUC 60-120 min, P<.05). However, gastric emptying rates, plasma CCK, GIP, insulin, and glucose concentrations were unaffected. There was a correlation between the increase in plasma phenylalanine and decrease in desire to eat after the liquid meal following the constituent amino acids (r=-.9774, P=.004). In conclusion, it is unlikely that aspartame increases satiety via CCK- or GLP-1-mediated mechanisms, but small changes in circulating phenylalanine concentrations may influence appetite.

  1. Distinct docking mechanisms mediate interactions between the Msg5 phosphatase and mating or cell integrity mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae.

    PubMed

    Palacios, Lorena; Dickinson, Robin J; Sacristán-Reviriego, Almudena; Didmon, Mark P; Marín, María José; Martín, Humberto; Keyse, Stephen M; Molina, María

    2011-12-01

    MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif ((102)IYT(104)) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274-373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity. PMID:22006927

  2. Mechanics of Dynamin-Mediated Membrane Fission

    PubMed Central

    Morlot, Sandrine; Roux, Aurélien

    2013-01-01

    In eukaryotic cells, membrane compartments are split into two by membrane fission. This ensures discontinuity of membrane containers and thus proper compartmentalization. The first proteic machinery implicated in catalyzing membrane fission was dynamin. Dynamin forms helical collars at the neck of endocytic buds. This structural feature suggested that the helix of dynamin could constrict in order to promote fission of the enclosed membrane. However, verifying this hypothesis revealed itself to be a challenge, which inspired many in vitro and in vivo studies. The primary goal of this review is to discuss recent structural and physical data from biophysical studies that have refined our understanding of the dynamin mechanism. In addition to the constriction hypothesis, other models have been proposed to explain how dynamin induces membrane fission. We present experimental data supporting these various models and assess which model is the most probable. PMID:23541160

  3. The mechanics of cell protrusion

    NASA Astrophysics Data System (ADS)

    Danuser, Gaudenz

    2006-03-01

    The protrusion of the cell edge is the first step in a cycle of molecular processes that drive cell movements during development, immune responses, wound healing and many other physiological functions. It is also the earliest pathological event observed during metastasis of cancer. Textbook models associate protrusion with the assembly of an actin polymer network subadjacent to the cell plasma membrane. However, for this process to be transformed into edge advancement, polymerization-induced forces need to be balanced by adhesion complexes that link the actin network to the extracellular domain. Also, the effectiveness of network assembly in mediating forward movement of the cell edge depends on how contraction forces pull the network in the cell front retrogradly towards the cell center. Thus, what is observed in a microscope as cell protrusion reflects the kinematic output of at least three space- and time-modulated mechanisms of force generation. The coordination of these machineries is thought to be regulated by a complex network of mechano-chemical signals. Our goal is to establish the contributions of each those mechanisms and their control by reconstructing the spatiotemporal distribution of intracellular forces via inverse dynamics and molecular intervention with the relevant signalling pathways. To this end, we have developed quantitative Fluorescent Speckle Microscopy (qFSM) which provides high-resolution spatiotemporal measurements of actin network deformation and material properties in migrating cells. In addition, qFSM delivers maps of cytoskeleton assembly and disassembly, so that we can infer the plasticity of the material in situ. Together, this data allows us to deduce intracellular force distributions from the constitutive laws of strain and stress in the actin polymer network. Using this approach we discovered that unperturbed cells protrude in a dynamic steady state where periodic patterns of network assembly, adhesion formation, and

  4. Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: putative anticancer mechanism of antioxidants.

    PubMed

    Zubair, H; Khan, H Y; Sohail, A; Azim, S; Ullah, M F; Ahmad, A; Sarkar, F H; Hadi, S M

    2013-06-06

    Plant-derived dietary antioxidants have attracted considerable interest in recent past for their chemopreventive and cancer therapeutic abilities in animal models. Thymoquinone (TQ) is the major bioactive constituent of volatile oil of Nigella sativa and has been shown to exert various pharmacological properties, such as anti-inflammatory, cardiovascular, analgesic, anti-neoplastic, anticancer and chemopreventive. Although several mechanisms have been suggested for the chemopreventive and anticancer activity of TQ, a clear mechanism of action of TQ has not been elucidated. TQ is a known antioxidant at lower concentrations and most of the studies elucidating the mechanism have centered on the antioxidant property. However, recent publications have shown that TQ may act as a prooxidant at higher concentrations. It is well known that plant-derived antioxidants can switch to prooxidants even at low concentrations in the presence of transition metal ions such as copper. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Copper is an important metal ion present in the chromatin and is closely associated with DNA bases, particularly guanine. Using human peripheral lymphocytes and comet assay, we first show that TQ is able to cause oxidative cellular DNA breakage. Such a DNA breakage can be inhibited by copper-chelating agents, neocuproine and bathocuproine, and scavengers of reactive oxygen species. Further, it is seen that TQ targets cellular copper in prostate cancer cell lines leading to a prooxidant cell death. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of plant-derived antioxidants.

  5. Mechanisms of Gefitinib-mediated reversal of tamoxifen resistance in MCF-7 breast cancer cells by inducing ERα re-expression.

    PubMed

    Zhang, Xia; Zhang, Bin; Liu, Jie; Liu, Jiwei; Li, Changzheng; Dong, Wei; Fang, Shu; Li, Minmin; Song, Bao; Tang, Bo; Wang, Zhehai; Zhang, Yang

    2015-02-03

    Estrogen receptor (ER)-positive breast cancer patients may turn ER-negative and develop acquired drug resistance, which compromises the efficacy of endocrine therapy. By investigating the phenomenon that gefitinib can re-sensitise tamoxifen (TAM)-resistant MCF-7 breast cancer cells (MCF-7/TAM) to TAM, the present study verified that gefitinib could reverse the acquired drug resistance in endocrine therapy and further explored the underlying mechanism.ERα-negative MCF-7/TAM cells were established. Upon treating the cells with gefitinib, the mRNA and protein levels of ERα and ERβ, as well as the expression of molecules involved in the MAPK pathway, were examined using the RT-PCR and immunocytochemistry. The RT-PCR results showed that the mRNA levels of ERα and ERβ in MCF-7/TAM cells were up-regulated following gefitinib treatment; specifically, ERα was re-expressed, and ERβ expression was up-regulated. The expression of molecules involved in the MAPK pathway, including RAS, MEK1/2, and p-ERK1/2, in MCF-7/TAM cells was significantly up-regulated, compared with MCF-7 cells. After the gefitinib treatment, the expression levels of MEK1/2 and p-ERK1/2 were significantly down-regulated. ERα loss is the primary cause for TAM resistance. Gefitinib reverses TAM resistance primarily by up-regulating the ERα mRNA level and inducing the re-expression of ERα. The MAPK pathway plays a key role in ERα re-expression.

  6. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.

    PubMed

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-07-06

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications.

  7. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism

    PubMed Central

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-01-01

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)–nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications. PMID:27380727

  8. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.

    PubMed

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-01-01

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications. PMID:27380727

  9. In vivo iron and zinc deficiency diminished T- and B-selective mitogen stimulation of murine lymphoid cells through protein kinase C-mediated mechanism.

    PubMed

    Klecha, A J; Salgueiro, J; Wald, M; Boccio, J; Zubillaga, M; Leonardi, N M; Gorelik, G; Cremaschi, G A

    2005-05-01

    Zinc and iron are crucial mineral components of human diet, because their deficiency leads to several disorders, including alterations of the immune function. It has been demonstrated, in both humans and rodents, that a diminished number of lymphoid cells and a loss of lymphocyte activity accompany deprivation of these essential minerals. The aim of this work was to analyze if iron and/or zinc imbalances regulate lymphocyte activity and the intracellular signals involved in the effect. Mice from the BALB/c strain were fed with iron- and/or zinc-deficient or mineral-supplemented diets, according to the American Institute of Nutrition Rodent Diets. Levels of iron and zinc were assessed in blood, liver, or bone samples. Selective mitogen stimulation of T- and B-lymphocytes were performed. We found a diminished proliferative response in T- and B-lymphocytes from zinc- and/or iron-deficient animals with respect to controls. These effects were related to decreased mitogen-induced translocation of protein kinase C (PKC) activity to cell membranes on both cell types from all animals fed with deficient diets. Our results demonstrate that iron and zinc deficiencies affect both T- and B-lymphocyte function by PKC-dependent mechanisms.

  10. Transcriptional regulator Id2 mediates CD8+ T cell immunity.

    PubMed

    Cannarile, Michael A; Lind, Nicholas A; Rivera, Richard; Sheridan, Alison D; Camfield, Kristin A; Wu, Bei Bei; Cheung, Kitty P; Ding, Zhaoqing; Goldrath, Ananda W

    2006-12-01

    Transcriptional programs that initiate and sustain the proliferation, differentiation and survival of CD8(+) T cells during immune responses are not completely understood. Here we show that inhibitor of DNA binding 2 (Id2), an antagonist of E protein transcription factors, was upregulated in CD8(+) T cells during infection and that expression of Id2 was maintained in memory CD8(+) T cells. Although Id2-deficient naive CD8(+) T cells recognized antigen and proliferated normally early after infection, effector CD8(+) T cells did not accumulate because the cells were highly susceptible to apoptosis. Id2-deficient CD8(+) T cells responding to infection had changes in the expression of genes that influence survival and had altered memory formation. Our data emphasize the importance of Id2 in regulating gene expression by CD8(+) T cells and the magnitude of effector responses, suggesting a mechanism involving Id protein- and E protein-mediated survival and differentiation of mature T cells.

  11. Fibronectin fragments containing the RGDS cell-binding domain mediate monocyte migration into the rabbit lung. A potential mechanism for C5 fragment-induced monocyte lung accumulation.

    PubMed Central

    Doherty, D E; Henson, P M; Clark, R A

    1990-01-01

    Many inflammatory processes are characterized by an early phase of neutrophil migration and a later phase of monocyte migration into the inflammatory site. Mechanisms that govern the transition between phases are the subject of these investigations. Acute lung inflammation induced by C5 fragments in the rabbit leads to an initial neutrophil influx and plasma leakage into the alveolar space, followed by monocyte influx that we have previously shown to be dependent on prior emigration of neutrophils. Neutrophil enzymes are known to cleave intact fibronectin into fragments that are monocyte chemotaxins in vitro. Accordingly, generation of appropriate fibronectin fragments in situ by proteolytic enzymes from infiltrating neutrophils might represent a potential mechanism for attraction of monocytes into the lung. The studies reported herein demonstrate that a 120-kD fragment of fibronectin containing the RGDS fibroblast cell-binding domain induced monocyte migration into the rabbit lung in vivo. Intact fibronectin was inactive. A significant proportion of the monocyte migration was neutrophil independent. Intact fibronectin was present in bronchoalveolar lavage fluid from C5 fragment-treated animals rendered neutropenic, but absent in lavage from normal C5 fragment-treated animals. Fibronectin fragments were present in bronchoalveolar lavage fluid from both C5 fragment-treated and control rabbits. In addition, the amount of fibronectin was significantly increased in lavage of C5 fragment-treated normal but not neutropenic animals. Monoclonal antibodies directed against an epitope of fibronectin containing the RGDS cell-binding domain significantly inhibited the C5 fragment-induced monocyte migration, but not neutrophil migration. These studies suggest that chemotactic fibronectin fragments may in part be responsible for the recruitment of monocytes into areas of acute lung inflammation. Images PMID:2212000

  12. Cell-Cell Interactions Mediate the Response of Vascular Smooth Muscle Cells to Substrate Stiffness

    PubMed Central

    Sazonova, Olga V.; Lee, Kristen L.; Isenberg, Brett C.; Rich, Celeste B.; Nugent, Matthew A.; Wong, Joyce Y.

    2011-01-01

    The vessel wall experiences progressive stiffening with age and the development of cardiovascular disease, which alters the micromechanical environment experienced by resident vascular smooth muscle cells (VSMCs). In vitro studies have shown that VSMCs are sensitive to substrate stiffness, but the exact molecular mechanisms of their response to stiffness remains unknown. Studies have also shown that cell-cell interactions can affect mechanotransduction at the cell-substrate interface. Using flexible substrates, we show that the expression of proteins associated with cell-matrix adhesion and cytoskeletal tension is regulated by substrate stiffness, and that an increase in cell density selectively attenuates some of these effects. We also show that cell-cell interactions exert a strong effect on cell morphology in a substrate-stiffness dependent manner. Collectively, the data suggest that as VSMCs form cell-cell contacts, substrate stiffness becomes a less potent regulator of focal adhesion signaling. This study provides insight into the mechanisms by which VSMCs respond to the mechanical environment of the blood vessel wall, and point to cell-cell interactions as critical mediators of VSMC response to vascular injury. PMID:21806930

  13. Cytolysis of oligodendrocytes is mediated by killer (K) cells but not by natural killer (NK) cells.

    PubMed

    Satoh, J; Kim, S U; Kastrukoff, L F

    1991-03-01

    The cytotoxic activity of killer (K) cells against enriched cultures of bovine oligodendrocytes (BOL) was investigated in multiple sclerosis (MS) and controls. Human K cells mediated cytotoxicity to primary cultures of BOL in the presence of anti-BOL antiserum in all study groups, while BOL were resistant to human natural killer (NK) cells. Cytotoxic activity was significantly reduced in MS when compared to age-matched normal controls but not when compared to other neurologic disease (OND) patients. K cell-mediated lysis of BOL could also be induced with anti-galactocerebroside antibody but not with other antibodies including those specific for OL antigens (myelin basic protein, proteolipid apoprotein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase). Enrichment of the effector population indicated that antibody-dependent cell-mediated cytotoxicity (ADCC) to BOL was mediated by large granular lymphocytes, and the effector population was further characterized by flow cytometry. The effector cells mediating ADCC could be inhibited by protein A of Staphylococcus aureus, and by K562 cells in cold competition assay. These observations indicate that oligodendrocytes are resistant to NK cells but are susceptible to cytolysis mediated by K cells. This may represent a potentially important immune mechanism in the pathogenesis of MS.

  14. In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures

    PubMed Central

    Ni, Chao; Chen, Yuhui; Zeng, Musheng; Pei, Rongjuan; Du, Yong; Tang, Linquan; Wang, Mengyi; Hu, Yazhuo; Zhu, Hanyu; He, Meifang; Wei, Xiawei; Wang, Shan; Ning, Xiangkai; Wang, Manna; Wang, Jufang; Ma, Li; Chen, Xinwen; Sun, Qiang; Tang, Hong; Wang, Ying; Wang, Xiaoning

    2015-01-01

    Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel “in-cell infection” mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified “in-cell infection” as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that “in-cell infection” is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs. PMID:25916549

  15. Mediators and mechanisms of relaxation in rabbit urethral smooth muscle

    PubMed Central

    Waldeck, Kristian; Ny, Lars; Persson, Katarina; Andersson, Karl-Erik

    1998-01-01

    Electrophysiological and mechanical experiments were performed to investigate whether the nitric oxide (NO)-mediated relaxation of rabbit urethral smooth muscle is associated with a hyperpolarization of the membrane potential. In addition, a possible role for vasoactive intestinal peptide (VIP) and carbon monoxide (CO) as relaxant agents in rabbit urethra was investigated. Immunohistochemical experiments were performed to characterize the NO-synthase (NOS) and VIP innervation. Possible target cells for NO were studied by using antisera against cyclic GMP. The cyclic GMP-immunoreactivity was investigated on tissues pretreated with 1 mM IBMX, 0.1 mM zaprinast and 1 mM sodium nitroprusside. Intracellular recordings of the membrane potential in the circular smooth muscle layer revealed two types of spontaneous depolarizations, slow waves with a duration of 3–4 s and an amplitude of 30–40 mV, and faster (0.5–1 s), more irregular depolarizations with an amplitude of 5–15 mV. The resting membrane potential was 39±1 mV (n=12). Application of NO (30 μM), CO (30 μM) or VIP (1 μM) did not change the resting membrane potential. Both NO (1–100 μM) and VIP (1 nM–1 μM) produced concentration-dependent relaxations amounting to 87±4% and 97±2% (n=6), respectively. The relaxant effect of CO (1–30 μM) amounted to 27±4% (n=5) at the highest concentration used. Immunohistochemical experiments revealed a rich supply of NOS-immunoreactive nerve fibres in the smooth muscle layers. Numerous spinous cyclic GMP-immunoreactive cells were found interspersed between the smooth muscle bundles, mainly localized in the outer layer. These cells had long processes forming a network surrounding the smooth muscle bundles. VIP-immunoreactivity was sparse in comparison to NOS-immunoreactive nerves. The rich supply of NOS-immunoreactive nerve fibres supports the view that NO is an important NANC-mediator in the rabbit urethra. In contrast to several

  16. Mechanisms of Defense against Intracellular Pathogens Mediated by Human Macrophages.

    PubMed

    Bloom, Barry R; Modlin, Robert L

    2016-06-01

    The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the human host?" The origins of this work derive from a long-standing interest in the mechanisms of protection against two such paradigmatic intracellular pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, that have brilliantly adapted to the human host. It was obvious that these pathogens, which cause chronic diseases and persist in macrophages, must have acquired subtle strategies to resist host microbicidal mechanisms, yet since the vast majority of individuals infected with M. tuberculosis do not develop disease, there must be some potent human antimicrobial mechanisms. What follows is not a comprehensive review of the vast literature on the role of human macrophages in protection against infectious disease, but a summary of the research in our two laboratories with collaborators that we hope has contributed to some understanding of mechanisms of resistance and pathogenesis. While mouse models revealed some necessary conditions for protection, e.g., innate immunity, Th1 cells and their cytokines, and major histocompatibility complex class I-restricted T cells, here we emphasize multiple antimicrobial mechanisms that exist in human macrophages that differ from those of most experimental animals. Prominent here is the vitamin D-dependent antimicrobial pathway common to human macrophages activated by innate and acquired immune responses, mediated by antimicrobial peptides, e.g., cathelicidin, through an interleukin-15- and interleukin-32-dependent common pathway that is necessary for macrophage killing of M. tuberculosis in vitro. PMID:27337485

  17. PEROXIDASE-MEDIATED MAMMALIAN CELL CYTOTOXICITY

    PubMed Central

    Edelson, Paul J.; Cohn, Zanvil A.

    1973-01-01

    Lactoperoxidase, in the presence of hydrogen peroxide and iodide is cytotoxic for human and mouse lymphoid cells, and human erythrocytes. Myeloperoxidase, in amounts equivalent to 1.5 x 106 neutrophils, readily replaces lactoperoxidase, and allows the substitution of the iodide ion by chloride. The myeloperoxidase-mediated reaction is rapid, and highly efficient, leading to 85–90% cell death in 90 min, as measured by 51chromium release and dye exclusion. The mixture of granulocytes. monocytes, and lymphocytes present in an inflammatory exudate, and the intimate cell-to-cell association characteristic of cytotoxic phenomena may provide the in vivo requirements for such a system. PMID:4717124

  18. Secretome identification of immune cell factors mediating metastatic cell homing

    PubMed Central

    Aguado, Brian A.; Wu, Jia J.; Azarin, Samira M.; Nanavati, Dhaval; Rao, Shreyas S.; Bushnell, Grace G.; Medicherla, Chaitanya B.; Shea, Lonnie D.

    2015-01-01

    Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics. PMID:26634905

  19. Cell-mediated Protection in Influenza Infection

    PubMed Central

    Thomas, Paul G.; Keating, Rachael; Hulse-Post, Diane J.

    2006-01-01

    Current vaccine strategies against influenza focus on generating robust antibody responses. Because of the high degree of antigenic drift among circulating influenza strains over the course of a year, vaccine strains must be reformulated specifically for each influenza season. The time delay from isolating the pandemic strain to large-scale vaccine production would be detrimental in a pandemic situation. A vaccine approach based on cell-mediated immunity that avoids some of these drawbacks is discussed here. Specifically, cell-mediated responses typically focus on peptides from internal influenza proteins, which are far less susceptible to antigenic variation. We review the literature on the role of CD4+ and CD8+ T cell–mediated immunity in influenza infection and the available data on the role of these responses in protection from highly pathogenic influenza infection. We discuss the advantages of developing a vaccine based on cell-mediated immune responses toward highly pathogenic influenza virus and potential problems arising from immune pressure. PMID:16494717

  20. Circadian rhythm of mechanically mediated differentiation of osteoblasts.

    PubMed

    Roberts, W E; Klingler, E; Mozsary, P G

    1984-01-01

    Rats entrained to alternating 12 h light/dark periods were sacrificed at hourly intervals over one complete circadian cycle. Each animal was injected with 3H-Thymidine 1 h before death. Autoradiographs of serial sections of maxillary first molar periodontal ligament (PDL) were prepared. Nuclear volume was determined for labeled fibroblastlike PDL cells along a physiological bone forming surface. Preosteoblasts (large nuclei), the immediate proliferating precursors of osteoblasts, were found to synthesize DNA primarily during the environmental light period and divide during the subsequent dark cycle. Less differentiated precursor cells (small nuclei), the proliferating predecessors of preosteoblasts, were in S phase primarily during the dark period and divided in the following light cycle. Since previous studies have indicated, the stress/strain-mediated increase in nuclear size to form preosteoblasts also requires about 8-12 h, the least complex osteoblast differentiation model, which is consistent with the present data, is a 60 h sequence involving at least four cell types and five alternating dark/light cycles. The principal rate-limiting step in osteoblast differentiation is the mechanically related shift in nuclear size (change in genomic expression) associated with formation of preosteoblasts. PMID:6430524

  1. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. PMID:26601950

  2. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

  3. Cell-mediated destruction of cells grown on artificial capillaries.

    PubMed

    Zwilling, B S; Clayman, D A

    1978-11-01

    This investigation was designed to determine the conditions required to assess cell-mediated destruction of target L-cells grown on artificial capillaries. In control cultures that contained L-cells alone, solid nodules with a diameter of 1 mm as well as dense cellular growth could be visually observed by the 12th day of culture. Alloimmune spleen cells from both immunized and normal C57BL/10 mice were shown to be capable of destroying tritiated thymidine-labeled L-cells growing on artificial capillaries. The destruction of target cells grown as monolayers in capillary culture correlated well with monolayer cultures incubated in 16-mm plastic tissue culture wells. When target cells were grown in capillary culture for 5 days before the addition of effector cells, significant destruction by normal effector cells was not observed until the 15th day of culture whereas that mediated by immune cells was observed by the 7th day. The possible effects of cell-culturing conditions on the kinetics of cell-mediated destruction in capillary chambers are discussed.

  4. The Adenovirus Type 5 E1B-55K Oncoprotein Actively Shuttles in Virus-Infected Cells, Whereas Transport of E4orf6 Is Mediated by a CRM1-Independent Mechanism

    PubMed Central

    Dosch, Tanja; Horn, Florian; Schneider, Grit; Krätzer, Friedrich; Dobner, Thomas; Hauber, Joachim; Stauber, Roland H.

    2001-01-01

    The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the “driving force” for adenoviral late mRNA export. PMID:11356976

  5. Mechanism of enhanced responses after combination photodynamic therapy (cPDT) in carcinoma cells involves C/EBP-mediated transcriptional upregulation of the coproporphyrinogen oxidase (CPO) gene

    NASA Astrophysics Data System (ADS)

    Anand, Sanjay; Hasan, Tayyaba; Maytin, Edward V.

    2013-03-01

    Photodynamic therapy (PDT) with aminolevulinate (ALA) is widely accepted as an effective treatment for superficial carcinomas and pre-cancers. However, PDT is still suboptimal for deeper tumors, mainly due to inadequate ALA penetration and subsequent conversion to PpIX. We are interested in improving the effectiveness of photodynamic therapy (PDT) for deep tumors, using a combination approach (cPDT) in which target protoporphyrin (PpIX) levels are significantly enhanced by differentiation caused by giving Vitamin D or methotrexate (MTX) for 3 days prior to ALAPDT. In LNCaP and MEL cells, a strong correlation between inducible differentiation and expression of C/EBP transcription factors, as well as between differentiation and mRNA levels of CPO (a key heme-synthetic enzyme), indicates the possibility of CPO transcriptional regulation by the C/EBPs. Sequence analysis of the first 1300 base pairs of the murine CPO upstream region revealed 15 consensus C/EBP binding sites. Electrophoretic Mobility Shift Assays (EMSA) proved that these sites form specific complexes that have strong, moderate or weak affinities for C/EBPs. However, in the context of the full-length CPO promoter, inactivation of any type of site (strong or weak) reduced CPO promoter activity (luciferase assay) to nearly the same extent, suggesting cooperative interactions. A comparative analysis of murine and human CPO promoters revealed possible protein-protein interactions between C/EBPs and several neighboring transcription factors such as NFkB, Sp1, AP-1, CBP/p300 and CREB (an enhanceosome complex). Overall, these results confirm that C/EBP's are important for CPO expression via complex mechanisms which upregulate PpIX and enhance the outcome of cPDT.

  6. Dual pH-Mediated Mechanized Hollow Zirconia Nanospheres.

    PubMed

    Wang, MingDong; Gong, GuangCai; Feng, Jing; Wang, Ting; Ding, ChenDi; Zhou, BaoJing; Jiang, Wei; Fu, JiaJun

    2016-09-01

    We demonstrate for the first time how to assemble mechanized hollow zirconia nanospheres (MHzNs), consisting of hollow mesoporous zirconia nanospheres (HMZNs) as nanoscaffolds and supramolecular switches anchored on the exterior surface of HMZNs. The remarkable advantage of substitution of HMZNs for conventional mesoporous silica nanoscaffolds is that HMZNs can suffer the hot alkaline reaction environment, which provides a novel strategy for functionalization and thus achieve dual pH-mediated controlled release functions by simple and practicable assembly procedure. Under neutral solution, cucurbituril[7] (CB[7]) macrocycles complexed with propanone bis(2-aminoethyl)ketal (PBAEK) to form [2]pseudorotaxanes as supramolecular switches, blocking the pore orifices and preventing the undesirable leakage of cargoes. When solution pH was adjusted to alkaline range, CB[7] macrocycles, acting as caps, disassociated from PBAEK stalks and opened the switches due to the dramatic decrease of ion-dipole interactions. While under acidic conditions, PBAEK stalks were broken on account of the cleavage of ketal groups, resulting in the collapse of supramolecular switches and subsequent release of encapsulated cargoes. MHzNs owning dual pH-mediated controlled release characteristic are expected to apply in many fields. In this work, the feasibility of doxorubicin (DOX)-loaded MHzNs as targeted drug delivery systems was evaluated. In vitro cellular studies demonstrate that DOX-loaded MHzNs can be easily taken up by SMMC-7721 cells, can rapidly release DOX intracellularly, and can enhance cytotoxicity against tumor cells, proving their potential for chemotherapy. PMID:27523904

  7. Environmental properties set cell mechanics and morphology

    NASA Astrophysics Data System (ADS)

    Janmey, Paul

    2012-02-01

    Many cell types are sensitive to mechanical signals that are produced either by application of exogenous force to their surfaces, or by the resistance that their surroundings place on forces generated by the cells themselves. Cell morphology, motility, proliferation, and protein expression all change in response to substrate stiffness. Changing the elastic moduli of substrates alters the formation of focal adhesions, the assembly of actin filaments into bundles, and the stability of intermediate filaments. The range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. Mechanosensing depends on the type of adhesion receptor by which the cell binds, and therefore on both the molecular composition of the extracellular matrix and the nature of its link to the cytoskeleton. Many cell types can alter their own stiffness to match that of the substrate to which they adhere. The maximal elastic modulus that cells such as fibroblasts can attain is similar to that of crosslinked actin networks at the concentrations in the cell cortex. The precise mechanisms of mechanosensing are not well defined, but they presumably require an elastic connection between cell and substrate, mediated by transmembrane proteins. The viscoelastic properties of different extracellular matrices and cytoskeletal elements strongly influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening, leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. Cell cortical elasticity is dominated by cytoskeletal polymer networks and can be modulated by internal tension. Simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular

  8. Mast Cell-Derived Histamine Mediates Cystitis Pain

    PubMed Central

    Rudick, Charles N.; Bryce, Paul J.; Guichelaar, Laura A.; Berry, Ruth E.; Klumpp, David J.

    2008-01-01

    Background Mast cells trigger inflammation that is associated with local pain, but the mechanisms mediating pain are unclear. Interstitial cystitis (IC) is a bladder disease that causes debilitating pelvic pain of unknown origin and without consistent inflammation, but IC symptoms correlate with elevated bladder lamina propria mast cell counts. We hypothesized that mast cells mediate pelvic pain directly and examined pain behavior using a murine model that recapitulates key aspects of IC. Methods and Findings Infection of mice with pseudorabies virus (PRV) induces a neurogenic cystitis associated with lamina propria mast cell accumulation dependent upon tumor necrosis factor alpha (TNF), TNF-mediated bladder barrier dysfunction, and pelvic pain behavior, but the molecular basis for pelvic pain is unknown. In this study, both PRV-induced pelvic pain and bladder pathophysiology were abrogated in mast cell-deficient mice but were restored by reconstitution with wild type bone marrow. Pelvic pain developed normally in TNF- and TNF receptor-deficient mice, while bladder pathophysiology was abrogated. Conversely, genetic or pharmacologic disruption of histamine receptor H1R or H2R attenuated pelvic pain without altering pathophysiology. Conclusions These data demonstrate that mast cells promote cystitis pain and bladder pathophysiology through the separable actions of histamine and TNF, respectively. Therefore, pain is independent of pathology and inflammation, and histamine receptors represent direct therapeutic targets for pain in IC and other chronic pain conditions. PMID:18461160

  9. Redox polymer mediation for enzymatic biofuel cells

    NASA Astrophysics Data System (ADS)

    Gallaway, Joshua

    Mediated biocatalytic cathodes prepared from the oxygen-reducing enzyme laccase and redox-conducting osmium hydrogels were characterized for use as cathodes in enzymatic biofuel cells. A series of osmium-based redox polymers was synthesized with redox potentials spanning the range from 0.11 V to 0.85 V (SHE), and the resulting biocatalytic electrodes were modeled to determine reaction kinetic constants using the current response, measured osmium concentration, and measured apparent electron diffusion. As in solution-phase systems, the bimolecular rate constant for mediation was found to vary greatly with mediator potential---from 250 s-1M-1 when mediator and enzyme were close in potential to 9.4 x 10 4 s-1M-1 when this overpotential was large. Optimum mediator potential for a cell operating with a non-limiting platinum anode and having no mass transport limitation from bulk solution was found to be 0.66 V (SHE). Redox polymers were synthesized under different concentrations, producing osmium variation. An increase from 6.6% to 7.2% osmium increased current response from 1.2 to 2.1 mA/cm2 for a planar film in 40°C oxygen-saturated pH 4 buffer, rotating at 900 rpm. These results translated to high surface area electrodes, nearly doubling current density to 13 mA/cm2, the highest to date for such an electrode. The typical fungal laccase from Trametes versicolor was replaced by a bacterially-expressed small laccase from Streptomyces coelicolor, resulting in biocatalytic films that reduced oxygen at increased pH, with full functionality at pH 7, producing 1.5 mA/cm 2 in planar configuration. Current response was biphasic with pH, matching the activity profile of the free enzyme in solution. The mediated enzyme electrode system was modeled with respect to apparent electron diffusion, mediator concentration, and transport of oxygen from bulk solution, all of which are to some extent controlled by design. Each factor was found to limit performance in certain circumstances

  10. In Search of the Molecular Mechanisms Mediating the Inhibitory Effect of the GnRH Antagonist Degarelix on Human Prostate Cell Growth

    PubMed Central

    Sakai, Monica; Martinez-Arguelles, Daniel B.; Patterson, Nathan H.; Chaurand, Pierre; Papadopoulos, Vassilios

    2015-01-01

    Degarelix is a gonadrotropin-releasing hormone (GnRH) receptor (GnRHR) antagonist used in patients with prostate cancer who need androgen deprivation therapy. GnRHRs have been found in extra-pituitary tissues, including prostate, which may be affected by the GnRH and GnRH analogues used in therapy. The direct effect of degarelix on human prostate cell growth was evaluated. Normal prostate myofibroblast WPMY-1 and epithelial WPE1-NA22 cells, benign prostatic hyperplasia (BPH)-1 cells, androgen-independent PC-3 and androgen-dependent LNCaP prostate cancer cells, as well as VCaP cells derived from a patient with castration-resistant prostate cancer were used. Discriminatory protein and lipid fingerprints of normal, hyperplastic, and cancer cells were generated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The investigated cell lines express GNRHR1 and GNRHR2 and their endogenous ligands. Degarelix treatment reduced cell viability in all prostate cell lines tested, with the exception of the PC-3 cells; this can be attributed to increased apoptosis, as indicated by increased caspase 3/7, 8 and 9 levels. WPE1-NA22, BPH-1, LNCaP, and VCaP cell viability was not affected by treatment with the GnRH agonists leuprolide and goserelin. Using MALDI MS, we detected changes in m/z signals that were robust enough to create a complete discriminatory profile induced by degarelix. Transcriptomic analysis of BPH-1 cells provided a global map of genes affected by degarelix and indicated that the biological processes affected were related to cell growth, G-coupled receptors, the mitogen-activated protein kinase (MAPK) pathway, angiogenesis and cell adhesion. Taken together, these data demonstrate that (i) the GnRH antagonist degarelix exerts a direct effect on prostate cell growth through apoptosis; (ii) MALDI MS analysis provided a basis to fingerprint degarelix-treated prostate cells; and (iii) the clusters of genes affected by degarelix suggest that

  11. Molecular mechanism of statin-mediated LOX-1 inhibition

    PubMed Central

    Biocca, Silvia; Iacovelli, Federico; Matarazzo, Sara; Vindigni, Giulia; Oteri, Francesco; Desideri, Alessandro; Falconi, Mattia

    2015-01-01

    Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins. PMID:25950192

  12. Pathophysiological mechanisms of catecholamine and cocaine-mediated cardiotoxicity.

    PubMed

    Liaudet, Lucas; Calderari, Belinda; Pacher, Pal

    2014-11-01

    Overactivation of the sympatho-adrenergic system is an essential mechanism providing short-term adaptation to the stressful conditions of critical illnesses. In the same way, the administration of exogenous catecholamines is mandatory to support the failing circulation in acutely ill patients. In contrast to these short-term benefits, prolonged adrenergic stress is detrimental to the cardiovascular system by initiating a series of adverse effects triggering significant cardiotoxicity, whose pathophysiological mechanisms are complex and only partially elucidated. In addition to the development of myocardial oxygen supply/demand imbalance induced by the sustained activation of adrenergic receptors, catecholamines can damage cardiomyocytes by fostering mitochondrial dysfunction, via two main mechanisms. The first one is calcium overload, consecutive to β-adrenergic receptor-mediated activation of protein kinase A and subsequent phosphorylation of multiple Ca(2+)-cycling proteins. The second one is oxidative stress, primarily related to the transformation of catecholamines into "aminochromes," which undergo redox cycling in mitochondria to generate copious amounts of oxygen-derived free radicals. In turn, calcium overload and oxidative stress promote mitochondrial permeability transition and cardiomyocyte cell death, both via the apoptotic and necrotic pathways. Comparable mechanisms of myocardial toxicity, including marked oxidative stress and mitochondrial dysfunction, have been reported with the use of cocaine, a common recreational drug with potent sympathomimetic activity. The aim of the current review is to present in detail the pathophysiological processes underlying the development of catecholamine and cocaine-induced cardiomyopathy, as such conditions may be frequently encountered in the clinical practice of cardiologists and ICU specialists. PMID:24398587

  13. Monocular and binocular mechanisms mediating flicker adaptation.

    PubMed

    Zhuang, Xiaohua; Shevell, Steven K

    2015-12-01

    Flicker adaptation reduces subsequent temporal contrast sensitivity. Recent studies show that this adaptation likely results from neural changes in the magnocellular visual pathway, but whether this adaptation occurs at a monocular or a binocular level, or both, is unclear. Here, two experiments address this question. The first experiment exploits the observation that flicker adaptation is stronger at higher than lower temporal frequencies. Observers' two eyes adapted to 3Hz flicker with an incremental pulse at 1/4 duty cycle, either in-phase or out-of-phase in the two eyes. At the binocular level, the flicker rate was 6Hz in the out-of-phase condition if the two eyes' pulse trains sum. Similar sensitivity reduction was found in both phase conditions, as expected for independent monocular adapting mechanisms. The second experiment tested for interocular transfer of adaptation between eyes. Results showed that (1) flicker adaptation was strongest with adapting and test fields in only the same eye, (2) adaptation can be partially transferred interocularly with adaptation in only the opposite eye, and (3) adaptation was weakened when both eyes were adapted simultaneously at different contrasts, compared to test-eye adaptation alone. Taken together, the findings are consistent with mechanisms of flicker adaptation at both the monocular and binocular level. PMID:26505684

  14. Nature of "memory" in T-cell-mediated antibacterial immunity: anamnestic production of mediator T cells.

    PubMed Central

    North, R J

    1975-01-01

    Mice that survived an immunizing infection with Listeria monocytogenes remained specifically resistant to lethal secondary infection for several months. This acquired, long-lived state of resistance was not dependent on activated macrophages that remained after the primary response. It depended, instead, on an acquired long-lived capacity on the part of immunized mice for generating mediator T cells faster and in larger numbers than normal mice. The number of mediator T cells generated in response to secondary infection was proportional to the level of infection. The results suggest that the accelerated production of mediator T cells that occurs in response to secondary infection represents the expression of a state of immunological T-cell memory. PMID:811558

  15. IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

    PubMed

    Braun, Matthias; Ress, Marie L; Yoo, Young-Eun; Scholz, Claus J; Eyrich, Matthias; Schlegel, Paul G; Wölfl, Matthias

    2016-07-01

    Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1. PMID:27622043

  16. Single-cell force spectroscopy of pili-mediated adhesion

    NASA Astrophysics Data System (ADS)

    Sullan, Ruby May A.; Beaussart, Audrey; Tripathi, Prachi; Derclaye, Sylvie; El-Kirat-Chatel, Sofiane; Li, James K.; Schneider, Yves-Jacques; Vanderleyden, Jos; Lebeer, Sarah; Dufrêne, Yves F.

    2013-12-01

    Although bacterial pili are known to mediate cell adhesion to a variety of substrates, the molecular interactions behind this process are poorly understood. We report the direct measurement of the forces guiding pili-mediated adhesion, focusing on the medically important probiotic bacterium Lactobacillus rhamnosus GG (LGG). Using non-invasive single-cell force spectroscopy (SCFS), we quantify the adhesion forces between individual bacteria and biotic (mucin, intestinal cells) or abiotic (hydrophobic monolayers) surfaces. On hydrophobic surfaces, bacterial pili strengthen adhesion through remarkable nanospring properties, which - presumably - enable the bacteria to resist high shear forces under physiological conditions. On mucin, nanosprings are more frequent and adhesion forces larger, reflecting the influence of specific pili-mucin bonds. Interestingly, these mechanical responses are no longer observed on human intestinal Caco-2 cells. Rather, force curves exhibit constant force plateaus with extended ruptures reflecting the extraction of membrane nanotethers. These single-cell analyses provide novel insights into the molecular mechanisms by which piliated bacteria colonize surfaces (nanosprings, nanotethers), and offer exciting avenues in nanomedicine for understanding and controlling the adhesion of microbial cells (probiotics, pathogens).

  17. Dscam-Mediated Cell Recognition Regulates Neural Circuit Formation

    PubMed Central

    Hattori, Daisuke; Millard, S. Sean; Wojtowicz, Woj M.; Zipursky, S. Lawrence

    2009-01-01

    The Dscam family of immunoglobulin cell surface proteins mediates recognition events between neurons that play an essential role in the establishment of neural circuits. The Drosophila Dscam1 locus encodes tens of thousands of cell surface proteins via alternative splicing. These isoforms exhibit exquisite isoform-specific binding in vitro that mediates homophilic repulsion in vivo. These properties provide the molecular basis for self-avoidance, an essential developmental mechanism that allows axonal and dendritic processes to uniformly cover their synaptic fields. In a mechanistically similar fashion, homophilic repulsion mediated by Drosophila Dscam2 prevents processes from the same class of cells from occupying overlapping synaptic fields through a process called tiling. Genetic studies in the mouse visual system support the view that vertebrate DSCAM also promotes both self-avoidance and tiling. By contrast, DSCAM and DSCAM-L promote layer-specific targeting in the chick visual system, presumably through promoting homophilic adhesion. The fly and mouse studies underscore the importance of homophilic repulsion in regulating neural circuit assembly, whereas the chick studies suggest that DSCA Mproteins may mediate a variety of different recognition events during wiring in a context-dependent fashion. PMID:18837673

  18. N-Cadherin-Mediated Signaling Regulates Cell Phenotype for Nucleus Pulposus Cells of the Intervertebral Disc

    PubMed Central

    Hwang, Priscilla Y.; Jing, Liufang; Michael, Keith W.; Richardson, William J.; Chen, Jun; Setton, Lori A.

    2015-01-01

    Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell–cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration. Therefore, it is important to understand the mechanisms governing juvenile NP cell cluster behavior towards the goal of revealing factors that can promote juvenile, healthy NP cell phenotypes. N-cadherin has been identified as a cell–cell adhesion marker that is present in juvenile NP cells, but disappears with age. The goal of this study was to reveal the importance of N-cadherin in regulating cell–cell interactions in juvenile NP cell cluster formation and test for a regulatory role in maintaining a juvenile NP phenotype in vitro. Juvenile porcine IVD cells, of notochordal origin, were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype. Additionally, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Findings reveal N-cadherin-mediated cell–cell contacts promote cell clustering behavior and regulate NP cell matrix production and preservation of NP-specific markers. Inhibition of N-cadherin-mediated contacts resulted in loss of all features of the juvenile NP cell. These results establish a regulatory role for N-cadherin in juvenile NP cells, and suggest that preservation of the N-cadherin mediated cell–cell contact is important for preserving juvenile NP cell phenotype and morphology. PMID:25848407

  19. Cell mediated autoimmune granulocytopenia in a case of Felty's syndrome.

    PubMed Central

    Slavin, S; Liang, M H

    1980-01-01

    A variety of mechanisms have been demonstrated or suggested to explain the neutropenia that accompanies Felty's syndrome. This case report presents with Felty's syndrome with recurrent infections with initially had a clinical response to splenectomy. Eleven years later profound neutropenia recurred. In-vitro evidence for cell mediated autosensitisation of peripheral blood lymphocytes to autologous bone marrow cells was found. The cellular abnormalities improved after high-dose corticosteroids but not lithium. However, there did not appear to be a reduction in the incidence of clinical infections. The finding suggests that granulocytopenia in some patients with Felty's syndrome may be an autoimmune phenomenon. PMID:7436567

  20. Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

    PubMed

    Xing, Yalan; Su, Tin Tin; Ruohola-Baker, Hannele

    2015-01-01

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer. PMID:25959206

  1. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells.

    PubMed

    Harmon, Jamie S; Stein, Roland; Robertson, R Paul

    2005-03-25

    Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5

  2. Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells.

    PubMed

    Harmon, Jamie S; Stein, Roland; Robertson, R Paul

    2005-03-25

    Glucose toxicity in pancreatic islet beta cells causes loss of insulin gene expression, content, and secretion due to loss of binding of transcription factors, most notably PDX-1 and RIPE-3b1 activator, to the promoter region of the insulin gene. Recently, RIPE-3b1 activator was cloned and identified as the mammalian homologue of avian MafA/Maf-L (MafA). This enabled us to carry out more extensive studies of the role of MafA in glucotoxicity than were hitherto possible. Northern analysis of glucotoxic HIT-T15 cells revealed normal amounts of MafA mRNA, but Western analysis demonstrated a 97 +/- 1% reduction in MafA protein (p < 0.0001). The proteasome is a likely site for MafA degradation as lactacystin, an irreversible proteasome inhibitor, caused an accumulation of MafA protein. Antioxidants have previously been shown to prevent the adverse effects of glucose toxicity on beta cell function both in vivo and in vitro. In the current study, chronic culturing of HIT-T15 cells with the antioxidant N-acetylcysteine (NAC) prevented loss of MafA protein (late passage = 18.9 +/- 10.4% of early passage, p < 0.001; late passage with NAC = 68.7 +/- 19.7% of early passage, p = not significant) and loss of DNA binding (late passage = 63.7 +/- 9% of early passage, p < 0.02; late passage with NAC = 116 +/- 10% of early passage, p = not significant). Additionally, transient transfection of PDX-1 or MafA cDNA into glucotoxic cells increased PDX-1 and MafA protein levels and individually increased insulin promoter activity (untreated = 34%, PDX-1 = 70%, MafA = 78%; percentage of activity of early passage cells), whereas the combined transfection of MafA and PDX-1 completely restored insulin promoter activity. This recovery of promoter activity following transient transfection had no effect on endogenous insulin mRNA. However, adenoviral infection of MafA and PDX-1 significantly increased endogenous insulin mRNA levels by 93% (121 +/- 9 versus 233 +/- 18 density light units; n = 5

  3. Cell-mediated immunity in anorexia nervosa.

    PubMed Central

    Cason, J; Ainley, C C; Wolstencroft, R A; Norton, K R; Thompson, R P

    1986-01-01

    Twelve patients with anorexia nervosa were studied for cell-mediated immunity in terms of delayed hypersensitivity reactions to recall antigens, lymphocyte transformation responses to T-cell mitogens, and numbers of circulating leucocytes and T-cell subpopulations. Compared to controls, all patients had reduced cutaneous reactions and four were anergic. There was a mild leucopenia in patients and both T4+ and T3+ numbers were slightly reduced. Mean peak transformation responses for patients were slightly lower than controls for phytohaemagglutinin, but not for concanavalin A; however, patients required greater doses of mitogens to elicit peak transformation responses. Plasmas from patients did not contain inhibitors of transformation responses. We conclude that there are functional cellular abnormalities associated with the under-nutrition of anorexia nervosa. PMID:3742879

  4. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  5. Mediated coalescence: a possible mechanism for tumor cellular heterogeneity

    PubMed Central

    Ambrose, Joseph; Livitz, Michelle; Wessels, Deborah; Kuhl, Spencer; Lusche, Daniel F; Scherer, Amanda; Voss, Edward; Soll, David R

    2015-01-01

    Recently, we demonstrated that tumorigenic cell lines and fresh tumor cells seeded in a 3D Matrigel model, first grow as clonal islands (primary aggregates), then coalesce through the formation and contraction of cellular cables. Non-tumorigenic cell lines and cells from normal tissue form clonal islands, but do not form cables or coalesce. Here we show that as little as 5% tumorigenic cells will actively mediate coalescence between primary aggregates of majority non-tumorigenic or non-cancerous cells, by forming cellular cables between them. We suggest that this newly discovered, specialized characteristic of tumorigenic cells may explain, at least in part, why tumors contain primarily non-tumorigenic cells. PMID:26807328

  6. Distinct Neural Mechanisms Mediate Olfactory Memory Formation at Different Timescales

    ERIC Educational Resources Information Center

    McNamara, Ann Marie; Magidson, Phillip D.; Linster, Christiane; Wilson, Donald A.; Cleland, Thomas A.

    2008-01-01

    Habituation is one of the oldest forms of learning, broadly expressed across sensory systems and taxa. Here, we demonstrate that olfactory habituation induced at different timescales (comprising different odor exposure and intertrial interval durations) is mediated by different neural mechanisms. First, the persistence of habituation memory is…

  7. Inferring RBP-Mediated Regulation in Lung Squamous Cell Carcinoma

    PubMed Central

    Lafzi, Atefeh; Kazan, Hilal

    2016-01-01

    RNA-binding proteins (RBPs) play key roles in post-transcriptional regulation of mRNAs. Dysregulations in RBP-mediated mechanisms have been found to be associated with many steps of cancer initiation and progression. Despite this, previous studies of gene expression in cancer have ignored the effect of RBPs. To this end, we developed a lasso regression model that predicts gene expression in cancer by incorporating RBP-mediated regulation as well as the effects of other well-studied factors such as copy-number variation, DNA methylation, TFs and miRNAs. As a case study, we applied our model to Lung squamous cell carcinoma (LUSC) data as we found that there are several RBPs differentially expressed in LUSC. Including RBP-mediated regulatory effects in addition to the other features significantly increased the Spearman rank correlation between predicted and measured expression of held-out genes. Using a feature selection procedure that accounts for the adaptive search employed by lasso regularization, we identified the candidate regulators in LUSC. Remarkably, several of these candidate regulators are RBPs. Furthermore, majority of the candidate regulators have been previously found to be associated with lung cancer. To investigate the mechanisms that are controlled by these regulators, we predicted their target gene sets based on our model. We validated the target gene sets by comparing against experimentally verified targets. Our results suggest that the future studies of gene expression in cancer must consider the effect of RBP-mediated regulation. PMID:27186987

  8. Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells.

    PubMed

    Hiwarkar, Prashant; Qasim, Waseem; Ricciardelli, Ida; Gilmour, Kimberly; Quezada, Sergio; Saudemont, Aurore; Amrolia, Persis; Veys, Paul

    2015-12-24

    Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

  9. NKG2D ligands mediate immunosurveillance of senescent cells.

    PubMed

    Sagiv, Adi; Burton, Dominick G A; Moshayev, Zhana; Vadai, Ezra; Wensveen, Felix; Ben-Dor, Shifra; Golani, Ofra; Polic, Bojan; Krizhanovsky, Valery

    2016-02-01

    Cellular senescence is a stress response mechanism that limits tumorigenesis and tissue damage. Induction of cellular senescence commonly coincides with an immunogenic phenotype that promotes self-elimination by components of the immune system, thereby facilitating tumor suppression and limiting excess fibrosis during wound repair. The mechanisms by which senescent cells regulate their immune surveillance are not completely understood. Here we show that ligands of an activating Natural Killer (NK) cell receptor (NKG2D), MICA and ULBP2 are consistently up-regulated following induction of replicative senescence, oncogene-induced senescence and DNA damage - induced senescence. MICA and ULBP2 proteins are necessary for efficient NK-mediated cytotoxicity towards senescent fibroblasts. The mechanisms regulating the initial expression of NKG2D ligands in senescent cells are dependent on a DNA damage response, whilst continuous expression of these ligands is regulated by the ERK signaling pathway. In liver fibrosis, the accumulation of senescent activated stellate cells is increased in mice lacking NKG2D receptor leading to increased fibrosis. Overall, our results provide new insights into the mechanisms regulating the expression of immune ligands in senescent cells and reveal the importance of NKG2D receptor-ligand interaction in protecting against liver fibrosis. PMID:26878797

  10. Suppression of autophagy exacerbates Mefloquine-mediated cell death.

    PubMed

    Shin, Ji Hyun; Park, So Jung; Jo, Yoon Kyung; Kim, Eun Sung; Kang, Hee; Park, Ji-Ho; Lee, Eunjoo H; Cho, Dong-Hyung

    2012-05-01

    Mefloquine is an effective treatment drug for malaria. However, it can cause several adverse side effects, and the precise mechanism associated with the adverse neurological effects of Mefloquine is not clearly understood. In this study, we investigated the effect of Mefloquine on autophagy in neuroblastoma cells. Mefloquine treatment highly induced the formation of autophagosomes and the conversion of LC3I into LC3II. Moreover, Mefloquine-induced autophagy was efficiently suppressed by an autophagy inhibitor and by down regulation of ATG6. The autophagy was also completely blocked in ATG5 deficient mouse embryonic fibroblast cells. Moreover, suppression of autophagy significantly intensified Mefloquine-mediated cytotoxicity in SH-SY5Y cells. Our findings suggest that suppression of autophagy may exacerbate Mefloquine toxicity in neuroblastoma cells.

  11. Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

    SciTech Connect

    Martin, Claire; Lafosse, Jean-Michel; Malavaud, Bernard; Cuvillier, Olivier

    2010-01-01

    Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

  12. Modeling the capture rate by a radially oscillating spherical bubble. A bio-mimetic model for studying the mechanically-mediated uptake by cells

    NASA Astrophysics Data System (ADS)

    Raudino, Antonio; Raciti, Domenica; Grassi, Antonio

    2016-11-01

    The surface of living cells constitutes a dynamic environment submitted to complex oscillatory motions. Oscillations may modify the uptake of incoming molecules. In this study we explored a bio-mimetic system formed by oscillating bubbles suspended in a sea of randomly distributed diffusants. We investigated by a time-dependent Fokker-Planck equation the effect of the periodic motions on the adsorption of a diffusant onto the moving bubble surface. We introduced both direct interactions between the diffusant and the fluctuating surface and indirect interactions due to the hydrodynamic motions around a vibrating surface. Results are expressed in terms of oscillation frequencies and amplitudes. An overall reduction of the bound diffusant at the bubble surface was observed.

  13. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  14. Heterodimerization of apelin receptor and neurotensin receptor 1 induces phosphorylation of ERK(1/2) and cell proliferation via Gαq-mediated mechanism.

    PubMed

    Bai, Bo; Cai, Xin; Jiang, Yunlu; Karteris, Emmanouil; Chen, Jing

    2014-10-01

    Dimerization of G protein-coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin-converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C-terminal residues of vasoactive peptides including apelin-13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co-immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK(1/2) activation and increased proliferation via activation of Gq α-subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK(1/2) activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.

  15. [Mechanism of mediators in human skin. Pharmacoanalysis and therapeutic aspects].

    PubMed

    Stüttgen, G

    1977-06-01

    Under physiological and pathological conditions, the skin is influenced by tissue hormones (biogenic amines, kinines, prostaglandines), neuro-hormones (acetylcholine, catecholamines) and by proteins as complement. The combination of neurological stimulation with a series of self-propagating metabolic changes, shows new aspects of tissue reaction. Histamine triggers a serie of mediators as kinines and prostaglandines. Due to their influence on mediators and their effects on regulatory mechanisms the cyclic nucleotides can serve as model substances for the development of therapeutic processes for dermatological as well as for other disorders. The influence on mediator-systems by topical treatment and proven permeability of such substances is of extreme importance for the future development of local dermatological therapy.

  16. PTEN mediates Notch-dependent stalk cell arrest in angiogenesis.

    PubMed

    Serra, Helena; Chivite, Iñigo; Angulo-Urarte, Ana; Soler, Adriana; Sutherland, James D; Arruabarrena-Aristorena, Amaia; Ragab, Anan; Lim, Radiance; Malumbres, Marcos; Fruttiger, Marcus; Potente, Michael; Serrano, Manuel; Fabra, Àngels; Viñals, Francesc; Casanovas, Oriol; Pandolfi, Pier Paolo; Bigas, Anna; Carracedo, Arkaitz; Gerhardt, Holger; Graupera, Mariona

    2015-07-31

    Coordinated activity of VEGF and Notch signals guides the endothelial cell (EC) specification into tip and stalk cells during angiogenesis. Notch activation in stalk cells leads to proliferation arrest via an unknown mechanism. By using gain- and loss-of-function gene-targeting approaches, here we show that PTEN is crucial for blocking stalk cell proliferation downstream of Notch, and this is critical for mouse vessel development. Endothelial deletion of PTEN results in vascular hyperplasia due to a failure to mediate Notch-induced proliferation arrest. Conversely, overexpression of PTEN reduces vascular density and abrogates the increase in EC proliferation induced by Notch blockade. PTEN is a lipid/protein phosphatase that also has nuclear phosphatase-independent functions. We show that both the catalytic and non-catalytic APC/C-Fzr1/Cdh1-mediated activities of PTEN are required for stalk cells' proliferative arrest. These findings define a Notch-PTEN signalling axis as an orchestrator of vessel density and implicate the PTEN-APC/C-Fzr1/Cdh1 hub in angiogenesis.

  17. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    NASA Astrophysics Data System (ADS)

    Song, Zhen

    Life-threatening cardiac arrhythmias continue to pose a major health problem. Ventricular fibrillation, which is a complex form of electrical wave turbulence in the lower chambers of the heart, stops the heart from pumping and is the largest cause of natural death in the United States. Atrial fibrillation, a related form of wave turbulence in the upper heart chambers, is in turn the most common arrhythmia diagnosed in clinical practice. Despite extensive research to date, mechanisms of cardiac arrhythmias remain poorly understood. It is well established that both spatial disorder of the refractory period of heart cells and triggered activity (TA) jointly contribute to the initiation and maintenance of arrhythmias. TA broadly refers to the abnormal generation of a single or a sequence of abnormal excitation waves from a small submillimeter region of the heart in the interval of time between two normal waves generated by the heart's natural pacemaker (the sinoatrial node). TA has been widely investigated experimentally and occurs in several pathological conditions where the intracellular concentration of free Ca2+ ions in heart cells becomes elevated. Under such conditions, Ca2+ can be spontaneously released from intracellular stores, thereby driving an electrogenic current that exchanges 3Na+ ions for one Ca2+ ion across the cell membrane. This current in turn depolarizes the membrane of heart cells after a normal excitation. If this calcium-mediated "delayed after depolarization'' (DAD) is sufficiently large, it can generate an action potential. While the arrhythmogenic importance of spontaneous Ca2+ release and DADs is well appreciated, the conditions under which they occur in heart pathologies remain poorly understood. Calcium overload is only one factor among several other factors that can promote DADs, including sympathetic nerve stimulation, different expression levels of membrane ion channels and calcium handling proteins, and different mutations of those

  18. Modeling mechanisms of cell secretion.

    PubMed

    Tsaneva-Atanasova, Krasimira; Osinga, Hinke M; Tabak, Joël; Pedersen, Morten Gram

    2010-12-01

    Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Anomalies in the processes involved in secretion contribute to the development and progression of diseases such as diabetes and other hormonal disorders. To unravel the mechanisms that govern such diseases, it is essential to understand how hormones, growth factors and neurotransmitters are synthesized and processed, and how their signals are recognized, amplified and transmitted by intracellular signaling pathways in the target cells. Here, we discuss diverse aspects of the detailed mechanisms involved in secretion based on mathematical models. The models range from stochastic ones describing the trafficking of secretory vesicles to deterministic ones investigating the regulation of cellular processes that underlie hormonal secretion. In all cases, the models are closely related to experimental results and suggest theoretical predictions for the secretion mechanisms.

  19. Role of complement and NK cells in antibody mediated rejection.

    PubMed

    Akiyoshi, Takurin; Hirohashi, Tsutomu; Alessandrini, Alessandro; Chase, Catherine M; Farkash, Evan A; Neal Smith, R; Madsen, Joren C; Russell, Paul S; Colvin, Robert B

    2012-12-01

    Despite extensive research on T cells and potent immunosuppressive regimens that target cellular mediated rejection, few regimens have been proved to be effective on antibody-mediated rejection (AMR), particularly in the chronic setting. C4d deposition in the graft has been proved to be a useful marker for AMR; however, there is an imperfect association between C4d and AMR. While complement has been considered as the main player in acute AMR, the effector mechanisms in chronic AMR are still debated. Recent studies support the role of NK cells and direct effects of antibody on endothelium cells in a mechanism suggesting the presence of a complement-independent pathway. Here, we review the history, currently available systems and progress in experimental animal research. Although there are consistent findings from human and animal research, transposing the experimental results from rodent to human has been hampered by the differences in endothelial functions between species. We briefly describe the findings from patients and compare them with results from animals, to propose a combined perspective.

  20. Hypoxia-mediated regulation of gene expression in mammalian cells

    PubMed Central

    Shih, Shu-Ching; Claffey, Kevin P.

    1998-01-01

    The molecular mechanism underlying oxygen sensing in mammalian cells has been extensively investigated in the areas of glucose transport, glycolysis, erythropoiesis, angiogenesis and catecholamine metabolism. Expression of functionally operative representative proteins in these specific areas, such as the glucose transporter 1, glycolytic enzymes, erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase are all induced by hypoxia. Recent studies demonstrated that both transcriptional activation and post-transcriptional mechanisms are important to the hypoxia-mediated regulation of gene expression. In this article, the cis-acting elements and trans-acting factors involved in the transcriptional activation of gene expression will be reviewed. In addition, the mechanisms of post-transcriptional mRNA stabilization will also be addressed. We will discuss whether these two processes of regulation of hypoxia-responsive genes are mechanistically linked and co-operative in nature. PMID:10319016

  1. On tensegrity in cell mechanics.

    PubMed

    Volokh, K Y

    2011-09-01

    All models are wrong, but some are useful. This famous saying mirrors the situation in cell mechanics as well. It looks like no particular model of the cell deformability can be unconditionally preferred over others and different models reveal different aspects of the mechanical behavior of living cells. The purpose of the present work is to discuss the so-called tensegrity models of the cell cytoskeleton. It seems that the role of the cytoskeleton in the overall mechanical response of the cell was not appreciated until Donald Ingber put a strong emphasis on it. It was fortunate that Ingber linked the cytoskeletal structure to the fascinating art of tensegrity architecture. This link sparked interest and argument among biologists, physicists, mathematicians, and engineers. At some point the enthusiasm regarding tensegrity perhaps became overwhelming and as a reaction to that some skepticism built up. To demystify Ingber's ideas the present work aims at pinpointing the meaning of tensegrity and its role in our understanding of the importance of the cytoskeleton for the cell deformability and motility. It should be noted also that this paper emphasizes basic ideas rather than carefully follows the chronology of the development of tensegrity models. The latter can be found in the comprehensive review by Dimitrije Stamenovic (2006) to which the present work is complementary. PMID:21977516

  2. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis

    PubMed Central

    Poon, Ivan KH; Baxter, Amy A; Lay, Fung T; Mills, Grant D; Adda, Christopher G; Payne, Jennifer AE; Phan, Thanh Kha; Ryan, Gemma F; White, Julie A; Veneer, Prem K; van der Weerden, Nicole L; Anderson, Marilyn A; Kvansakul, Marc; Hulett, Mark D

    2014-01-01

    Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique ‘cationic grip’ configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001 PMID:24692446

  3. Mechanosensitive pannexin-1 channels mediate microvascular metastatic cell survival.

    PubMed

    Furlow, Paul W; Zhang, Steven; Soong, T David; Halberg, Nils; Goodarzi, Hani; Mangrum, Creed; Wu, Y Gloria; Elemento, Olivier; Tavazoie, Sohail F

    2015-07-01

    During metastatic progression, circulating cancer cells become lodged within the microvasculature of end organs, where most die from mechanical deformation. Although this phenomenon was first described over a half-century ago, the mechanisms enabling certain cells to survive this metastasis-suppressive barrier remain unknown. By applying whole-transcriptome RNA-sequencing technology to isogenic cancer cells of differing metastatic capacities, we identified a mutation encoding a truncated form of the pannexin-1 (PANX1) channel, PANX1(1-89), as recurrently enriched in highly metastatic breast cancer cells. PANX1(1-89) functions to permit metastatic cell survival during traumatic deformation in the microvasculature by augmenting ATP release from mechanosensitive PANX1 channels activated by membrane stretch. PANX1-mediated ATP release acts as an autocrine suppressor of deformation-induced apoptosis through P2Y-purinergic receptors. Finally, small-molecule therapeutic inhibition of PANX1 channels is found to reduce the efficiency of breast cancer metastasis. These data suggest a molecular basis for metastatic cell survival on microvasculature-induced biomechanical trauma. PMID:26098574

  4. Roles of cell signaling pathways in cell-to-cell contact-mediated Epstein-Barr virus transmission.

    PubMed

    Nanbo, Asuka; Terada, Haruna; Kachi, Kunihiro; Takada, Kenzo; Matsuda, Tadashi

    2012-09-01

    Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells. PMID:22718812

  5. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    PubMed

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  6. Novel Lipid Mediators and Resolution Mechanisms in Acute Inflammation

    PubMed Central

    Serhan, Charles N.

    2010-01-01

    Because inflammation is appreciated as a unifying basis of many widely occurring diseases, the mechanisms involved in its natural resolution are of considerable interest. Using contained, self-limited inflammatory exudates and a systems approach, novel lipid-derived mediators and pathways were uncovered in the resolution of inflammatory exudates. These new families of local mediators control both the duration and magnitude of acute inflammation as well as the return of the site to homeostasis in the process of catabasis. This new genus of specialized proresolving mediators (SPM) includes essential fatty acid–derived lipoxins, resolvins, protectins, and, most recently, maresins. These families were named based on their unique structures and potent stereoselective actions. The temporally initiated biosynthesis of SPM and their direct impact on leukocyte trafficking and macrophage-directed clearance mechanisms provide clear evidence that resolution is an active, programmed response at the tissue level. Moreover, SPM that possess anti-inflammatory (ie, limiting PMN infiltration) and proresolving (enhance macrophage uptake and clearance of apoptotic PMN and microbial particles) actions as well as stimulating mucosal antimicrobial responses demonstrate that anti-inflammation and proresolution are different responses of the host and novel defining properties of these molecules. The mapping of new resolution circuits has opened the possibility for understanding mechanisms that lead from acute to chronic inflammation, or to the resolution thereof, as well as to potential, resolution-based immunopharmacological therapies. PMID:20813960

  7. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

    PubMed Central

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno; Smith, Amos B.; Shaw, George M.; Hahn, Beatrice H.; Sodroski, Joseph; Kaufmann, Daniel E.; Finzi, Andrés

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells. PMID:26870823

  8. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity.

    PubMed

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R; Melillo, Bruno; Smith, Amos B; Shaw, George M; Hahn, Beatrice H; Sodroski, Joseph; Kaufmann, Daniel E; Finzi, Andrés

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells.

  9. Calcium signaling as a mediator of cell energy demand and a trigger to cell death.

    PubMed

    Bhosale, Gauri; Sharpe, Jenny A; Sundier, Stephanie Y; Duchen, Michael R

    2015-09-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury.

  10. Calcium signaling as a mediator of cell energy demand and a trigger to cell death

    PubMed Central

    Bhosale, Gauri; Sharpe, Jenny A.; Sundier, Stephanie Y.

    2015-01-01

    Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury. PMID:26375864

  11. Biomineralization mediated by anaerobic methane-consuming cell consortia

    PubMed Central

    Chen, Ying; Li, Yi-Liang; Zhou, Gen-Tao; Li, Han; Lin, Yang-Ting; Xiao, Xiang; Wang, Feng-Ping

    2014-01-01

    Anaerobic methanotrophic archaea (ANME) play a significant role in global carbon cycles. These organisms consume more than 90% of ocean-derived methane and influence the landscape of the seafloor by stimulating the formation of carbonates. ANME frequently form cell consortia with sulfate-reducing bacteria (SRB) of the family Deltaproteobacteria. We investigated the mechanistic link between ANME and the natural consortium by examining anaerobic oxidation of methane (AOM) metabolism and the deposition of biogenetic minerals through high-resolution imaging analysis. All of the cell consortia found in a sample of marine sediment were encrusted by a thick siliceous envelope consisting of laminated and cementing substances, whereas carbonate minerals were not found attached to cells. Beside SRB cells, other bacteria (such as Betaproteobacteria) were found to link with the consortia by adhering to the siliceous crusts. Given the properties of siliceous minerals, we hypothesize that ANME cell consortia can interact with other microorganisms and their substrates via their siliceous envelope, and this mechanism of silicon accumulation may serve in clay mineral formation in marine sedimentary environments. A mechanism for biomineralization mediated by AOM consortia was suggested based on the above observations. PMID:25027246

  12. Biomineralization mediated by anaerobic methane-consuming cell consortia.

    PubMed

    Chen, Ying; Li, Yi-Liang; Zhou, Gen-Tao; Li, Han; Lin, Yang-Ting; Xiao, Xiang; Wang, Feng-Ping

    2014-01-01

    Anaerobic methanotrophic archaea (ANME) play a significant role in global carbon cycles. These organisms consume more than 90% of ocean-derived methane and influence the landscape of the seafloor by stimulating the formation of carbonates. ANME frequently form cell consortia with sulfate-reducing bacteria (SRB) of the family Deltaproteobacteria. We investigated the mechanistic link between ANME and the natural consortium by examining anaerobic oxidation of methane (AOM) metabolism and the deposition of biogenetic minerals through high-resolution imaging analysis. All of the cell consortia found in a sample of marine sediment were encrusted by a thick siliceous envelope consisting of laminated and cementing substances, whereas carbonate minerals were not found attached to cells. Beside SRB cells, other bacteria (such as Betaproteobacteria) were found to link with the consortia by adhering to the siliceous crusts. Given the properties of siliceous minerals, we hypothesize that ANME cell consortia can interact with other microorganisms and their substrates via their siliceous envelope, and this mechanism of silicon accumulation may serve in clay mineral formation in marine sedimentary environments. A mechanism for biomineralization mediated by AOM consortia was suggested based on the above observations. PMID:25027246

  13. Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

    PubMed Central

    Burugula, Balabharathi; Ganesh, Bhagyalaxmi S.

    2011-01-01

    Purpose. Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs. Methods. Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 μM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 μM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB). Results. Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression. Conclusions. The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions. PMID:21498608

  14. Molecular basis of sidekick-mediated cell-cell adhesion and specificity

    PubMed Central

    Goodman, Kerry M; Yamagata, Masahito; Jin, Xiangshu; Mannepalli, Seetha; Katsamba, Phinikoula S; Ahlsén, Göran; Sergeeva, Alina P; Honig, Barry; Sanes, Joshua R; Shapiro, Lawrence

    2016-01-01

    Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1–4), arranged in a horseshoe conformation. These Ig1–4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1–4, with Ig1–2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions. DOI: http://dx.doi.org/10.7554/eLife.19058.001 PMID:27644106

  15. The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

    PubMed

    Barbosa-Lorenzi, Valéria Cintra; Buranello, Patrícia Andressa de Almeida; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance; Pereira-da-Silva, Gabriela

    2011-12-16

    Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

  16. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation

    PubMed Central

    Wu, Yongkang; van Besouw, Nicole M.; Shi, Yunying; Hoogduijn, Martin J.; Wang, Lanlan; Baan, Carla C.

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection.

  17. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation

    PubMed Central

    Wu, Yongkang; van Besouw, Nicole M.; Shi, Yunying; Hoogduijn, Martin J.; Wang, Lanlan; Baan, Carla C.

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection. PMID:27602031

  18. The Biological Effects of IL-21 Signaling on B-Cell-Mediated Responses in Organ Transplantation.

    PubMed

    Wu, Yongkang; van Besouw, Nicole M; Shi, Yunying; Hoogduijn, Martin J; Wang, Lanlan; Baan, Carla C

    2016-01-01

    Antibody-mediated rejection has emerged as one of the major issues limiting the success of organ transplantation. It exerts a highly negative impact on graft function and outcome, and effective treatment is lacking. The triggers for antibody development, and the mechanisms leading to graft dysfunction and failure, are incompletely understood. The production of antibodies is dependent on instructions from various immunocytes including CD4 T-helper cells that secrete interleukin (IL)-21 and interact with antigen-specific B-cells via costimulatory molecules. In this article, we discuss the role of IL-21 in the activation and differentiation of B-cells and consider the mechanisms of IL-21 and B-cell interaction. An improved understanding of the biological mechanisms involved in antibody-mediated complications after organ transplantation could lead to the development of novel therapeutic strategies, which control humoral alloreactivity, potentially preventing and treating graft-threatening antibody-mediated rejection. PMID:27602031

  19. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    NASA Astrophysics Data System (ADS)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  20. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    NASA Astrophysics Data System (ADS)

    Song, Zhen

    Life-threatening cardiac arrhythmias continue to pose a major health problem. Ventricular fibrillation, which is a complex form of electrical wave turbulence in the lower chambers of the heart, stops the heart from pumping and is the largest cause of natural death in the United States. Atrial fibrillation, a related form of wave turbulence in the upper heart chambers, is in turn the most common arrhythmia diagnosed in clinical practice. Despite extensive research to date, mechanisms of cardiac arrhythmias remain poorly understood. It is well established that both spatial disorder of the refractory period of heart cells and triggered activity (TA) jointly contribute to the initiation and maintenance of arrhythmias. TA broadly refers to the abnormal generation of a single or a sequence of abnormal excitation waves from a small submillimeter region of the heart in the interval of time between two normal waves generated by the heart's natural pacemaker (the sinoatrial node). TA has been widely investigated experimentally and occurs in several pathological conditions where the intracellular concentration of free Ca2+ ions in heart cells becomes elevated. Under such conditions, Ca2+ can be spontaneously released from intracellular stores, thereby driving an electrogenic current that exchanges 3Na+ ions for one Ca2+ ion across the cell membrane. This current in turn depolarizes the membrane of heart cells after a normal excitation. If this calcium-mediated "delayed after depolarization'' (DAD) is sufficiently large, it can generate an action potential. While the arrhythmogenic importance of spontaneous Ca2+ release and DADs is well appreciated, the conditions under which they occur in heart pathologies remain poorly understood. Calcium overload is only one factor among several other factors that can promote DADs, including sympathetic nerve stimulation, different expression levels of membrane ion channels and calcium handling proteins, and different mutations of those

  1. Mechanisms mediating nitroglycerin-induced delayed-onset hyperalgesia in the rat.

    PubMed

    Ferrari, L F; Levine, J D; Green, P G

    2016-03-11

    Nitroglycerin (glycerol trinitrate, GTN) induces headache in migraineurs, an effect that has been used both diagnostically and in the study of the pathophysiology of this neurovascular pain syndrome. An important feature of this headache is a delay from the administration of GTN to headache onset that, because of GTN's very rapid metabolism, cannot be due to its pharmacokinetic profile. It has recently been suggested that activation of perivascular mast cells, which has been implicated in the pathophysiology of migraine, may contribute to this delay. We reported that hyperalgesia induced by intradermal GTN has a delay to onset of ∼ 30 min in male and ∼ 45 min in female rats. This hyperalgesia was greater in females, was prevented by pretreatment with the anti-migraine drug, sumatriptan, as well as by chronic pretreatment with the mast cell degranulator, compound 48/80. The acute administration of GTN and compound 48/80 both induced hyperalgesia that was prevented by pretreatment with octoxynol-9, which attenuates endothelial function, suggesting that GTN and mast cell-mediated hyperalgesia are endothelial cell-dependent. Furthermore, A-317491, a P2X3 antagonist, which inhibits endothelial cell-dependent hyperalgesia, also prevents GTN and mast cell-mediated hyperalgesia. We conclude that delayed-onset mechanical hyperalgesia induced by GTN is mediated by activation of mast cells, which in turn release mediators that stimulate endothelial cells to release ATP, to act on P2X3, a ligand-gated ion channel, in perivascular nociceptors. A role of the mast and endothelial cell in GTN-induced hyperalgesia suggests potential novel risk factors and targets for the treatment of migraine.

  2. Mechanical strain promotes osteoblastic differentiation through integrin-β1-mediated β-catenin signaling.

    PubMed

    Yan, Yuxian; Sun, Haoyang; Gong, Yuanwei; Yan, Zhixiong; Zhang, Xizheng; Guo, Yong; Wang, Yang

    2016-08-01

    As integrins are mechanoresponsive, there exists an intimate relationship between integrins and mechanical strain. Integrin-β1 mediates the impact of mechanical strain on bone. Mechanical strain induces bone formation through the activation of β-catenin pathways, which suggests that integrin-β1 mediates β-catenin signaling in osteoblasts in response to mechanical strain. In the present study, we examined the role of integrin-β1 in Wnt/β-catenin signal transduction in mechanically strained osteoblasts. MC3T3-E1 osteoblastic cells were transfected with integrin-β1 small interfering RNA (si-Itgβ1), and exposed to mechanical tensile strain of 2,500 microstrain (µε) using a four-point bending device. The mechanical strain enhanced the mRNA expression of integrin-β1, the protein levels of phosphorylated (p-) glycogen synthase kinase-3β (GSK‑3β) and β-catenin, simultaneously increased the mRNA levels of runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), the protein levels of bone morphogenetic protein (BMP)-2 and -4 and enhanced the alkaline phosphatase (ALP) activity of the ME3T3-E1 cells. The elevations were inhibited by si-Itgβ1. Additionally, the mechanical strain induced the nuclear translocation of β-catenin into the nucleus, which was also inhibited by si-Itgβ1. These findings indicated that mechanical strain promoted osteoblastic differentiation through integrin‑β1‑mediated β-catenin signaling.

  3. Cadherin-11 mediates contact inhibition of locomotion during Xenopus neural crest cell migration.

    PubMed

    Becker, Sarah F S; Mayor, Roberto; Kashef, Jubin

    2013-01-01

    Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC.

  4. Altered heme-mediated modulation of dendritic cell function in sickle cell alloimmunization

    PubMed Central

    Godefroy, Emmanuelle; Liu, Yunfeng; Shi, Patricia; Mitchell, W. Beau; Cohen, Devin; Chou, Stella T.; Manwani, Deepa; Yazdanbakhsh, Karina

    2016-01-01

    Transfusions are the main treatment for patients with sickle cell disease. However, alloimmunization remains a major life-threatening complication for these patients, but the mechanism underlying pathogenesis of alloimmunization is not known. Given the chronic hemolytic state characteristic of sickle cell disease, resulting in release of free heme and activation of inflammatory cascades, we tested the hypothesis that anti-inflammatory response to heme is compromised in alloimmunized sickle patients, increasing their risk of alloimmunization. Heme-exposed monocyte-derived dendritic cells from both non-alloimmunized sickle patients and healthy donors inhibited priming of pro-inflammatory CD4+ type 1 T cells, and exhibited significantly reduced levels of the maturation marker CD83. In contrast, in alloimmunized patients, heme did not reverse priming of pro-inflammatory CD4+ cells by monocyte-derived dendritic cells or their maturation. Furthermore, heme dampened NF-κB activation in non-alloimmunized, but not in alloimmunized monocyte-derived dendritic cells. Heme-mediated CD83 inhibition depended on Toll-like receptor 4 but not heme oxygenase 1. These data suggest that extracellular heme limits CD83 expression on dendritic cells in non-alloimmunized sickle patients through a Toll-like receptor 4-mediated pathway, involving NF-κB, resulting in dampening of pro-inflammatory responses, but that in alloimmunized patients this pathway is defective. This opens up the possibility of developing new therapeutic strategies to prevent sickle cell alloimmunization. PMID:27229712

  5. Altered heme-mediated modulation of dendritic cell function in sickle cell alloimmunization.

    PubMed

    Godefroy, Emmanuelle; Liu, Yunfeng; Shi, Patricia; Mitchell, W Beau; Cohen, Devin; Chou, Stella T; Manwani, Deepa; Yazdanbakhsh, Karina

    2016-09-01

    Transfusions are the main treatment for patients with sickle cell disease. However, alloimmunization remains a major life-threatening complication for these patients, but the mechanism underlying pathogenesis of alloimmunization is not known. Given the chronic hemolytic state characteristic of sickle cell disease, resulting in release of free heme and activation of inflammatory cascades, we tested the hypothesis that anti-inflammatory response to heme is compromised in alloimmunized sickle patients, increasing their risk of alloimmunization. Heme-exposed monocyte-derived dendritic cells from both non-alloimmunized sickle patients and healthy donors inhibited priming of pro-inflammatory CD4(+) type 1 T cells, and exhibited significantly reduced levels of the maturation marker CD83. In contrast, in alloimmunized patients, heme did not reverse priming of pro-inflammatory CD4(+) cells by monocyte-derived dendritic cells or their maturation. Furthermore, heme dampened NF-κB activation in non-alloimmunized, but not in alloimmunized monocyte-derived dendritic cells. Heme-mediated CD83 inhibition depended on Toll-like receptor 4 but not heme oxygenase 1. These data suggest that extracellular heme limits CD83 expression on dendritic cells in non-alloimmunized sickle patients through a Toll-like receptor 4-mediated pathway, involving NF-κB, resulting in dampening of pro-inflammatory responses, but that in alloimmunized patients this pathway is defective. This opens up the possibility of developing new therapeutic strategies to prevent sickle cell alloimmunization.

  6. Nanomedicine-mediated cancer stem cell therapy.

    PubMed

    Shen, Song; Xia, Jin-Xing; Wang, Jun

    2016-01-01

    Circumstantial evidence suggests that most tumours are heterogeneous and contain a small population of cancer stem cells (CSCs) that exhibit distinctive self-renewal, proliferation and differentiation capabilities, which are believed to play a crucial role in tumour progression, drug resistance, recurrence and metastasis in multiple malignancies. Given that the existence of CSCs is a primary obstacle to cancer therapy, a tremendous amount of effort has been put into the development of anti-CSC strategies, and several potential approaches to kill therapeutically-resistant CSCs have been explored, including inhibiting ATP-binding cassette transporters, blocking essential signalling pathways involved in self-renewal and survival of CSCs, targeting CSCs surface markers and destroying the tumour microenvironment. Meanwhile, an increasing number of therapeutic agents (e.g. small molecule drugs, nucleic acids and antibodies) to selectively target CSCs have been screened or proposed in recent years. Drug delivery technology-based approaches hold great potential for tackling the limitations impeding clinical applications of CSC-specific agents, such as poor water solubility, short circulation time and inconsistent stability. Properly designed nanocarrier-based therapeutic agents (or nanomedicines) offer new possibilities of penetrating CSC niches and significantly increasing therapeutic drug accumulation in CSCs, which are difficult for free drug counterparts. In addition, intelligent nanomedicine holds great promise to overcome pump-mediated multidrug resistance which is driven by ATP and to decrease detrimental effects on normal somatic stem cells. In this review, we summarise the distinctive biological processes related to CSCs to highlight strategies against inherently drug-resistant CSCs. We then focus on some representative examples that give a glimpse into state-of-the-art nanomedicine approaches developed for CSCs elimination. A perspective on innovative therapeutic

  7. Mechanisms of endonuclease-mediated mRNA decay.

    PubMed

    Schoenberg, Daniel R

    2011-01-01

    Endonuclease cleavage was one of the first identified mechanisms of mRNA decay but until recently it was thought to play a minor role to the better-known processes of deadenylation, decapping, and exonuclease-catalyzed decay. Most of the early examples of endonuclease decay came from studies of a particular mRNA whose turnover changed in response to hormone, cytokine, developmental, or nutritional stimuli. Only a few of these examples of endonuclease-mediated mRNA decay progressed to the point where the enzyme responsible for the initiating event was identified and studied in detail. The discovery of microRNAs and RISC-catalyzed endonuclease cleavage followed by the identification of PIN (pilT N-terminal) domains that impart endonuclease activity to a number of the proteins involved in mRNA decay has led to a resurgence of interest in endonuclease-mediated mRNA decay. PIN domains show no substrate selectivity and their involvement in a number of decay pathways highlights a recurring theme that the context in which an endonuclease function is a primary factor in determining whether any given mRNA will be targeted for decay by this or the default exonuclease-mediated decay processes.

  8. Detection of cell mediated immune response to avian influenza viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In birds, lymphomyeloid tissues develop from epithelial (Bursa of Fabricus or thymus) or mesenchymal tissue which are populated by heamatopoietic stem cells. These stem cells develop directly into immunologically competent B (bursa) and T (thymus) cells. Cell-mediated immunity (CMI) is a part of the...

  9. Mechanism and regulation of the nonsense-mediated decay pathway

    PubMed Central

    Hug, Nele; Longman, Dasa; Cáceres, Javier F.

    2016-01-01

    The Nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs) but also regulates the abundance of a large number of cellular RNAs. The central role of NMD in the control of gene expression requires the existence of buffering mechanisms that tightly regulate the magnitude of this pathway. Here, we will focus on the mechanism of NMD with an emphasis on the role of RNA helicases in the transition from NMD complexes that recognize a PTC to those that promote mRNA decay. We will also review recent strategies aimed at uncovering novel trans-acting factors and their functional role in the NMD pathway. Finally, we will describe recent progress in the study of the physiological role of the NMD response. PMID:26773057

  10. Stem and progenitor cell-mediated tumor selective gene therapy.

    PubMed

    Aboody, K S; Najbauer, J; Danks, M K

    2008-05-01

    The poor prognosis for patients with aggressive or metastatic tumors and the toxic side effects of currently available treatments necessitate the development of more effective tumor-selective therapies. Stem/progenitor cells display inherent tumor-tropic properties that can be exploited for targeted delivery of anticancer genes to invasive and metastatic tumors. Therapeutic genes that have been inserted into stem cells and delivered to tumors with high selectivity include prodrug-activating enzymes (cytosine deaminase, carboxylesterase, thymidine kinase), interleukins (IL-2, IL-4, IL-12, IL-23), interferon-beta, apoptosis-promoting genes (tumor necrosis factor-related apoptosis-inducing ligand) and metalloproteinases (PEX). We and others have demonstrated that neural and mesenchymal stem cells can deliver therapeutic genes to elicit a significant antitumor response in animal models of intracranial glioma, medulloblastoma, melanoma brain metastasis, disseminated neuroblastoma and breast cancer lung metastasis. Most studies reported reduction in tumor volume (up to 90%) and increased survival of tumor-bearing animals. Complete cures have also been achieved (90% disease-free survival for >1 year of mice bearing disseminated neuroblastoma tumors). As we learn more about the biology of stem cells and the molecular mechanisms that mediate their tumor-tropism and we identify efficacious gene products for specific tumor types, the clinical utility of cell-based delivery strategies becomes increasingly evident.

  11. Lico A Enhances Nrf2-Mediated Defense Mechanisms against t-BHP-Induced Oxidative Stress and Cell Death via Akt and ERK Activation in RAW 264.7 Cells

    PubMed Central

    Lv, Hongming; Ren, Hua; Wang, Lidong; Chen, Wei; Ci, Xinxin

    2015-01-01

    Licochalcone A (Lico A) exhibits various biological properties, including anti-inflammatory and antioxidant activities. In this study, we investigated the antioxidative potential and mechanisms of Lico A against tert-butyl hydroperoxide- (t-BHP-) induced oxidative damage in RAW 264.7 cells. Our results indicated that Lico A significantly inhibited t-BHP-induced cytotoxicity, apoptosis, and reactive oxygen species (ROS) generation and reduced glutathione (GSH) depletion but increased the glutamate-cysteine ligase modifier (GCLM) subunit and the glutamate-cysteine ligase catalytic (GCLC) subunit genes expression. Additionally, Lico A dramatically upregulated the antioxidant enzyme heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), which were associated with inducing Nrf2 nuclear translocation, decreasing Keap1 protein expression and increasing antioxidant response element (ARE) promoter activity. Lico A also obviously induced the activation of serine/threonine kinase (Akt) and extracellular signal-regulated kinase (ERK), but PI3K/Akt and ERK inhibitors treatment displayed clearly decreased levels of LicoA-induced Nrf2 nuclear translocation and HO-1 expression, respectively. Furthermore, Lico A treatment markedly attenuated t-BHP-induced oxidative damage, which was reduced by treatment with PI3K/Akt, ERK, and HO-1 inhibitors. Therefore, Lico A might have a protective role against t-BHP-induced cytotoxicity by modulating HO-1 and by scavenging ROS via the activation of the PI3K/Akt and ERK/Nrf2 signaling pathways. PMID:26576227

  12. Visfatin Mediates SCLC Cells Migration across Brain Endothelial Cells through Upregulation of CCL2.

    PubMed

    Liu, Tingting; Miao, Ziwei; Jiang, Jiusheng; Yuan, Shuai; Fang, Wengang; Li, Bo; Chen, Yuhua

    2015-05-18

    Small-cell lung cancer (SCLC) is characterized as an aggressive tumor with brain metastasis. Although preventing SCLC metastasis to the brain is immensely important for survival, the molecular mechanisms of SCLC cells penetrating the blood-brain barrier (BBB) are largely unknown. Recently, visfatin has been considered as a novel pro-inflammatory adipocytokine involved in various cancers. Herein, we present evidence that elevated levels of visfatin in the serum of SCLC patients were associated with brain metastasis, and visfain was increased in NCI-H446 cells, a SCLC cell line, during interacting with human brain microvascular endothelial cells (HBMEC). Using in vitro BBB model, we found that visfatin could promote NCI-H446 cells migration across HBMEC monolayer, while the effect was inhibited by knockdown of visfatin. Furthermore, our findings indicated that CC chemokine ligand 2 (CCL2) was involved in visfatin-mediated NCI-H446 cells transendothelial migtation. Results also showed that the upregulation of CCL2 in the co-culture system was reversed by blockade of visfatin. In particular, visfatin-induced CCL2 was attenuated by specific inhibitor of PI3K/Akt signaling in NCI-H446 cells. Taken together, we demonstrated that visfatin was a prospective target for SCLC metastasis to brain, and understanding the molecular mediators would lead to effective strategies for inhibition of SCLC brain metastasis.

  13. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  14. Pili-mediated Interactions between Neisseria Gonorrhoeae Bacteria are the Driving Mechanism of Microcolony Merging

    NASA Astrophysics Data System (ADS)

    Poenisch, Wolfram; Weber, Christoph; Alzurqa, Khaled; Nasrollahi, Hadi; Biais, Nicolas; Zaburdaev, Vasily; Collective Dynamics of Cells Team; Mechano-Micro-Biology Lab Team

    2015-03-01

    During the early infection with Neisseria gonorrhoeae the bacteria form microcolonies consisting of a few hundreds to a few thousands of cells. The formation of colonies is mediated by type IV pili, thin and long filaments that are also involved in the motion of single cells over a substrate. A related process causes attractive cell-cell-interactions. While the motion of single cells has been extensively studied during the past years, the physical principles driving the growth of these colonies are poorly understood. One key mechanism of colony growth is coalescence of smaller colonies. Therefore we experimentally examine the process of merging of two Neisseria gonorrhoeae colonies. We develop a theoretical microscopic model of single cells interacting solely by their pili. The experimental data and the results obtained from our model are in excellent quantitative agreement. We observe a fast initial approach of the two merging colonies within a few minutes, that is followed by a slow relaxation of the colony shape with a characteristic time of several hours. These findings suggest that pili-mediated interactions are the primary driving mechanism of the microcolony merging process.

  15. Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells.

    PubMed

    Sosna, Justyna; Philipp, Stephan; Fuchslocher Chico, Johaiber; Saggau, Carina; Fritsch, Jürgen; Föll, Alexandra; Plenge, Johannes; Arenz, Christoph; Pinkert, Thomas; Kalthoff, Holger; Trauzold, Anna; Schmitz, Ingo; Schütze, Stefan; Adam, Dieter

    2016-10-15

    Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H(+)-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches. PMID:27528614

  16. Cell mediated immunity to fungi: a reassessment.

    PubMed

    Romani, Luigina

    2008-09-01

    Protective immunity against fungal pathogens is achieved by the integration of two distinct arms of the immune system, the innate and adaptive responses. Innate and adaptive immune responses are intimately linked and controlled by sets of molecules and receptors that act to generate the most effective form of immunity for protection against fungal pathogens. The decision of how to respond will still be primarily determined by interactions between pathogens and cells of the innate immune system, but the actions of T cells will feed back into this dynamic equilibrium to regulate the balance between tolerogenic and inflammatory responses. In the last two decades, the immunopathogenesis of fungal infections and fungal diseases was explained primarily in terms of Th1/Th2 balance. Although Th1 responses driven by the IL-12/IFN-gamma axis are central to protection against fungi, other cytokines and T cell-dependent pathways have come of age. The newly described Th17 developmental pathway may play an inflammatory role previously attributed to uncontrolled Th1 responses and serves to accommodate the seemingly paradoxical association of chronic inflammatory responses with fungal persistence in the face of an ongoing inflammation. Regulatory T cells in their capacity to inhibit aspects of innate and adaptive antifungal immunity have become an integral component of immune resistance to fungi, and provide the host with immune defense mechanisms adequate for protection, without necessarily eliminating fungal pathogens which would impair immune memory--or causing an unacceptable level of tissue damage. The enzyme indoleamine 2,3-dioxygenase and tryptophan metabolites contribute to immune homeostasis by inducing Tregs and taming overzealous or heightened inflammatory responses.

  17. Cell mechanics in biomedical cavitation

    PubMed Central

    Wang, Qianxi; Manmi, Kawa; Liu, Kuo-Kang

    2015-01-01

    Studies on the deformation behaviours of cellular entities, such as coated microbubbles and liposomes subject to a cavitation flow, become increasingly important for the advancement of ultrasonic imaging and drug delivery. Numerical simulations for bubble dynamics of ultrasound contrast agents based on the boundary integral method are presented in this work. The effects of the encapsulating shell are estimated by adapting Hoff's model used for thin-shell contrast agents. The viscosity effects are estimated by including the normal viscous stress in the boundary condition. In parallel, mechanical models of cell membranes and liposomes as well as state-of-the-art techniques for quantitative measurement of viscoelasticity for a single cell or coated microbubbles are reviewed. The future developments regarding modelling and measurement of the material properties of the cellular entities for cutting-edge biomedical applications are also discussed. PMID:26442142

  18. Restoring E-cadherin-mediated cell-cell adhesion increases PTEN protein level and stability in human breast carcinoma cells

    SciTech Connect

    Li Zengxia; Wang Liying; Zhang Wen; Fu Yi; Zhao Hongbo; Hu Yali; Prins, Bram Peter; Zha Xiliang

    2007-11-09

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a well-characterized tumor suppressor that negatively regulates cell growth and survival. Despite the critical role of PTEN in cell signaling, the mechanisms of its regulation are still under investigation. We reported here that PTEN expression could be controlled by overexpression or knock-down of E-cadherin in several mammary carcinoma cell lines. Furthermore, we showed that the accumulation of PTEN protein in E-cadherin overexpressing cells was due to increased PTEN protein stability rather than the regulation of its transcription. The proteasome-dependent PTEN degradation pathway was impaired after restoring E-cadherin expression. Moreover, maintenance of E-cadherin mediated cell-cell adhesion was necessary for its regulating PTEN. Altogether, our results suggested that E-cadherin mediated cell-cell adhesion was essential for preventing the proteasome degradation of PTEN, which might explain how breast carcinoma cells which lost cell-cell contact proliferate rapidly and are prone to metastasis.

  19. ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

    PubMed

    Fujikura, Daisuke; Toyomane, Kochi; Kamiya, Kozue; Mutoh, Memi; Mifune, Etsuko; Ohnuma, Miyuki; Higashi, Hideaki

    2016-09-01

    Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells. PMID:27170489

  20. NK Cells and γδ T Cells Mediate Resistance to Polyomavirus–Induced Tumors

    PubMed Central

    Mishra, Rabinarayan; Chen, Alex T.; Welsh, Raymond M.; Szomolanyi-Tsuda, Eva

    2010-01-01

    NK and γδ T cells can eliminate tumor cells in many experimental models, but their effect on the development of tumors caused by virus infections in vivo is not known. Polyomavirus (PyV) induces tumors in neonatally infected mice of susceptible strains and in adult mice with certain immune deficiencies, and CD8+ αβ T cells are regarded as the main effectors in anti-tumor immunity. Here we report that adult TCRβ knockout (KO) mice that lack αβ but have γδ T cells remain tumor-free after PyV infection, whereas TCRβ×δ KO mice that lack all T cells develop tumors. In addition, E26 mice, which lack NK and T cells, develop the tumors earlier than TCRβ×δ KO mice. These observations implicate γδ T and NK cells in the resistance to PyV-induced tumors. Cell lines established from PyV-induced tumors activate NK and γδ T cells both in culture and in vivo and express Rae-1, an NKG2D ligand. Moreover, these PyV tumor cells are killed by NK cells in vitro, and this cytotoxicity is prevented by treatment with NKG2D-blocking antibodies. Our findings demonstrate a protective role for NK and γδ T cells against naturally occurring virus-induced tumors and suggest the involvement of NKG2D-mediated mechanisms. PMID:20523894

  1. Cell penetrating peptide-mediated transport enables the regulated secretion of accumulated cargoes from mast cells.

    PubMed

    Howl, John; Jones, Sarah

    2015-03-28

    The in vivo utility of technologies employing cell penetrating peptides and bioportides may be compromised by the general capacity of polycationic peptides to activate mast cell secretion. Moreover, the same technologies could be exploited in a clinical setting either to directly modulate intrinsic exocytotic mechanisms or to load mast cells with bioactive cargoes. Comparative investigations identified two cell penetrating vectors, Tat and C105Y, which readily translocate into mast cells without inducing receptor-independent exocytosis. Efficient Tat transduction also enabled the intracellular delivery and accumulation of cargoes within discrete intracellular compartments. A tetramethylrhodamine-Tat conjugate is effectively translocated into the secretory lysosomes of RBL-2H3 cells. In contract, the intracellular delivery of avidin, as a non-covalent complex with a biotinylated Tat vector, is also efficient but the protein is predominantly accumulated outside of secretory lysosomes. Significantly, both cargoes can be subsequently released following mast cell stimulation either by mastoparan, a wasp venom secretagogue, or by the physiological mechanism of antigen-induced aggregation of high affinity IgE receptors. These studies indicate that mast cells could be exploited to direct the delivery of bioactive agents to disease sites as an innovative cell-mediated therapy. PMID:25660072

  2. Cell mediated immunity in Antarctic wintering personnel; 1984-1992.

    PubMed

    Muller, H K; Lugg, D J; Quinn, D

    1995-08-01

    Cell-mediated immune responses were studied in 12 Antarctic and sub-Antarctic wintering groups at quarterly intervals over the period 1984-1992, using the cutaneous CMI Multitest. These populations are among the most isolated on earth. While the sub-Antarctic population at Macquarie Island had levels of responsiveness and hypoergy (9%) comparable to healthy populations in temperate zones, the Antarctic Continental group showed a level of hypoergy of 36%. There was no seasonal variation in the pattern of responses. It is concluded that the extreme and isolated environment and stress factors are responsible for the decreased immunological responsiveness but the mechanisms are presently unclear. On review, one factor appears to be perceived anxiety. The high rate of hypoergy in Antarctica, where medical care is limited, may have health implications. These groups provide an excellent analogue for immunological investigations in longer term space flight.

  3. TLR5 mediates CD172α+ intestinal lamina propria dendritic cell induction of Th17 cells

    PubMed Central

    Liu, Han; Chen, Feidi; Wu, Wei; Cao, Anthony T; Xue, Xiaochang; Yao, Suxia; Evans-Marin, Heather L; Li, Yan-Qing; Cong, Yingzi

    2016-01-01

    Multiple mechanisms exist in regulation of host responses to massive challenges from microbiota to maintain immune homeostasis in the intestines. Among these is the enriched Th17 cells in the intestines, which regulates intestinal homeostasis through induction of antimicrobial peptides and secretory IgA among others. However, the means by which Th17 cells develop in response to microbiota is still not completely understood. Although both TLR5 and CD172α+ lamina propria dendritic cells (LPDC) have been shown to promote Th17 cell development, it is still unclear whether TLR5 mediates the CD172α+LPDC induction of Th17 cells. By using a microbiota antigen-specific T cell reporter mouse system, we demonstrated that microbiota antigen-specific T cells developed into Th17 cells in the intestinal LP, but not in the spleen when transferred into TCRβxδ−/− mice. LPDCs expressed high levels of TLR5, and most CD172α+LPDCs also co-expressed TLR5. LPDCs produced high levels of IL-23, IL-6 and TGFβ when stimulated with commensal flagellin and promoted Th17 cell development when cultured with full-length CBir1 flagellin but not CBir1 peptide. Wild-type CD172α+, but not CD172α−, LPDCs induced Th17 cells, whereas TLR5-deficient LPDC did not induce Th17 cells. Our data thereby demonstrated that TLR5 mediates CD172α+LPDC induction of Th17 cells in the intestines. PMID:26907705

  4. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    SciTech Connect

    Choi, Yoon Jung; Lee, Jue Yeon; Lee, Seung Jin; Chung, Chong-Pyoung; Park, Yoon Jeong

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  5. A possible mechanism in DHEA-mediated protection against osteoarthritis.

    PubMed

    Li, Wei-Jun; Tang, Lu-Ping; Xiong, Yan; Chen, Wei-Ping; Zhou, Xin-Die; Ding, Qian-Hai; Wu, Li-Dong

    2014-11-01

    Dehydroepiandrosterone (DHEA) and its ester form, DHEA-S, are the most abundant steroids in human plasma. Our previous studies showed that DHEA protects against osteoarthritis (OA). The aim of this paper was to explore the possible mechanisms that underlie DHEA-mediated protection against OA. We tested the expression of β-catenin, it was increased significantly in OA. Rabbit cartilage was treated with various concentrations of DHEA in both IL-1β-induced rabbit chondrocytes and in rabbit cartilage from the anterior cruciate ligament transaction-induced OA model. We found DHEA decreased the expression of β-catenin. Then we further activated Wnt/β-catenin signaling by β-catenin transfection and inactivated it by the inhibitor Dickkopf1 in chondrocytes to reveal its role in the pathogenesis of OA. It turns out the protective effect of DHEA was significantly decreased when Wnt/β-catenin signaling was activated, while inactivating Wnt/β-catenin signaling enhanced the effects of DHEA. Therefore, we hypothesize that DHEA probably exerted its chondroprotective effect by regulating Wnt/β-catenin signaling. Our findings demonstrate the critical role of Wnt/β-catenin signaling in DHEA-mediated protection against OA. PMID:25065588

  6. Mast cell mediators: their differential release and the secretory pathways involved.

    PubMed

    Moon, Tae Chul; Befus, A Dean; Kulka, Marianna

    2014-01-01

    Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host-environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved.

  7. Mast Cell Mediators: Their Differential Release and the Secretory Pathways Involved

    PubMed Central

    Moon, Tae Chul; Befus, A. Dean; Kulka, Marianna

    2014-01-01

    Mast cells (MC) are widely distributed throughout the body and are common at mucosal surfaces, a major host–environment interface. MC are functionally and phenotypically heterogeneous depending on the microenvironment in which they mature. Although MC have been classically viewed as effector cells of IgE-mediated allergic diseases, they are also recognized as important in host defense, innate and acquired immunity, homeostatic responses, and immunoregulation. MC activation can induce release of pre-formed mediators such as histamine from their granules, as well as release of de novo synthesized lipid mediators, cytokines, and chemokines that play diverse roles, not only in allergic reactions but also in numerous physiological and pathophysiological responses. Indeed, MC release their mediators in a discriminating and chronological manner, depending upon the stimuli involved and their signaling cascades (e.g., IgE-mediated or Toll-like receptor-mediated). However, the precise mechanisms underlying differential mediator release in response to these stimuli are poorly known. This review summarizes our knowledge of MC mediators and will focus on what is known about the discriminatory release of these mediators dependent upon diverse stimuli, MC phenotypes, and species of origin, as well as on the intracellular synthesis, storage, and secretory processes involved. PMID:25452755

  8. Mechanisms and Representations of Language-Mediated Visual Attention

    PubMed Central

    Huettig, Falk; Mishra, Ramesh Kumar; Olivers, Christian N. L.

    2011-01-01

    The experimental investigation of language-mediated visual attention is a promising way to study the interaction of the cognitive systems involved in language, vision, attention, and memory. Here we highlight four challenges for a mechanistic account of this oculomotor behavior: the levels of representation at which language-derived and vision-derived representations are integrated; attentional mechanisms; types of memory; and the degree of individual and group differences. Central points in our discussion are (a) the possibility that local microcircuitries involving feedforward and feedback loops instantiate a common representational substrate of linguistic and non-linguistic information and attention; and (b) that an explicit working memory may be central to explaining interactions between language and visual attention. We conclude that a synthesis of further experimental evidence from a variety of fields of inquiry and the testing of distinct, non-student, participant populations will prove to be critical. PMID:22291672

  9. Mechanisms of abscisic acid-mediated control of stomatal aperture.

    PubMed

    Munemasa, Shintaro; Hauser, Felix; Park, Jiyoung; Waadt, Rainer; Brandt, Benjamin; Schroeder, Julian I

    2015-12-01

    Drought stress triggers an increase in the level of the plant hormone abscisic acid (ABA), which initiates a signaling cascade to close stomata and reduce water loss. Recent studies have revealed that guard cells control cytosolic ABA concentration through the concerted actions of biosynthesis, catabolism as well as transport across membranes. Substantial progress has been made at understanding the molecular mechanisms of how the ABA signaling core module controls the activity of anion channels and thereby stomatal aperture. In this review, we focus on our current mechanistic understanding of ABA signaling in guard cells including the role of the second messenger Ca(2+) as well as crosstalk with biotic stress responses. PMID:26599955

  10. Cell crawling mediates collective cell migration to close undamaged epithelial gaps

    PubMed Central

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C.; Sheetz, Michael P.; Trepat, Xavier; Ladoux, Benoît

    2012-01-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  11. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benoît

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  12. Mechanism of HSV infection through soluble adapter-mediated virus bridging to the EGF receptor

    SciTech Connect

    Nakano, Kenji; Kobayashi, Masatoshi; Nakamura, Kei-ichiro; Nakanishi, Takeshi; Asano, Ryutaro; Kumagai, Izumi; Tahara, Hideaki; Kuwano, Michihiko; Cohen, Justus B.; Glorioso, Joseph C.

    2011-04-25

    Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.

  13. Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

    PubMed

    Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

    2015-09-30

    Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

  14. P-cadherin-mediated Rho GTPase regulation during collective cell migration

    PubMed Central

    Plutoni, Cédric; Bazellières, Elsa; Gauthier-Rouvière, Cécile

    2016-01-01

    ABSTRACT This commentary addresses the role of P-cadherin in collective cell migration (CCM), a cooperative and coordinated migration mode, used by cells during normal and pathological migration processes. We discuss how cadherin-mediated cell-cell junctions (CCJs) play a critical role in CCM through their ability to regulate Rho GTPase-dependent pathways and how this leads to the generation and orientation of mechanical forces. We will also highlight the key function of P-cadherin (a poor prognostic marker in several tumors) in promoting collective cell movement in epithelial and mesenchymal cells. PMID:27152729

  15. BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms

    PubMed Central

    Hatzi, Katerina; Nance, J. Philip; Kroenke, Mark A.; Bothwell, Marcella; Haddad, Elias K.

    2015-01-01

    Follicular helper T cells (Tfh cells) are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh cells have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration and repression of alternative cell fates. Interestingly, although some BCL6-bound genes possessed BCL6 DNA–binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and BCL6 depends on AP1 for recruitment to BCL6-binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology and provide insight into how this master regulator mediates distinct cell context–dependent phenotypes. PMID:25824819

  16. 3D culture model of fibroblast-mediated collagen creep to identify abnormal cell behaviour.

    PubMed

    Kureshi, A K; Afoke, A; Wohlert, S; Barker, S; Brown, R A

    2015-11-01

    Native collagen gels are important biomimetic cell support scaffolds, and a plastic compression process can now be used to rapidly remove fluid to any required collagen density, producing strong 3D tissue-like models. This study aimed to measure the mechanical creep properties of such scaffolds and to quantify any enhanced creep occurring in the presence of cells (cell-mediated creep). The test rig developed applies constant creep tension during culture and measures real-time extension due to cell action. This was used to model extracellular matrix creep, implicated in the transversalis fascia (TF) in inguinal hernia. Experiments showed that at an applied tension equivalent to 15% break strength, cell-mediated creep over 24-h culture periods was identified at creep rates of 0.46 and 0.38%/h for normal TF and human dermal fibroblasts, respectively. However, hernia TF fibroblasts produced negligible cell-mediated creep levels under the same conditions. Raising the cell culture temperature from 4 to 37 °C was used to demonstrate live cell dependence of this creep. This represents the first in vitro demonstration of TF cell-mediated collagen creep and to our knowledge the first demonstration of a functional, hernia-related cell abnormality. PMID:25862069

  17. Autophagy mediates phase transitions from cell death to life.

    PubMed

    Han, Kyungreem; Kim, Jinwoong; Choi, MooYoung

    2015-09-01

    Autophagy is a lysosomal degradation pathway, which is critical for maintaining normal cellular functions. Despite considerable advances in defining the specific molecular mechanism governing the autophagy pathway during the last decades, we are still far from understanding the underlying principle of the autophagy machinery and its complex role in human disease. As an alternative attempt to reinvigorate the search for the principle of the autophagy pathway, we in this study make use of the computer-aided analysis, complementing current molecular-level studies of autophagy. Specifically, we propose a hypothesis that autophagy mediates cellular phase transitions and demonstrate that the autophagic phase transitions are essential to the maintenance of normal cellular functions and critical in the fate of a cell, i.e., cell death or survival. This study should provide valuable insight into how interactions of sub-cellular components such as genes and protein modules/complexes regulate autophagy and then impact on the dynamic behaviors of living cells as a whole, bridging the microscopic molecular-level studies and the macroscopic cellular-level and physiological approaches. PMID:27441218

  18. Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration

    PubMed Central

    Dudakov, Jarrod A.; Jenq, Robert R.; Velardi, Enrico; Young, Lauren F.; Smith, Odette M.; Lawrence, Gillian; Ivanov, Juliet A.; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L.; O'Rourke, Kevin P.; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas; Nieuwenhuis, Edward E.; Shroyer, Noah F.; Liu, Chen; Kolesnick, Richard

    2015-01-01

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  19. Ca-Mediated Electroformation of Cell-Sized Lipid Vesicles

    PubMed Central

    Tao, Fei; Yang, Peng

    2015-01-01

    Cell-sized lipid giant unilamellar vesicles (GUVs) are formed when lipid molecules self-assemble to construct a single bilayer compartment with similar morphology to living cells. The physics of self-assembly process is only generally understood and the size distribution of GUVs tends to be very polydisperse. Herein we report a strategy for the production of controlled size distributions of GUVs by a novel mechanism dissecting the mediation ability of calcium (Ca) on the conventional electroformation of GUVs. We finely construct both of the calcium ion (Ca2+) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca2+ Slip plane polarized by alternating electric field could induce a pattern of electroosmotic flow across the surface, and thus confine the fusion and growth of GUVs to facilitate the formation of uniform GUVs. The model is further improved by directly using CaCO3 that is in situ formed on a lipid film surface, providing a GUV population with narrow polydispersity. The two models deciphers the new biological function of calcium on the birth of cell-like lipid vesicles, and thus might be potentially relevant to the construction of new model to elucidate the cellular development process. PMID:25950604

  20. Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death.

    PubMed

    Xia, Hong-Guang; Najafov, Ayaz; Geng, Jiefei; Galan-Acosta, Lorena; Han, Xuemei; Guo, Yuan; Shan, Bing; Zhang, Yaoyang; Norberg, Erik; Zhang, Tao; Pan, Lifeng; Liu, Junli; Coloff, Jonathan L; Ofengeim, Dimitry; Zhu, Hong; Wu, Kejia; Cai, Yu; Yates, John R; Zhu, Zhengjiang; Yuan, Junying; Vakifahmetoglu-Norberg, Helin

    2015-08-31

    Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

  1. Mast cell chymase induces smooth muscle cell apoptosis by disrupting NF-{kappa}B-mediated survival signaling

    SciTech Connect

    Leskinen, Markus J.; Heikkilae, Hanna M.; Speer, Mei Y.; Hakala, Jukka K.; Laine, Mika; Kovanen, Petri T.; Lindstedt, Ken A. . E-mail: ken.lindstedt@wri.fi

    2006-05-01

    Chymase released from activated mast cells induces apoptosis of vascular smooth muscle cells (SMCs) in vitro by degrading the pericellular matrix component fibronectin, so causing disruption of focal adhesion complexes and Akt dephosphorylation, which are necessary for cell adhesion and survival. However, the molecular mechanisms of chymase-mediated apoptosis downstream of Akt have remained elusive. Here, we show by means of RT-PCR, Western blotting, EMSA, immunocytochemistry and confocal microscopy, that chymase induces SMC apoptosis by disrupting NF-{kappa}B-mediated survival signaling. Following chymase treatment, the translocation of active NF-{kappa}B/p65 to the nucleus was partly abolished and the amount of nuclear p65 was reduced. Pretreatment of SMCs with chymase also inhibited LPS- and IL-1{beta}-induced nuclear translocation of p65. The chymase-induced degradation of p65 was mediated by active caspases. Loss of NF-{kappa}B-mediated transactivation resulted in downregulation of bcl-2 mRNA and protein expression, leading to mitochondrial swelling and release of cytochrome c. The apoptotic process involved activation of both caspase 9 and caspase 8. The results reveal that, by disrupting the NF-{kappa}B-mediated survival-signaling pathway, activated chymase-secreting mast cells can mediate apoptosis of cultured arterial SMCs. Since activated mast cells colocalize with apoptotic SMCs in vulnerable areas of human atherosclerotic plaques, they may participate in the weakening and rupture of atherosclerotic plaques.

  2. Eph/ephrin-B-mediated cell-to-cell interactions govern MTS20(+) thymic epithelial cell development.

    PubMed

    Montero-Herradón, Sara; García-Ceca, Javier; Sánchez Del Collado, Beatriz; Alfaro, David; Zapata, Agustín G

    2016-08-01

    Thymus development is a complex process in which cell-to-cell interactions between thymocytes and thymic epithelial cells (TECs) are essential to allow a proper maturation of both thymic cell components. Although signals that control thymocyte development are well known, mechanisms governing TEC maturation are poorly understood, especially those that regulate the maturation of immature TEC populations during early fetal thymus development. In this study, we show that EphB2-deficient, EphB2LacZ and EphB3-deficient fetal thymuses present a lower number of cells and delayed maturation of DN cell subsets compared to WT values. Moreover, deficits in the production of chemokines, known to be involved in the lymphoid seeding into the thymus, contribute in decreased proportions of intrathymic T cell progenitors (PIRA/B(+)) in the mutant thymuses from early stages of development. These features correlate with increased proportions of MTS20(+) cells but fewer MTS20(-) cells from E13.5 onward in the deficient thymuses, suggesting a delayed development of the first epithelial cells. In addition, in vitro the lack of thymocytes or the blockade of Eph/ephrin-B-mediated cell-to-cell interactions between either thymocytes-TECs or TECs-TECs in E13.5 fetal thymic lobes coursed with increased proportions of MTS20(+) TECs. This confirms, for the first time, that the presence of CD45(+) cells, corresponding at these stages to DN1 and DN2 cells, and Eph/ephrin-B-mediated heterotypic or homotypic cell interactions between thymocytes and TECs, or between TECs and themselves, contribute to the early maturation of MTS20(+) TECs.

  3. Continuing to illuminate the mechanisms underlying UV-mediated melanomagenesis

    PubMed Central

    Dellinger, Ryan W.; Liu-Smith, Feng; Meyskens, Frank L.

    2014-01-01

    The incidence of melanoma is one of the fastest growing of all tumor types in the United States and the number of cases worldwide has doubled in the past 30 years. Melanoma, which arises from melanocytes, is an extremely aggressive tumor that invades the vascular and lymphatic systems to establish tumors elsewhere in the body. Melanoma is a particularly resilient cancer and systemic therapy approaches have achieved minimal success against metastatic melanoma resulting in only a few FDA-approved treatments with limited benefit. Leading treatments offer minimal efficacy with response rates generally under 15% in the long term with no clear effect on melanoma-related mortality. Even the recent success of the specific BRAF mutant inhibitor vemurafenib has been tempered somewhat since acquired resistance is rapidly observed. Thus, understanding the mechanism(s) of melanoma carcinogenesis is paramount to combating this deadly disease. Not only for the treatment of melanoma but, ultimately, for prevention. In this report, we will summarize our work to date regarding the characterization of ultraviolet radiation (UVR)-mediated melanomagenesis and highlight several promising avenues of ongoing research. PMID:25022944

  4. Minireview: mechanisms of growth hormone-mediated gene regulation.

    PubMed

    Chia, Dennis J

    2014-07-01

    GH exerts a diverse array of physiological actions that include prominent roles in growth and metabolism, with a major contribution via stimulating IGF-1 synthesis. GH achieves its effects by influencing gene expression profiles, and Igf1 is a key transcriptional target of GH signaling in liver and other tissues. This review examines the mechanisms of GH-mediated gene regulation that begin with signal transduction pathways activated downstream of the GH receptor and continue with chromatin events at target genes and additionally encompasses the topics of negative regulation and cross talk with other cellular inputs. The transcription factor, signal transducer and activator of transcription 5b, is regarded as the major signaling pathway by which GH achieves its physiological effects, including in stimulating Igf1 gene transcription in liver. Recent studies exploring the mechanisms of how activated signal transducer and activator of transcription 5b accomplishes this are highlighted, which begin to characterize epigenetic features at regulatory domains of the Igf1 locus. Further research in this field offers promise to better understand the GH-IGF-1 axis in normal physiology and disease and to identify strategies to manipulate the axis to improve human health.

  5. The canonical Wg signaling modulates Bsk-mediated cell death in Drosophila

    PubMed Central

    Zhang, S; Chen, C; Wu, C; Yang, Y; Li, W; Xue, L

    2015-01-01

    Cell death is an essential regulatory mechanism for removing unneeded cells in animal development and tissue homeostasis. The c-Jun N-terminal kinase (JNK) pathway has pivotal roles in the regulation of cell death in response to various intrinsic and extrinsic stress signals. The canonical Wingless (Wg) signaling has been implicated in cell proliferation and cell fate decisions, whereas its role in cell death remains largely elusive. Here, we report that activated Bsk (the Drosophila JNK homolog) induced cell death is mediated by the canonical Wg signaling. First, loss of Wg signaling abrogates Bsk-mediated caspase-independent cell death. Second, activation of Wg signaling promotes cell death in a caspase-independent manner. Third, activation of Bsk signaling results in upregulated transcription of wingless (wg) gene. Finally, Wg pathway participates in the physiological function of Bsk signaling in development. These findings not only reveal a previously undiscovered role of Wg signaling in Bsk-mediated cell death, but also provide a novel mechanism for the interplay between the two important signaling pathways in development. PMID:25855961

  6. Regulatory mechanisms in cell-mediated immune responses. Role of I-J and I-C determinants in the activation of H-2I and H-2K/D alloantigen- specific suppressor T cells

    PubMed Central

    1983-01-01

    The role of individual H-2I subregion determinants in the activation of H-2I alloantigen-primed mixed leukocyte response suppressor T cells (MLR Ts), as well as their possible expression on stimulator cells required to trigger primed H-2K- or D-specific MLR Ts, was addressed in these studies. Both genetic and serologic studies demonstrated that MLR Ts potentially primed to alloantigens encoded by the entire H-2I region were triggered to MLR Ts factor production only by stimulator cells bearing the priming I-J and/or I-C, but not I-A or I-E alloantigens. The relevant I-J and I-C determinants were demonstrated on a single antigen-presenting cell population that is used in common by independent I-J-specific and I-C-specific MLR Ts. Unexpectedly, the stimulator cell population necessary to trigger MLR Ts primed to class I H-2K or D alloantigens expressed not only the priming class I determinant, but in addition, I-C alloantigens syngeneic with the MLR Ts haplotype. Stimulator populations bearing the appropriate H-2K or D alloantigen but serologically depleted of I-C+ cells or genetically constructed to display MLR Ts-disparate I-C determinants were ineffective stimulators of class I antigen-primed MLR Ts. Thus these data suggest that as allogeneic determinants, I-J- and I-C-encoded molecules are together the major triggering elements for MLR Ts primed to disparate H-2I region determinants. In addition, self-I-C molecule recognition appears to constitute an important feature of the triggering, and by implication, priming process of H-2 class I antigen- specific Ts cells. PMID:6193232

  7. Cell-mediated fiber recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

    PubMed Central

    Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

    2015-01-01

    To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices (ECM), we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibers, and bulk contraction of the material. While increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation, increasing fiber stiffness instead suppressed spreading and proliferation depending on network architecture. Lower fiber stiffness permitted active cellular forces to recruit nearby fibers, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signaling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fiber recruitment as a novel mechanism by which cells probe and respond to mechanics in fibrillar matrices. PMID:26461445

  8. A simple retinal mechanism contributes to perceptual interactions between rod- and cone-mediated responses in primates.

    PubMed

    Grimes, William N; Graves, Logan R; Summers, Mathew T; Rieke, Fred

    2015-01-01

    Visual perception across a broad range of light levels is shaped by interactions between rod- and cone-mediated signals. Because responses of retinal ganglion cells, the output cells of the retina, depend on signals from both rod and cone photoreceptors, interactions occurring in retinal circuits provide an opportunity to link the mechanistic operation of parallel pathways and perception. Here we show that rod- and cone-mediated responses interact nonlinearly to control the responses of primate retinal ganglion cells; these nonlinear interactions, surprisingly, were asymmetric, with rod responses strongly suppressing subsequent cone responses but not vice-versa. Human psychophysical experiments revealed a similar perceptual asymmetry. Nonlinear interactions in the retinal output cells were well-predicted by linear summation of kinetically-distinct rod- and cone-mediated signals followed by a synaptic nonlinearity. These experiments thus reveal how a simple mechanism controlling interactions between parallel pathways shapes circuit output and perception. PMID:26098124

  9. Internalisation of the bleomycin molecules responsible for bleomycin toxicity: a receptor-mediated endocytosis mechanism.

    PubMed

    Pron, G; Mahrour, N; Orlowski, S; Tounekti, O; Poddevin, B; Belehradek, J; Mir, L M

    1999-01-01

    Bleomycin (BLM) does not diffuse through the plasma membrane but nevertheless displays cytotoxic activity due to DNA break generation. The aim of the study was to describe the mechanism of BLM internalisation. We previously provided evidence for the existence of BLM-binding sites at the surface of DC-3F Chinese hamster fibroblasts, as well as of their involvement in BLM cytotoxicity on DC-3F cells and related BLM-resistant sublines. Here we report that A253 human cells and their BLM-resistant subline C-10E also possessed a membrane protein of ca. 250 kDa specifically binding BLM. Part of this C-10E cell resistance could be explained by a decrease in the number of BLM-binding sites exposed at the cell surface with respect to A253 cells. The comparison between A253 and DC-3F cells exposing a similar number of BLM-binding sites revealed that the faster the fluid phase endocytosis, the greater the cell sensitivity to BLM. Moreover, the experimental modification of endocytotic vesicle size showed that BLM cytotoxicity was directly correlated with the flux of plasma membrane area engulfed during endocytosis rather than with the fluid phase volume incorporated. Thus, BLM would be internalised by a receptor-mediated endocytosis mechanism which would first require BLM binding to its membrane receptor and then the transfer of the complex into intracellular endocytotic vesicles, followed by BLM entry into the cytosol, probably from a nonacidic compartment.

  10. Mechanisms underlying heterologous skin scaffold-mediated tissue remodeling

    PubMed Central

    Mimura, Kallyne K. O.; Moraes, Andréia R.; Miranda, Aline C.; Greco, Rebecca; Ansari, Tahera; Sibbons, Paul; Greco, Karin V.; Oliani, Sonia M.

    2016-01-01

    Biocompatibility of two newly developed porcine skin scaffolds was assessed after 3, 14, 21 and 90 days of implantation in rats. Both scaffolds showed absence of cells, preservation of ECM and mechanical properties comparable to non-decellularised skin before implantation. Host cell infiltration was much prominent on both scaffolds when compared to Permacol (surgical control). At day 3, the grafts were surrounded by polymorphonuclear cells, which were replaced by a notable number of IL-6-positive cells at day 14. Simultaneously, the number of pro-inflammatory M1-macrophage was enhanced. Interestingly, a predominant pro-remodeling M2 response, with newly formed vessels, myofibroblasts activation and a shift on the type of collagen expression was sequentially delayed (around 21 days). The gene expression of some trophic factors involved in tissue remodeling was congruent with the cellular events. Our findings suggested that the responsiveness of macrophages after non-crosslinked skin scaffolds implantation seemed to intimately affect various cell responses and molecular events; and this range of mutually reinforcing actions was predictive of a positive tissue remodeling that was essential for the long-standing success of the implants. Furthermore, our study indicates that non-crosslinked biologic scaffold implantation is biocompatible to the host tissue and somehow underlying molecular events involved in tissue repair. PMID:27725772

  11. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    SciTech Connect

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A. . E-mail: Wayne_Tompkins@ncsu.edu

    2004-12-20

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.

  12. Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms

    PubMed Central

    Shi, Xiao-Jing; Yu, Bin; Wang, Jun-Wei; Qi, Ping-Ping; Tang, Kai; Huang, Xin; Liu, Hong-Min

    2016-01-01

    Cancer cells always have increased ROS levels, thus making them more vulnerable to persistent endogenous oxidative stress. The biochemical difference between cancer and normal cells could be exploited to achieve selective cancer cell killing by exogenous ROS-producing agents. Herein we described a structurally novel steroidal spirooxindole by241 and its anticancer efficacy. By241 exhibited potent inhibition against human cancer cells and less toxic to normal cells. By241 concentration-dependently induced apoptosis of MGC-803 and EC9706 cells, accompanied with the mitochondrial dysfunction and increased ROS levels. NAC can completely restore the decreased cell viability of MGC-803 cells caused by by241, suggesting ROS-mediated mechanisms. The expression levels of proteins involved in the mitochondrion-related pathways were detected, showing increased expression of proapoptotic proteins and decreased expression of anti-apoptotic proteins, and activation of caspases-9/-3, but without activating caspase-8 expression. Pretreatment with Z-VAD-FMK partially rescued by241-induced apoptosis of MGC-803 cells. Additionally, by241 inhibited mTOR, activated p53 and its downstream proteins, cleaved MDM2 and PI3K/AKT as well as NF-κB signaling pathway. In vivo experiments showed that by241 did not have significant acute oral toxicity and exerted good anticancer efficacy against MGC-803 bearing mice models. Therefore, by241 may serve as a lead for further development for cancer therapy. PMID:27527552

  13. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

    PubMed

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi; Antonescu, Costin N

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  14. Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms

    PubMed Central

    Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi

    2016-01-01

    Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted

  15. UCP2- and non-UCP2-mediated electric current in eukaryotic cells exhibits different properties.

    PubMed

    Wang, Ruihua; MoYung, K C; Zhang, M H; Poon, Karen

    2015-12-01

    Using live eukaryotic cells, including cancer cells, MCF-7 and HCT-116, normal hepatocytes and red blood cells in anode and potassium ferricyanide in cathode of MFC could generate bio-based electric current. Electrons and protons generated from the metabolic reaction in both cytosol and mitochondria contributing to the leaking would mediate the generation of electric current. Both resveratrol (RVT) and 2,4-dinitrophenol (DNP) used to induce proton leak in mitochondria were found to promote electric current production in all cells except red blood cells without mitochondria. Proton leak might be important for electric current production by bringing the charge balance in cells to enhance the further electron leak. The induced electric current by RVT can be blocked by Genipin, an inhibitor of UCP2-mediated proton leak, while that induced by DNP cannot. RVT could reduce reactive oxygen species (ROS) level in cells better than that of DNP. In addition, RVT increased mitochondrial membrane potential (MMP), while DNP decreased it. Results highly suggested the existence of at least two types of electric current that showed different properties. They included UCP2-mediated and non-UCP2-mediated electric current. UCP2-mediated electric current exhibited higher reactive oxygen species (ROS) reduction effect per unit electric current production than that of non-UCP2-mediated electric current. Higher UCP2-mediated electric current observed in cancer cells might contribute to the mechanism of drug resistence. Correlation could not be established between electric current production with either ROS and MMP without distinguishing the types of electric current.

  16. Mechanical communication in cardiac cell synchronized beating

    NASA Astrophysics Data System (ADS)

    Nitsan, Ido; Drori, Stavit; Lewis, Yair E.; Cohen, Shlomi; Tzlil, Shelly

    2016-05-01

    Cell-cell communication, which enables cells to coordinate their activity and is essential for growth, development and function, is usually ascribed a chemical or electrical origin. However, cells can exert forces and respond to environment elasticity and to mechanical deformations created by their neighbours. The extent to which this mechanosensing ability facilitates intercellular communication remains unclear. Here we demonstrate mechanical communication between cells directly for the first time, providing evidence for a long-range interaction that induces long-lasting alterations in interacting cells. We show that an isolated cardiac cell can be trained to beat at a given frequency by mechanically stimulating the underlying substrate. Deformations are induced using an oscillatory mechanical probe that mimics the deformations generated by a beating neighbouring cardiac cell. Unlike electrical field stimulation, the probe-induced beating rate is maintained by the cell for an hour after the stimulation stops, implying that long-term modifications occur within the cell. These long-term alterations provide a mechanism for cells that communicate mechanically to be less variable in their electromechanical delay. Mechanical coupling between cells therefore ensures that the final outcome of action potential pacing is synchronized beating. We further show that the contractile machinery is essential for mechanical communication.

  17. Mechanics and morphogenesis of fission yeast cells.

    PubMed

    Davì, Valeria; Minc, Nicolas

    2015-12-01

    The integration of biochemical and biomechanical elements is at the heart of morphogenesis. While animal cells are relatively soft objects which shape and mechanics is mostly regulated by cytoskeletal networks, walled cells including those of plants, fungi and bacteria are encased in a rigid cell wall which resist high internal turgor pressure. How these particular mechanical properties may influence basic cellular processes, such as growth, shape and division remains poorly understood. Recent work using the model fungal cell fission yeast, Schizosaccharomyces pombe, highlights important contribution of cell mechanics to various morphogenesis processes. We envision this genetically tractable system to serve as a novel standard for the mechanobiology of walled cell.

  18. Hypoxia-mediated autophagic flux inhibits silver nanoparticle-triggered apoptosis in human lung cancer cells

    PubMed Central

    Jeong, Jae-Kyo; Gurunathan, Sangiliyandi; Kang, Min-Hee; Han, Jae Woong; Das, Joydeep; Choi, Yun-Jung; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Song, Hyuk; Kim, Jin-Hoi

    2016-01-01

    Solid tumors are frequently associated with resistance to chemotherapy because the fraction of hypoxic tumor cells is substantial. To understand the underlying mechanism of hypoxia on silver nanoparticle (AgNPs)-induced apoptosis, the expression of hypoxia-inducible factor (HIF)-1α, a hallmark of hypoxia, was measured in the presence and absence of AgNPs. The results showed that HIF-1α expression was upregulated after AgNPs treatment under both hypoxic and normoxic conditions. Cell viability assays showed that AgNPs promoted cell death in cancer cells but not in non-cancer cells, as cancer cells are slightly more acidic than normal cells. However, reactive oxygen species generation induced by AgNPs in lung cancer cells caused high susceptibility to oxidative stress, whereas pre-exposure to hypoxia blocked AgNPs-induced oxidative stress. Notably, HIF-1α inhibited AgNPs-induced mitochondria-mediated apoptosis by regulating autophagic flux through the regulation of ATG5, LC3-II, and p62. Further, cell viability after treatment of cancer cells with AgNPs under hypoxic conditions was lower in HIF-1α siRNA-transfected cells than in control siRNA-transfected cells, indicating that HIF-1α knockdown enhances hypoxia induced decrease in cell viability. Our results suggest that hypoxia-mediated autophagy may be a mechanism for the resistance of AgNPs-induced apoptosis and that strategies targeting HIF-1α may be used for cancer therapy. PMID:26867977

  19. Immunoglobulin G-mediated regulation of the murine immune response to transfused red blood cells occurs in the absence of active immune suppression: implications for the mechanism of action of anti-D in the prevention of haemolytic disease of the fetus and newborn?

    PubMed Central

    Brinc, Davor; Le-Tien, Hoang; Crow, Andrew R; Siragam, Vinayakumar; Freedman, John; Lazarus, Alan H

    2008-01-01

    Anti-D has been widely and effectively used in Rhesus blood group D negative mothers for the prevention of haemolytic disease of the fetus and newborn; its mechanism of action however, often referred to as antibody-mediated immune suppression (AMIS), remains largely unresolved. We investigated, in a murine model, whether active immune suppression or clonal deletion mediated by anti-red blood cell (RBC) immunoglobulin G (IgG) could explain the phenomenon of AMIS. Transfusion of IgG-opsonized foreign RBCs (i.e. AMIS) strongly attenuated antibody responses compared to transfusion of untreated foreign RBCs. When the AMIS-mice were subsequently transfused with untreated RBCs, no immune suppression was observed at 5 and 35 days after AMIS induction; in fact, the mice responded to retransfusion with untreated RBCs in a manner that was characteristic of a secondary immune response. When IgG-opsonized RBCs were transfused concurrently with untreated RBCs, a dose-dependent reduction of the antibody response was observed. This work suggests that the attenuation of the antibody responsiveness by anti-RBC IgG is not associated with active immune suppression or clonal deletion at either the T-cell or B-cell level; rather, the effect appears more characteristic of B-cell unresponsiveness to IgG-opsonized RBCs. These results may have implications for the understanding of the mechanism of action of anti-D in haemolytic disease of the fetus and newborn. PMID:18266717

  20. Mechanical properties of cells and ageing.

    PubMed

    Starodubtseva, Maria N

    2011-01-01

    Mechanical properties are fundamental properties of the cells and tissues of living organisms. The mechanical properties of a single cell as a biocomposite are determined by the interdependent combination of cellular components mechanical properties. Quantitative estimate of the cell mechanical properties depends on a cell state, method of measurement, and used theoretical model. Predominant tendency for the majority of cells with ageing is an increase of cell stiffness and a decrease of cell ability to undergo reversible large deformations. The mechanical signal transduction in old cells becomes less effective than that in young cells, and with ageing, the cells lose the ability of the rapid functional rearrangements of cellular skeleton. The article reviews the theoretical and experimental facts touching the age-related changes of the mechanical properties of cellular components and cells in the certain systems of an organism (the blood, the vascular system, the musculoskeletal system, the lens, and the epithelium). In fact, the cell mechanical parameters (including elastic modulii) can be useful as specific markers of cell ageing.

  1. Rejection Triggers Liver Transplant Tolerance: Involvements of Mesenchyme-Mediated Immune Control Mechanisms

    PubMed Central

    Morita, Miwa; Joyce, Daniel; Miller, Charles; Fung, John J.; Lu, Lina; Qian, Shiguang

    2015-01-01

    Liver tolerance was initially recognized by the spontaneous acceptance of liver allograft in many species. The underlying mechanisms are not completely understood. We have been inspired by an unexpected phenomenon that the liver transplant tolerance absolutely requires interferon (IFN)-γ, a rejection-associated inflammatory cytokine. In this study, we investigate the rejection of liver allografts deficient in IFN-γ receptor and reveal that the liver graft is equipped with machineries capable of counterattacking the host immune response through a mesenchyme-mediated immune control (MMIC) mechanism. MMIC is triggered by T effectors (Tef) cell-derived IFN-γ to drive the expression of B7-H1 on graft mesenchymal cells leading to Tef cell apoptosis. We describe the negative feedback loop between graft mesenchymal and Tef cells that ultimately results in liver transplant tolerance. Comparable elevations of T regulatory cells and myeloid-derived suppressor cells are seen in both rejection and tolerance groups, and are not dependent on IFN-γ stimulation, suggesting a critical role of Tef cell elimination in tolerance induction. We identify potent MMIC activity in hepatic stellate cells and liver sinusoidal endothelial cells. MMIC is unlikely exclusive to the liver, as spontaneous acceptance of kidney allografts has been reported, although less commonly, probably reflecting variance in MMIC activity. MMCI may represent an important homeostatic mechanism that supports peripheral tolerance, and could be a target for the prevention and treatment of transplant rejection. This study highlights that the graft is actively participant in the equipoise between tolerance and rejection and warrants more attention in the search for tolerance biomarkers. PMID:25998530

  2. L-Asparaginase II Produced by Salmonella Typhimurium Inhibits T Cell Responses and Mediates Virulence

    PubMed Central

    Kullas, Amy L.; McClelland, Michael; Yang, Hee-Jeong; Tam, Jason W.; Torres, AnnMarie; Porwollik, Steffen; Mena, Patricio; McPhee, Joseph B.; Bogomolnaya, Lydia; Andrews-Polymenis, Helene; van der Velden, Adrianus W.M.

    2013-01-01

    SUMMARY Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses. PMID:23245323

  3. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    PubMed

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  4. CD13 mediates phagocytosis in human monocytic cells.

    PubMed

    Licona-Limón, Ileana; Garay-Canales, Claudia A; Muñoz-Paleta, Ofelia; Ortega, Enrique

    2015-07-01

    CD13 is a membrane-bound ectopeptidase, highly expressed on monocytes, macrophages, and dendritic cells. CD13 is involved in diverse functions, including degradation of peptide mediators, cellular adhesion, migration, viral endocytosis, signaling, and positive modulation of phagocytosis mediated by FcγRs and other phagocytic receptors. In this work, we explored whether besides acting as an accessory receptor, CD13 by itself is a primary phagocytic receptor. We found that hCD13 mediates efficient phagocytosis of large particles (erythrocytes) modified so as to interact with the cell only through CD13 in human macrophages and THP-1 monocytic cells. The extent of this phagocytosis is comparable with the phagocytosis mediated through the canonical phagocytic receptor FcγRI. Furthermore, we demonstrated that hCD13 expression in the nonphagocytic cell line HEK293 is sufficient to enable these cells to internalize particles bound through hCD13. CD13-mediated phagocytosis is independent of other phagocytic receptors, as it occurs in the absence of FcγRs, CR3, and most phagocytic receptors. Phagocytosis through CD13 is independent of its enzymatic activity but is dependent on actin rearrangement and activation of PI3K and is partially dependent on Syk activation. Moreover, the cross-linking of CD13 with antibodies rapidly induced pSyk in human macrophages. Finally, we observed that antibody-mediated cross-linking of hCD13, expressed in the murine macrophage-like J774 cell line, induces production of ROS. These results demonstrate that CD13 is a fully competent phagocytic receptor capable of mediating internalization of large particles.

  5. Mechanisms of the ultrasound-mediated intracellular delivery of liposomes and dextrans.

    PubMed

    Afadzi, Mercy; Strand, Sabina P; Nilssen, Esben A; Måsøy, Svein-Erik; Johansen, Tonni F; Hansen, Rune; Angelsen, Bjørn A; de L Davies, Catharina

    2013-01-01

    The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

  6. Surface charge of gold nanoparticles mediates mechanism of toxicity

    NASA Astrophysics Data System (ADS)

    Schaeublin, Nicole M.; Braydich-Stolle, Laura K.; Schrand, Amanda M.; Miller, John M.; Hutchison, Jim; Schlager, John J.; Hussain, Saber M.

    2011-02-01

    Recently gold nanoparticles (Au NPs) have shown promising biological and military applications due to their unique electronic and optical properties. However, little is known about their biocompatibility in the event that they come into contact with a biological system. In the present study, we have investigated whether modulating the surface charge of 1.5 nm Au NPs induced changes in cellular morphology, mitochondrial function, mitochondrial membrane potential (MMP), intracellular calcium levels, DNA damage-related gene expression, and of p53 and caspase-3 expression levels after exposure in a human keratinocyte cell line (HaCaT). The evaluation of three different Au NPs (positively charged, neutral, and negatively charged) showed that cell morphology was disrupted by all three NPs and that they demonstrated a dose-dependent toxicity; the charged Au NPs displayed toxicity as low as 10 µg ml-1 and the neutral at 25 µg ml-1. Furthermore, there was significant mitochondrial stress (decreases in MMP and intracellular Ca2+ levels) following exposure to the charged Au NPs, but not the neutral Au NPs. In addition to the differences observed in the MMP and Ca2+ levels, up or down regulation of DNA damage related gene expression suggested a differential cell death mechanism based on whether or not the Au NPs were charged or neutral. Additionally, increased nuclear localization of p53 and caspase-3 expression was observed in cells exposed to the charged Au NPs, while the neutral Au NPs caused an increase in both nuclear and cytoplasmic p53 expression. In conclusion, these results indicate that surface charge is a major determinant of how Au NPs impact cellular processes, with the charged NPs inducing cell death through apoptosis and neutral NPs leading to necrosis.Recently gold nanoparticles (Au NPs) have shown promising biological and military applications due to their unique electronic and optical properties. However, little is known about their biocompatibility in the

  7. Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

    NASA Technical Reports Server (NTRS)

    Enebo, D. J.; Fattaey, H. K.; Moos, P. J.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition.

  8. Retinoid-mediated transcriptional regulaton of keratin genes in human epidermal and squamous cell carcinoma cells

    SciTech Connect

    Stellmach, V.; Leask, A.; Fuchs, E. )

    1991-06-01

    Vitamin A and other retinoids profoundly inhibit morphological and biochemical heatures of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, the authors retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. The studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5{prime} upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors {alpha} and {gamma}, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.

  9. Conceptual Conditioning: Mechanisms Mediating Conditioning Effects on Pain.

    PubMed

    Jepma, Marieke; Wager, Tor D

    2015-11-01

    Classical conditioning can profoundly modify subsequent pain responses, but the mechanisms that drive this effect are unresolved. In pain-conditioning studies, cues are typically conditioned to primary aversive reinforcers; hence, subsequent pain modulation could reflect learned precognitive associations (i.e., those involving neural plasticity independent of expectations and other forms of conceptual thought) or conceptual expectancies. We isolated conceptual contributions using a thermal pain-conditioning procedure in which different conditioned stimulus (CS) cues were repeatedly paired with symbolic representations of high and low noxious heat. In a subsequent test phase, identical noxious stimuli evoked larger skin conductance responses (SCRs) and pain ratings when preceded by CS cues associated with high temperature than by those associated with low temperature. These effects were mediated by participants' self-reported expectancies. CS cues associated with high temperature also evoked larger anticipatory SCRs than did CS cues associated with low temperature, but larger anticipatory SCRs predicted smaller subsequent heat-evoked SCRs. These results provide novel evidence that conditioned modulation of pain physiology can be acquired through purely conceptual processes, and that self-reported expectancies and physiological threat responses have opposing effects on pain.

  10. Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

    PubMed

    Ikeda, Tokunori; Hirata, Shinya; Takamatsu, Koutaro; Haruta, Miwa; Tsukamoto, Hirotake; Ito, Takaaki; Uchino, Makoto; Ando, Yukio; Nagafuchi, Seiho; Nishimura, Yasuharu; Senju, Satoru

    2014-01-01

    We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases. PMID:25522369

  11. Monolithic and mechanical multijunction space solar cells

    NASA Technical Reports Server (NTRS)

    Jain, Raj K.; Flood, Dennis J.

    1992-01-01

    High-efficiency, lightweight, radiation-resistant solar cells are essential to meet the large power requirements of future space missions. Single-junction cells are limited in efficiency. Higher cell efficiencies could be realized by developing multijunction, multibandgap solar cells. Monolithic and mechanically stacked tandem solar cells surpassing single-junction cell efficiencies have been fabricated. This article surveys the current status of monolithic and mechanically stacked multibandgap space solar cells, and outlines problems yet to be resolved. The monolithic and mechanically stacked cells each have their own problems related to size, processing, current and voltage matching, weight, and other factors. More information is needed on the effect of temperature and radiation on the cell performance. Proper reference cells and full-spectrum range simulators are also needed to measure efficiencies correctly. Cost issues are not addressed, since the two approaches are still in the developmental stage.

  12. Dye-mediated photosensitization of murine neuroblastoma cells

    SciTech Connect

    Sieber, F.; Sieber-Blum, M.

    1986-04-01

    The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

  13. Ascites Specific Inhibition of CD1d-Mediated Activation of NKT cells

    PubMed Central

    Webb, Tonya J.; Giuntoli, Robert L.; Rogers, Ophelia; Schneck, Jonathan; Oelke, Mathias

    2009-01-01

    Purpose Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate anti-tumor immunity. However, it has been shown that tumor associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor associated and other ascites on CD1d-mediated antigen presentation to NKT cells. Experimental Design To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classical CD8+ T cells. Results Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the cells’ ability to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-GalCer loaded CD1d-Ig-based artificial Antigen Presenting Cells (aAPC) to activate NKT cells. In addition, our data demonstrate that treatment with ascites does not inhibit HLA-A2 mediated activation of classical CD8+ T cells. Conclusions Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system. PMID:19047090

  14. The double-edge role of B cells in mediating antitumor T-cell immunity: Pharmacological strategies for cancer immunotherapy.

    PubMed

    Wang, Jing-Zhang; Zhang, Yu-Hua; Guo, Xin-Hua; Zhang, Hong-Yan; Zhang, Yuan

    2016-07-01

    Emerging evidence reveals the controversial role of B cells in antitumor immunity, but the underlying mechanisms have to be explored. Three latest articles published in the issue 521 of Nature in 2015 reconfirmed the puzzling topic and put forward some explanations of how B cells regulate antitumor T-cell responses both positively and negatively. This paper attempts to demonstrate that different B-cell subpopulations have distinct immunological properties and that they are involved in either antitumor responses or immunosuppression. Recent studies supporting the positive and negative roles of B cells in tumor development were summarized comprehensively. Several specific B-cell subpopulations, such as IgG(+), IgA(+), IL-10(+), and regulatory B cells, were described in detail. The mechanisms underlying the controversial B-cell effects were mainly attributed to different B-cell subpopulations, different B-cell-derived cytokines, direct B cell-T cell interaction, different cancer categories, and different malignant stages, and the immunological interaction between B cells and T cells is mediated by dendritic cells. Promising B-cell-based antitumor strategies were proposed and novel B-cell regulators were summarized to present interesting therapeutic targets. Future investigations are needed to make sure that B-cell-based pharmacological strategies benefit cancer immunotherapy substantially.

  15. Regulation of cell proliferation and apoptosis by bioactive lipid mediators.

    PubMed

    Clària, Joan

    2006-11-01

    Bioactive lipid mediators are increasingly being recognized as important endogenous regulators of cell activation, signaling, apoptosis and proliferation. Most of these lipid mediators are originated from cleavage of constituents of cellular membranes under the activity of phospholipases and sphingomyelinases. One of the major cascades of bioactive lipid mediator production involves the release of arachidonic acid from membrane phospholipids followed by the formation of eicosanoids (i.e. prostaglandins, leukotrienes and lipoxins). These biologically active metabolites of arachidonic acid are emerging as key regulators of cell proliferation and neo-angiogenesis and agents that specifically target these lipid mediators are being investigated as potential anticancer drugs. On the other hand, the lysophospholipid family, which includes members of the sphingomyelin-ceramide-sphingosine-1-phosphate and lysophosphatidic acid subfamilies, has evolved as an important group of lipid signaling molecules implicated in cellular differentiation, cell growth and apoptosis. This article reviews the most recent patents in this field of research, covering the following strategies based on the modulation of bioactive lipid mediators: (1) prostaglandin H synthase-2 inhibitors, (2) lipoxin analogs and aspirin-triggered lipid mediators, and (3) lysophosphatidic acid and other lysophospholipids. PMID:18221047

  16. Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

    SciTech Connect

    Clarkin, Claire E. Garonna, Elena; Pitsillides, Andrew A.; Wheeler-Jones, Caroline P.D.

    2008-10-15

    In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to 'remotely located' ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE{sub 2} on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE{sub 2} increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF{sub 1{alpha}} release and EC proliferation. In contrast, PGE{sub 2} attenuated VEGF{sub 165}-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE{sub 2} restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH{sub 2} (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.

  17. Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae

    PubMed Central

    Sinha, Bidisha; Köster, Darius; Ruez, Richard; Gonnord, Pauline; Bastiani, Michele; Abankwa, Daniel; Stan, Radu. V.; Butler-Browne, Gillian; Vedie, Benoit; Johannes, Ludger; Morone, Nobuhiro; Parton, Robert G.; Raposo, Graça; Sens, Pierre; Lamaze, Christophe; Nassoy, Pierre

    2011-01-01

    SUMMARY The precise role of caveolae, the characteristic plasma membrane invaginations present in many cells, still remains debated. The high density of caveolae in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by cell osmotic swelling or by uniaxial stretching results in the immediate disappearance of caveolae, which is associated with a reduced caveolin/Cavin1 interaction, and an increase of free caveolins at the plasma membrane. Tether pulling force measurements in live cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin and ATP-independent cell response which buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that allows cells to quickly accommodate sudden and acute mechanical stresses. PMID:21295700

  18. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs. PMID:23965842

  19. Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

    PubMed

    Carestia, A; Rivadeneyra, L; Romaniuk, M A; Fondevila, C; Negrotto, S; Schattner, M

    2013-11-01

    Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.

  20. p38alpha and p38gamma mediate oncogenic ras-induced senescence through differential mechanisms.

    PubMed

    Kwong, Jinny; Hong, Lixin; Liao, Rong; Deng, Qingdong; Han, Jiahuai; Sun, Peiqing

    2009-04-24

    Oncogene-induced senescence is a tumor-suppressive defense mechanism triggered upon activation of certain oncogenes in normal cells. Recently, the senescence response to oncogene activation has been shown to act as a bona fide barrier to cancer development in vivo. Multiple previous studies have implicated the importance of the p38 MAPK pathway in oncogene-induced senescence. However, the contribution of each of the four p38 isoforms (encoded by different genes) to senescence induction is unclear. In the current study, we demonstrated that p38alpha and p38gamma, but not p38beta, play an essential role in oncogenic ras-induced senescence. Both p38alpha and p38gamma are expressed in primary human fibroblasts and are activated upon transduction of oncogenic ras. Small hairpin RNA-mediated silencing of p38alpha or p38gamma expression abrogated ras-induced senescence, whereas constitutive activation of p38alpha and p38gamma caused premature senescence. Furthermore, upon activation by oncogenic ras, p38gamma stimulated the transcriptional activity of p53 by phosphorylating p53 at Ser(33), suggesting that the ability of p38gamma to mediate senescence is at least partly achieved through p53. However, p38alpha contributed to ras-inducted senescence via a p53-indepdendent mechanism in cells by mediating ras-induced expression of p16(INK4A), another key senescence effector. These findings have identified p38alpha and p38gamma as essential components of the signaling pathway that regulates the tumor-suppressing senescence response, providing insights into the molecular mechanisms underlying the differential involvement of the p38 isoforms in senescence induction.

  1. Context-dependent regulation of Musashi-mediated mRNA translation and cell cycle regulation.

    PubMed

    MacNicol, Melanie C; Cragle, Chad E; MacNicol, Angus M

    2011-01-01

    Musashi-mediated mRNA translational control has been implicated in the promotion of physiological and pathological stem cell proliferation. During self-renewal of mammalian stem cells, Musashi has been proposed to act to repress the translation of mRNAs encoding inhibitors of cell cycle progression. By contrast, in maturing Xenopus oocytes Musashi activates translation of target mRNAs that encode proteins promoting cell cycle progression. The mechanisms directing Musashi to differentially control mRNA translation in mammalian stem cells and Xenopus oocytes is unknown. In this study, we demonstrate that the mechanisms defining Musashi function lie within the cellular context. Specifically, we show that murine Musashi acts as an activator of translation in maturing Xenopus oocytes while Xenopus Musashi functions as a repressor of target mRNA translation in mammalian cells. We further demonstrate that within the context of a primary mammalian neural stem/progenitor cell, Musashi can be converted from a repressor of mRNA translation to an activator of translation in response to extracellular stimuli. We present current models of Musashi-mediated mRNA translational control and discuss possible mechanisms for regulating Musashi function. An understanding of these mechanisms presents exciting possibilities for development of therapeutic targets to control physiological and pathological stem cell proliferation.

  2. Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation.

    PubMed

    ElKeeb, A M; Collier, M E W; Maraveyas, A; Ettelaie, C

    2015-08-01

    We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA-mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53. PMID:25903973

  3. Mechanisms of a novel anticancer therapeutic strategy involving atmospheric pressure plasma-mediated apoptosis and DNA strand break formation.

    PubMed

    Chung, Woo-Hyun

    2016-01-01

    Atmospheric pressure plasma has been developed for a variety of biomedical applications due to its chemically reactive components. Recently, the plasma has emerged as a promising novel cancer therapy based on its ability to selectively ablate cancer cells while leaving normal cells essentially unaffected. The therapeutic effect of plasma is attributed to intracellular generation of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondria-mediated apoptosis and to activation of the DNA damage checkpoint signaling pathway via severe DNA strand break formation. However, the biochemical mechanisms responsible for appropriate activation of these physiological events and which pathway is more crucial for plasma-mediated cytotoxicity have not been clarified. Understanding the molecular link between ROS/RNS-mediated apoptosis and DNA damage-involved chromosome instability is critical for the development of more efficacious therapeutic strategies for selective killing of diverse cancer cells.

  4. Aquaporin-1 Translocation and Degradation Mediates the Water Transportation Mechanism of Acetazolamide

    PubMed Central

    Gao, Junwei; Han, Jing; Pan, Xueyang; Pan, Yan; Tie, Lu; Li, Xuejun

    2012-01-01

    Background Diuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA. Methodology/Principal Findings For in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1−/−) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1−/− mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1. Conclusions/Significance Our results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering

  5. Key Physical Mechanisms in Nanostructured Solar Cells

    SciTech Connect

    Dr Stephan Bremner

    2010-07-21

    The objective of the project was to study both theoretically and experimentally the excitation, recombination and transport properties required for nanostructured solar cells to deliver energy conversion efficiencies well in excess of conventional limits. These objectives were met by concentrating on three key areas, namely, investigation of physical mechanisms present in nanostructured solar cells, characterization of loss mechanisms in nanostructured solar cells and determining the properties required of nanostructured solar cells in order to achieve high efficiency and the design implications.

  6. Dendritic Cells: Cellular Mediators for Immunological Tolerance

    PubMed Central

    Chung, Chun Yuen J.; Ysebaert, Dirk; Berneman, Zwi N.

    2013-01-01

    In general, immunological tolerance is acquired upon treatment with non-specific immunosuppressive drugs. This indiscriminate immunosuppression of the patient often causes serious side-effects, such as opportunistic infectious diseases. Therefore, the need for antigen-specific modulation of pathogenic immune responses is of crucial importance in the treatment of inflammatory diseases. In this perspective, dendritic cells (DCs) can have an important immune-regulatory function, besides their notorious antigen-presenting capacity. DCs appear to be essential for both central and peripheral tolerance. In the thymus, DCs are involved in clonal deletion of autoreactive immature T cells by presenting self-antigens. Additionally, tolerance is achieved by their interactions with T cells in the periphery and subsequent induction of T cell anergy, T cell deletion, and induction of regulatory T cells (Treg). Various studies have described, modulation of DC characteristics with the purpose to induce antigen-specific tolerance in autoimmune diseases, graft-versus-host-disease (GVHD), and transplantations. Promising results in animal models have prompted researchers to initiate first-in-men clinical trials. The purpose of current review is to provide an overview of the role of DCs in the immunopathogenesis of autoimmunity, as well as recent concepts of dendritic cell-based therapeutic opportunities in autoimmune diseases. PMID:23762100

  7. Ion mediated targeting of cells with nanoparticles

    NASA Astrophysics Data System (ADS)

    Maheshwari, Vivek; Fu, Jinlong

    2010-03-01

    In eukaryotic cells, Ca^2+ ions are necessary for intracellular signaling, in activity of mitochondria and a variety of other cellular process that have been linked to cell apoptosis, proteins synthesis and cell-cycle regulation. Here we show that Ca^2+ ions, serving as the bio-compatible interface can be used to target Saccharomyces cerevisiae (SaC, baker's yeast), a model eukaryotic cell, with Au nanoparticles (10 nm). The Ca^2+ ions bind to the carboxylic acid groups in the citrate functionalized Au nanoparticles. This transforms the nanoparticles into micron long 1-D branched chain assemblies due to inter-particle dipole-dipole interaction and inter-particle bonding due to the divalent nature of the Ca^2+ ion. A similar transformation is observed with the use of divalent ions Mg^2+, Cd^2+ and Fe^2+. The 1-D assembly aids the interfacing of ion-nanoparticles on the cell by providing multiple contact points. Further monovalent ions such as Na^+ are also effective for the targeting of the cell with nanoparticles. However Na-Au nanoparticles are limited in their deposition as they exist in solution as single particles. The cells remain alive after the deposition process and their vitality is unaffected by the interfacing with ion-nanoparticles.

  8. Obstructive renal injury: from fluid mechanics to molecular cell biology.

    PubMed

    Ucero, Alvaro C; Gonçalves, Sara; Benito-Martin, Alberto; Santamaría, Beatriz; Ramos, Adrian M; Berzal, Sergio; Ruiz-Ortega, Marta; Egido, Jesus; Ortiz, Alberto

    2010-04-22

    Urinary tract obstruction is a frequent cause of renal impairment. The physiopathology of obstructive nephropathy has long been viewed as a mere mechanical problem. However, recent advances in cell and systems biology have disclosed a complex physiopathology involving a high number of molecular mediators of injury that lead to cellular processes of apoptotic cell death, cell injury leading to inflammation and resultant fibrosis. Functional studies in animal models of ureteral obstruction using a variety of techniques that include genetically modified animals have disclosed an important role for the renin-angiotensin system, transforming growth factor-β1 (TGF-β1) and other mediators of inflammation in this process. In addition, high throughput techniques such as proteomics and transcriptomics have identified potential biomarkers that may guide clinical decision-making.

  9. Laser-mediated perforation of plant cells

    NASA Astrophysics Data System (ADS)

    Wehner, Martin; Jacobs, Philipp; Esser, Dominik; Schinkel, Helga; Schillberg, Stefan

    2007-07-01

    The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

  10. Connecting the mechanisms of T-cell regulation: dendritic cells as the missing link.

    PubMed

    Cobbold, Stephen P; Adams, Elizabeth; Nolan, Kathleen F; Regateiro, Frederico S; Waldmann, Herman

    2010-07-01

    A variety of different molecular mechanisms have been proposed to explain the suppressive action of regulatory T cells, including the production of anti-inflammatory cytokines, negative costimulatory ligands, indoleamine 2,3-dioxygenase-mediated tryptophan catabolism, CD73-mediated adenosine generation, and downregulation of antigen-presenting cells. Until now it has been unclear how important each of these different mechanisms might be and how they are coordinated. In this review, we examine the hypothesis that it is the interaction between regulatory T cells and dendritic cells that creates a local microenvironment depleted of essential amino acids and rich in adenosine that leads to the amplification of a range of different tolerogenic signals. These signals are all eventually integrated by mammalian target of rapamycin inhibition, which enables the induction of new forkhead box protein 3-expressing Tregs. If correct, this provides a molecular explanation for the in vivo phenomena of linked suppression and infectious tolerance. PMID:20636819

  11. Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells

    PubMed Central

    Narayanan, Poornima devi; Nandabalan, Sangeetha Kadapakkam

    2016-01-01

    Purpose In this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells. Methods EtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation. Results Exposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2. Conclusion The study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation. PMID:27382387

  12. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration

    PubMed Central

    Ma, Liang; Li, Ying; Peng, Junya; Wu, Danni; Zhao, Xiaoxin; Cui, Yitong; Chen, Lilian; Yan, Xiaojun; Du, Yanan; Yu, Li

    2015-01-01

    Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them “migrasomes”. We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism “migracytosis”. PMID:25342562

  13. Mechanics Regulates Fate Decisions of Human Embryonic Stem Cells

    PubMed Central

    Sun, Yubing; Villa-Diaz, Luis G.; Lam, Raymond H. W.; Chen, Weiqiang; Krebsbach, Paul H.; Fu, Jianping

    2012-01-01

    Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function. PMID:22615930

  14. Mast cells mediate malignant pleural effusion formation

    PubMed Central

    Giannou, Anastasios D.; Marazioti, Antonia; Spella, Magda; Kanellakis, Nikolaos I.; Apostolopoulou, Hara; Psallidas, Ioannis; Prijovich, Zeljko M.; Vreka, Malamati; Zazara, Dimitra E.; Lilis, Ioannis; Papaleonidopoulos, Vassilios; Kairi, Chrysoula A.; Patmanidi, Alexandra L.; Giopanou, Ioanna; Spiropoulou, Nikolitsa; Harokopos, Vaggelis; Aidinis, Vassilis; Spyratos, Dionisios; Teliousi, Stamatia; Papadaki, Helen; Taraviras, Stavros; Snyder, Linda A.; Eickelberg, Oliver; Kardamakis, Dimitrios; Iwakura, Yoichiro; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Kalomenidis, Ioannis; Blackwell, Timothy S.; Agalioti, Theodora; Stathopoulos, Georgios T.

    2015-01-01

    Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell–induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable. PMID:25915587

  15. Layers of dendritic cell-mediated T cell tolerance, their regulation and the prevention of autoimmunity

    PubMed Central

    Mayer, Christian T.; Berod, Luciana; Sparwasser, Tim

    2012-01-01

    The last decades of Nobel prize-honored research have unequivocally proven a key role of dendritic cells (DCs) at controlling both T cell immunity and tolerance. A tight balance between these opposing DC functions ensures immune homeostasis and host integrity. Its perturbation could explain pathological conditions such as the attack of self tissues, chronic infections, and tumor immune evasion. While recent insights into the complex DC network help to understand the contribution of individual DC subsets to immunity, the tolerogenic functions of DCs only begin to emerge. As these consist of many different layers, the definition of a “tolerogenic DC” is subjected to variation. Moreover, the implication of DCs and DC subsets in the suppression of autoimmunity are incompletely resolved. In this review, we point out conceptual controversies and dissect the various layers of DC-mediated T cell tolerance. These layers include central tolerance, Foxp3+ regulatory T cells (Tregs), anergy/deletion and negative feedback regulation. The mode and kinetics of antigen presentation is highlighted as an additional factor shaping tolerance. Special emphasis is given to the interaction between layers of tolerance as well as their differential regulation during inflammation. Furthermore, potential technical caveats of DC depletion models are considered. Finally, we summarize our current understanding of DC-mediated tolerance and its role for the suppression of autoimmunity. Understanding the mechanisms of DC-mediated tolerance and their complex interplay is fundamental for the development of selective therapeutic strategies, e.g., for the modulation of autoimmune responses or for the immunotherapy of cancer. PMID:22783257

  16. Intracellular Drug Delivery: Mechanisms for Cell Entry.

    PubMed

    Garnacho, Carmen

    2016-01-01

    Over the last half century, the delivery of pharmacologically active substances, such as synthetic drugs, natural compounds, gene material and many other pharmaceutical products, has been widely studied. Understanding the interactions of drug carriers with cells and how these interactions influence the cellular uptake is of paramount importance, since targets for many therapeutic agents against several disorders are localized in the subcellular compartments. Besides, the route of drug carrier entry (direct or via endocytosis) often defines the efficiency, kinetics and final destination of the drug itself. Although classical endocytic pathways such as phagocytosis, macropinocytosis, clathrin-mediated and caveola-dependent pathways are well characterized, their control for pharmaceutical drug delivery applications is still a challenging issue. Also, better knowledge of non-classical endocytic pathways may help optimize targeted drug delivery systems for intracellular delivery. Therefore, this review focuses on mechanisms of intracellular delivery, including direct internalization and endocytosis, as well as factors such as targeting moiety, target receptor, and size, shape, and surface properties of the drug carrier that can influence uptake process. PMID:26675221

  17. SUMOylation-mediated regulation of cell cycle progression and cancer

    PubMed Central

    Eifler, Karolin; Vertegaal, Alfred C.O.

    2016-01-01

    SUMOylation plays critical roles during cell cycle progression. Many important cell cycle regulators, including many oncogenes and tumor suppressors, are functionally regulated via SUMOylation. The dynamic SUMOylation pattern observed throughout the cell cycle is ensured via distinct spatial and temporal regulation of the SUMO machinery. Additionally, SUMOylation cooperates with other post-translational modifications to mediate cell cycle progression. Deregulation of these SUMOylation and deSUMOylation enzymes causes severe defects in cell proliferation and genome stability. Different types of cancers were recently shown to be dependent on a functioning SUMOylation system, a finding that could potentially be exploited in anti-cancer therapies. PMID:26601932

  18. Cell mechanics: principles, practices, and prospects

    PubMed Central

    Moeendarbary, Emad; Harris, Andrew R

    2014-01-01

    Cells generate and sustain mechanical forces within their environment as part of their normal physiology. They are active materials that can detect mechanical stimulation by the activation of mechanosensitive signaling pathways, and respond to physical cues through cytoskeletal re-organization and force generation. Genetic mutations and pathogens that disrupt the cytoskeletal architecture can result in changes to cell mechanical properties such as elasticity, adhesiveness, and viscosity. On the other hand, perturbations to the mechanical environment can affect cell behavior. These transformations are often a hallmark and symptom of a variety of pathologies. Consequently, there are now a myriad of experimental techniques and theoretical models adapted from soft matter physics and mechanical engineering to characterize cell mechanical properties. Interdisciplinary research combining modern molecular biology with advanced cell mechanical characterization techniques now paves the way for furthering our fundamental understanding of cell mechanics and its role in development, physiology, and disease. We describe a generalized outline for measuring cell mechanical properties including loading protocols, tools, and data interpretation. We summarize recent advances in the field and explain how cell biomechanics research can be adopted by physicists, engineers, biologists, and clinicians alike. PMID:25269160

  19. Probing cell mechanical properties with microfluidic devices

    NASA Astrophysics Data System (ADS)

    Rowat, Amy

    2012-02-01

    Exploiting flow on the micron-scale is emerging as a method to probe cell mechanical properties with 10-1000x advances in throughput over existing technologies. The mechanical properties of cells and the cell nucleus are implicated in a wide range of biological contexts: for example, the ability of white blood cells to deform is central to immune response; and malignant cells show decreased stiffness compared to benign cells. We recently developed a microfluidic device to probe cell and nucleus mechanical properties: cells are forced to deform through a narrow constrictions in response to an applied pressure; flowing cells through a series of constrictions enables us to probe the ability of hundreds of cells to deform and relax during flow. By tuning the constriction width so it is narrower than the width of the cell nucleus, we can specifically probe the effects of nuclear physical properties on whole cell deformability. We show that the nucleus is the rate-limiting step in cell passage: inducing a change in its shape to a multilobed structure results in cells that transit more quickly; increased levels of lamin A, a nuclear protein that is key for nuclear shape and mechanical stability, impairs the passage of cells through constrictions. We are currently developing a new class of microfluidic devices to simultaneously probe the deformability of hundreds of cell samples in parallel. Using the same soft lithography techniques, membranes are fabricated to have well-defined pore distribution, width, length, and tortuosity. We design the membranes to interface with a multiwell plate, enabling simultaneous measurement of hundreds of different samples. Given the wide spectrum of diseases where altered cell and nucleus mechanical properties are implicated, such a platform has great potential, for example, to screen cells based on their mechanical phenotype against a library of drugs.

  20. ECHIDNA-mediated post-Golgi trafficking of auxin carriers for differential cell elongation.

    PubMed

    Boutté, Yohann; Jonsson, Kristoffer; McFarlane, Heather E; Johnson, Errin; Gendre, Delphine; Swarup, Ranjan; Friml, Jirí; Samuels, Lacey; Robert, Stéphanie; Bhalerao, Rishikesh P

    2013-10-01

    The plant hormone indole-acetic acid (auxin) is essential for many aspects of plant development. Auxin-mediated growth regulation typically involves the establishment of an auxin concentration gradient mediated by polarly localized auxin transporters. The localization of auxin carriers and their amount at the plasma membrane are controlled by membrane trafficking processes such as secretion, endocytosis, and recycling. In contrast to endocytosis or recycling, how the secretory pathway mediates the localization of auxin carriers is not well understood. In this study we have used the differential cell elongation process during apical hook development to elucidate the mechanisms underlying the post-Golgi trafficking of auxin carriers in Arabidopsis. We show that differential cell elongation during apical hook development is defective in Arabidopsis mutant echidna (ech). ECH protein is required for the trans-Golgi network (TGN)-mediated trafficking of the auxin influx carrier AUX1 to the plasma membrane. In contrast, ech mutation only marginally perturbs the trafficking of the highly related auxin influx carrier LIKE-AUX1-3 or the auxin efflux carrier PIN-FORMED-3, both also involved in hook development. Electron tomography reveals that the trafficking defects in ech mutant are associated with the perturbation of secretory vesicle genesis from the TGN. Our results identify differential mechanisms for the post-Golgi trafficking of de novo-synthesized auxin carriers to plasma membrane from the TGN and reveal how trafficking of auxin influx carriers mediates the control of differential cell elongation in apical hook development. PMID:24043780

  1. Hepatocyte-mediated cytotoxicity and host defense mechanisms in the alcohol-injured liver.

    PubMed

    McVicker, Benita L; Thiele, Geoffrey M; Tuma, Dean J; Casey, Carol A

    2014-09-01

    The consumption of alcohol is associated with many health issues including alcoholic liver disease (ALD). The natural history of ALD involves the development of steatosis, inflammation (steatohepatitis), fibrosis and cirrhosis. During the stage of steatohepatitis, the combination of inflammation and cellular damage can progress to a severe condition termed alcoholic hepatitis (AH). Unfortunately, the pathogenesis of AH remains uncharacterized. Some modulations have been identified in host defense and liver immunity mechanisms during AH that highlight the role of intrahepatic lymphocyte accumulation and associated inflammatory cytokine responses. Also, it is hypothesized that alcohol-induced injury to liver cells may significantly contribute to the aberrant lymphocytic distribution that is seen in AH. In particular, the regulation of lymphocytes by hepatocytes may be disrupted in the alcoholic liver resulting in altered immunologic homeostasis and perpetuation of disease. In recent studies, it was demonstrated that the direct killing of activated T lymphocytes by hepatocytes is facilitated by the asialoglycoprotein receptor (ASGPR). The ASGPR is a well-characterized glycoprotein receptor that is exclusively expressed by hepatocytes. This hepatic receptor is known for its role in the clearance of desialylated glycoproteins or cells, yet neither its physiological function nor its role in disease states has been determined. Interestingly, alcohol markedly impairs ASGPR function; however, the effect alcohol has on ASGPR-mediated cytotoxicity of lymphocytes remains to be elucidated. This review discusses the contribution of hepatocytes in immunological regulation and, importantly, how pathological effects of ethanol disrupt hepatocellular-mediated defense mechanisms.

  2. The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

    PubMed

    Honti, Viktor; Csordás, Gábor; Kurucz, Éva; Márkus, Róbert; Andó, István

    2014-01-01

    In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom. PMID:23800719

  3. Calcium carbonate mineralization mediated by in vitro cultured mantle cells from Pinctada fucata.

    PubMed

    Kong, Wei; Li, Shiguo; Xiang, Liang; Xie, Liping; Zhang, Rongqing

    2015-08-01

    Formation of the molluscan shell is believed to be an extracellular event mediated by matrix proteins. We report calcium carbonate mineralization mediated by Pinctada fucata mantle cells. Crystals only appeared when mantle cells were present in the crystallization solution. These crystals were piled up in highly ordered units and showed the typical characteristics of biomineralization products. A thin organic framework was observed after dissolving the crystals in EDTA. Some crystals had etched surfaces with a much smoother appearance than other parts. Mantle cells were observed to be attached to some of these smooth surfaces. These results suggest that mantle cells may be directly involved in the nucleation and remodeling process of calcium carbonate mineralization. Our result demonstrate the practicability of studying the mantle cell mechanism of biomineralization and contribute to the overall understanding of the shell formation process.

  4. Challenges and Opportunities for T-Cell-Mediated Strategies to Eliminate HIV Reservoirs

    PubMed Central

    Brockman, Mark A.; Jones, R. Brad; Brumme, Zabrina L.

    2015-01-01

    HIV’s ability to establish latent reservoirs of reactivation-competent virus is the major barrier to cure. “Shock and kill” methods consisting of latency-reversing agents (LRAs) followed by elimination of reactivating cells through cytopathic effects are under active development. However, the clinical efficacy of LRAs remains to be established. Moreover, recent studies indicate that reservoirs may not be reduced efficiently by either viral cytopathic or CD8+ T-cell-mediated mechanisms. In this perspective, we highlight challenges to T-cell-mediated elimination of HIV reservoirs, including characteristics of responding T cells, aspects of the cellular reservoirs, and properties of the latent virus itself. We also discuss potential strategies to overcome these challenges by targeting the antiviral activity of T cells toward appropriate viral antigens following latency. PMID:26483795

  5. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists.

  6. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists. PMID:27528656

  7. Cellular mechanisms mediating rat renal microvascular constriction by angiotensin II.

    PubMed Central

    Takenaka, T; Suzuki, H; Fujiwara, K; Kanno, Y; Ohno, Y; Hayashi, K; Nagahama, T; Saruta, T

    1997-01-01

    To assess cellular mechanisms mediating afferent (AA) and efferent arteriolar (EA) constriction by angiotensin II (AngII), experiments were performed using isolated perfused hydronephrotic kidneys. In the first series of studies, AngII (0.3 nM) constricted AAs and EAs by 29+/-3 (n = 8, P < 0.01) and 27+/-3% (n = 8, P < 0.01), respectively. Subsequent addition of nifedipine restored AA but not EA diameter. Manganese (8 mM) reversed EA constriction by 65+/-9% (P < 0.01). In the second group, the addition of N-ethylmaleimide (10 microM), a Gi/Go protein antagonist, abolished AngII- induced EA (n = 6) but not AA constriction (n = 6). In the third series of experiments, treatment with 2-nitro-4-carboxyphenyl-N, N-diphenyl-carbamate (200 microM), a phospholipase C inhibitor, blocked both AA and EA constriction by AngII (n = 6 for each). In the fourth group, thapsigargin (1 microM) prevented AngII-induced AA constriction (n = 8) and attenuated EA constriction (8+/-2% decrease in EA diameter at 0.3 nM AngII, n = 8, P < 0.05). Subsequent addition of manganese (8 mM) reversed EA constriction. Our data provide evidence that in AAs, AngII stimulates phospholipase C with subsequent calcium mobilization that is required to activate voltage-dependent calcium channels. Our results suggest that AngII constricts EAs by activating phospholipase C via the Gi protein family, thereby eliciting both calcium mobilization and calcium entry. PMID:9329977

  8. Role of paracrine factors in stem and progenitor cell mediated cardiac repair and tissue fibrosis

    PubMed Central

    Burchfield, Jana S; Dimmeler, Stefanie

    2008-01-01

    A new era has begun in the treatment of ischemic disease and heart failure. With the discovery that stem cells from diverse organs and tissues, including bone marrow, adipose tissue, umbilical cord blood, and vessel wall, have the potential to improve cardiac function beyond that of conventional pharmacological therapy comes a new field of research aiming at understanding the precise mechanisms of stem cell-mediated cardiac repair. Not only will it be important to determine the most efficacious cell population for cardiac repair, but also whether overlapping, common mechanisms exist. Increasing evidence suggests that one mechanism of action by which cells provide tissue protection and repair may involve paracrine factors, including cytokines and growth factors, released from transplanted stem cells into the surrounding tissue. These paracrine factors have the potential to directly modify the healing process in the heart, including neovascularization, cardiac myocyte apoptosis, inflammation, fibrosis, contractility, bioenergetics, and endogenous repair. PMID:19014650

  9. Caloric restriction as a mechanism mediating resistance to environmental disease.

    PubMed Central

    Frame, L T; Hart, R W; Leakey, J E

    1998-01-01

    It has been observed that susceptibility to many degenerative diseases increases concurrently with industrialization and rising living standards. Although epidemiologic studies suggest that specific environmental and dietary factors may be important, caloric intake alone (as reflected in body size) may account for much of the differential risk observed among diverse human populations. It has been suggested from animal studies that caloric intake may be the primary effector for many hormonal, metabolic, physiologic, and behavioral responses that coordinate reproductive strategy to apparent availability of food. When caloric intake is excessive, particularly at critical developmental stages, physiologic priorities are set for body growth and fecundity rather than for endurance and longevity. The converse occurs during periods of famine, thus increasing the probability that sufficient individuals survive to restore the population when conditions improve. Calorically restricted rodents have significantly longer reproductive and total life spans than their ad libitum-fed controls and exhibit a spectrum of biochemical and physiologic alterations that characterize their adaptation to reduced intake. These include reduced stature, hypercorticism in the absence of elevated adrenocorticotropic hormone levels, increased metabolic efficiency, decreased mitogenic response coupled with increased rates of apoptosis, reduced inflammatory response, induction of stress proteins and DNA repair enzymes, altered drug-metabolizing enzyme expression, and modified cell-mediated immune function. The overall profile of these changes is one of improved defense against environmental stress. This has been suggested as the mechanistic basis for the protective effects of low body weight on radiation and chemically induced cancers in experimental animals. It may also explain the significantly higher thresholds of acute toxicity observed when calorically restricted rodents are exposed to certain

  10. Discovering mechanisms of signaling-mediated cysteine oxidation.

    PubMed

    Poole, Leslie B; Nelson, Kimberly J

    2008-02-01

    Accumulating evidence reveals hydrogen peroxide as a key player both as a damaging agent and, from emerging evidence over the past decade, as a second messenger in intracellular signaling. This rather mild oxidant acts upon downstream targets within signaling cascades to modulate the activity of a host of enzymes (e.g. phosphatases and kinases) and transcriptional regulators through chemoselective oxidation of cysteine residues. With the recent development of specific detection reagents for hydrogen peroxide and new chemical tools to detect the generation of the initial oxidation product, sulfenic acid, on reactive cysteines within target proteins, the scene is set to gain a better understanding of the mechanisms through which hydrogen peroxide acts as a second messenger in cell signaling.

  11. Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

    PubMed

    Blandin, Anne-Florence; Noulet, Fanny; Renner, Guillaume; Mercier, Marie-Cécile; Choulier, Laurence; Vauchelles, Romain; Ronde, Philippe; Carreiras, Franck; Etienne-Selloum, Nelly; Vereb, Gyorgy; Lelong-Rebel, Isabelle; Martin, Sophie; Dontenwill, Monique; Lehmann, Maxime

    2016-07-01

    Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma. PMID:27063097

  12. Cell-mediated immune responses to COPV early proteins.

    PubMed

    Jain, Suchitra; Moore, Richard A; Anderson, Davina M; Gough, Gerald W; Stanley, Margaret A

    Cell-mediated immunity plays a key role in the regression of papillomavirus-induced warts and intra-epithelial lesions but the target antigens that induce this response are not clear. Canine oral papillomavirus (COPV) infection of the oral cavity in dogs is a well-characterized model of mucosal papillomavirus infection that permits analysis of the immune events during the infectious cycle. In this study we show that during the COPV infectious cycle, systemic T cell responses to peptides of several early proteins particularly the E2 protein, as assayed by delayed type hypersensitivity, lymphoproliferation and IFN-gamma ELISPOT, can be detected. The maximal response occurs in a narrow time window that coincides with maximal viral DNA replication and wart regression: thereafter, systemic T cell responses to early proteins decline quite rapidly. Vaccination using particle-mediated immunotherapeutic delivery (PMID) of codon-modified COPV E2 and E1 genes induces strong antigen-specific cell-mediated immune responses in the vaccinated animals. These data show that therapeutic immunization by PMID with codon-modified E2 is completely effective, that to E1 is partially protective, that this correlates with the intensity of antigen-specific cell-mediated immune responses and, further, they emphasize the importance of these responses and the route of immunization in the generation of protective immunity. PMID:16949120

  13. Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan.

    PubMed Central

    Zimmerman, Ella; Geiger, Benjamin; Addadi, Lia

    2002-01-01

    A conceptual temporal and spatial gap exists between the first encounter of a cell with an adhesive substrate and the advanced stages of focal adhesion formation. Although ample information is available on focal adhesions structure and function, the mechanism of the first interaction events and the nature of the molecules mediating them are largely unknown. In this paper we identify cell-surface-associated hyaluronan as a mediator and modulator of the first steps of adhesion of A6 and other cells to conventional tissue culture substrates as well as to the surfaces of calcium-(R,R)-tartrate tetrahydrate crystals. Treatment of A6 cells with hyaluronidase suppresses their rapid interactions with these adhesive substrates, and incubation of either the hyaluronidase-treated cells or the substrate with hyaluronan restores cell adhesion. In contrast, excess hyaluronan on both the cells and the substrate strongly inhibits adhesion. We thus propose that cell-surface-associated hyaluronan can mediate and modulate cell-matrix adhesion at the very first encounter with the substrate. It may promote it through the establishment of exquisitely stereospecific chemical interactions or inhibit it by virtue of steric exclusion and/or electrostatic repulsion. PMID:11916844

  14. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

    PubMed Central

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology. PMID:27199668

  15. T-helper cell-mediated factors in drug-induced liver injury.

    PubMed

    Wang, Xinzhi; Zhang, Luyong; Jiang, Zhenzhou

    2015-07-01

    Drug-induced liver injury (DILI) leads to a large burden on the healthcare system due to its potential morbidity and mortality. The key for predicting and preventing DILI is to understand the underlying mechanisms. Hepatic inflammation is one of the most common features of DILI. The inflammation can be attributed to the innate immune response. The adaptive immune system is also affected by the innate immune response resulting in liver damage. T-helper cells are important regulators of acquired immunity. T-helper cell-mediated immune responses play pivotal roles in the pathogenesis of a variety of liver disorders. This review summarizes recent advances in the T-helper cell-mediated factors in DILI and potential mechanisms, which may lead to a better understanding of DILI. PMID:25752261

  16. Simvastatin may induce insulin resistance through a novel fatty acid mediated cholesterol independent mechanism

    PubMed Central

    Kain, Vasundhara; Kapadia, Bandish; Misra, Parimal; Saxena, Uday

    2015-01-01

    Statins are a class of oral drugs that are widely used for treatment of hypercholesterolemia. Recent clinical data suggest that chronic use of these drugs increases the frequency of new onset diabetes. Studies to define the risks of statin-induced diabetes and its underlying mechanisms are clearly necessary. We explored the possible mechanism of statin induced insulin resistance using a well-established cell based model and simvastatin as a prototype statin. Our data show that simvastatin induces insulin resistance in a cholesterol biosynthesis inhibition independent fashion but does so by a fatty acid mediated effect on insulin signaling pathway. These data may help design strategies for prevention of statin induced insulin resistance and diabetes in patients with hypercholesterolemia. PMID:26345110

  17. Mechanisms of complement-mediated clearance of bacteria from the murine lung.

    PubMed

    Heidbrink, P J; Toews, G B; Gross, G N; Pierce, A K

    1982-05-01

    Complement factors enhance host defense against bacterial challenges by attracting phagocytic cells to the site of the inoculum and by opsonizing bacteria for phagocytic ingestion. The relative contribution of these 2 mechanisms to in vivo clearance of bacteria from the lung has not been described. Hypocomplementemic and normal animals were challenged with various bacteria. Clearance of bacteria was studied by quantitative lung culture. Phagocytic response was determined by bronchoalveolar lavage. Staphylococci were cleared by macrophages without regard to the complement status of the host. Hypocomplementemic animals cleared pneumococci less efficiently than did control animals. This defect correlated with decreased neutrophil recruitment. Pseudomonas was not cleared in hypocomplementemic animals, but there was no difference in the number or type of phagocytes. This implies that an opsonic rather thn a chemotactic defect was responsible. These data suggest that the mechanism of complement-mediated defense against bacterial challenge varies with the type of pathogen present.

  18. Cancer cell glycocalyx mediates mechanotransduction and flow-regulated invasion.

    PubMed

    Qazi, Henry; Palomino, Rocio; Shi, Zhong-Dong; Munn, Lance L; Tarbell, John M

    2013-11-01

    Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) was investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiological levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP activity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx - a potential target for therapeutics.

  19. Slit2 involvement in glioma cell migration is mediated by Robo1 receptor.

    PubMed

    Mertsch, Sonja; Schmitz, Nicole; Jeibmann, Astrid; Geng, Jian-Guo; Paulus, Werner; Senner, Volker

    2008-03-01

    Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRT-PCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels. Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2 receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

  20. Molecular mechanisms of CRISPR-mediated microbial immunity.

    PubMed

    Gasiunas, Giedrius; Sinkunas, Tomas; Siksnys, Virginijus

    2014-02-01

    Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction–modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR–Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems. PMID:23959171

  1. Molecular mechanisms of CRISPR-mediated microbial immunity.

    PubMed

    Gasiunas, Giedrius; Sinkunas, Tomas; Siksnys, Virginijus

    2014-02-01

    Bacteriophages (phages) infect bacteria in order to replicate and burst out of the host, killing the cell, when reproduction is completed. Thus, from a bacterial perspective, phages pose a persistent lethal threat to bacterial populations. Not surprisingly, bacteria evolved multiple defense barriers to interfere with nearly every step of phage life cycles. Phages respond to this selection pressure by counter-evolving their genomes to evade bacterial resistance. The antagonistic interaction between bacteria and rapidly diversifying viruses promotes the evolution and dissemination of bacteriophage-resistance mechanisms in bacteria. Recently, an adaptive microbial immune system, named clustered regularly interspaced short palindromic repeats (CRISPR) and which provides acquired immunity against viruses and plasmids, has been identified. Unlike the restriction–modification anti-phage barrier that subjects to cleavage any foreign DNA lacking a protective methyl-tag in the target site, the CRISPR–Cas systems are invader-specific, adaptive, and heritable. In this review, we focus on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems.

  2. Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

    PubMed

    Yajima, Ichiro; Uemura, Noriyuki; Nizam, Saika; Khalequzzaman, Md; Thang, Nguyen D; Kumasaka, Mayuko Y; Akhand, Anwarul A; Shekhar, Hossain U; Nakajima, Tamie; Kato, Masashi

    2012-06-01

    Our fieldwork showed more than 1 μM (145.1 μg/L) barium in about 3 μM (210.7 μg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 μg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 μM) on arsenic (3 μM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

  3. Intratumoral oxygen gradients mediate sarcoma cell invasion.

    PubMed

    Lewis, Daniel M; Park, Kyung Min; Tang, Vitor; Xu, Yu; Pak, Koreana; Eisinger-Mathason, T S Karin; Simon, M Celeste; Gerecht, Sharon

    2016-08-16

    Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets. PMID:27486245

  4. Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation

    PubMed Central

    Du, Yibin; Zhang, Shuo; Yu, Tao; Du, Gongwen; Zhang, Hui; Yin, Zongsheng

    2016-01-01

    The present study aimed to determine whether co-culture with bone marrow-derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of β-tubulin III-positive cells were detected by microscopy. The wingless-type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway was analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC-mediated NSC proliferation and differentiation. The results revealed that co-culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co-culture with EPCs markedly induced the expression levels of Wnt3a and β-catenin and inhibited the phosphorylation of glycogen synthase kinase 3β (GSK-3β). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC-mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC-mediated expression of β-catenin, and its phosphorylation and activation of GSK-3β. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC-mediated NSC proliferation and differentiation. PMID:27484039

  5. Doxorubicin-mediated Apoptosis in Glioma Cells Requires NFAT3

    PubMed Central

    Gopinath, Sreelatha; Vanamala, Sravan K.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Dinh, Dzung H.; Rao, Jasti S.

    2009-01-01

    Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells. PMID:19784808

  6. Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis

    SciTech Connect

    Di Paolo, Julie A.; Huang, Tao; Balazs, Mercedesz; Barbosa, James; Barck, Kai H.; Bravo, Brandon J.; Carano, Richard A.D.; Darrow, James; Davies, Douglas R.; DeForge, Laura E.; Diehl, Lauri; Ferrando, Ronald; Gallion, Steven L.; Giannetti, Anthony M.; Gribling, Peter; Hurez, Vincent; Hymowitz, Sarah G.; Jones, Randall; Kropf, Jeffrey E.; Lee, Wyne P.; Maciejewski, Patricia M.; Mitchell, Scott A.; Rong, Hong; Staker, Bart L.; Whitney, J. Andrew; Yeh, Sherry; Young, Wendy B.; Yu, Christine; Zhang, Juan; Reif, Karin; Currie, Kevin S.

    2011-09-20

    Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes Fc{gamma}RIII-induced TNF{alpha}, IL-1{beta} and IL-6 production. Accordingly, in myeloid- and Fc{gamma}R-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

  7. Active cell mechanics: Measurement and theory.

    PubMed

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  8. Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration

    PubMed Central

    Knight, Laura M.; Stakaityte, Gabriele; Wood, Jennifer, J.; Abdul-Sada, Hussein; Griffiths, David A.; Howell, Gareth J.; Wheat, Rachel; Blair, G. Eric; Steven, Neil M.; Macdonald, Andrew; Blackbourn, David J.

    2014-01-01

    ABSTRACT Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule

  9. Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

    PubMed Central

    Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin. PMID:20498856

  10. Tryptophan depletion as a mechanism of gamma interferon-mediated chlamydial persistence.

    PubMed Central

    Beatty, W L; Belanger, T A; Desai, A A; Morrison, R P; Byrne, G I

    1994-01-01

    Previous studies have shown that the immune-regulated cytokine gamma interferon (IFN-gamma) activates host cells to restrict intracellular growth of the bacterial pathogen Chlamydia trachomatis by induction of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Recently, subinhibitory levels of IFN-gamma were used to generate an in vitro persistent chlamydial infection characterized by large aberrant, noninfectious reticulate bodies from which infectious progeny could be recovered following the removal of IFN-gamma. Studies were done to determine if the mechanism functioning to induce chlamydiae to enter a persistent state in the presence of low levels of IFN-gamma was similar to that reported to inhibit chlamydial growth. Host cells treated with levels of IFN-gamma required to induce persistence were assessed for IDO activity by high-performance liquid chromatography analysis of tryptophan and its catabolic products. Substantial tryptophan catabolism was detected in acid-soluble cellular pools, indicating that the intracellular availability of this essential amino acid was limited under these conditions. In addition, a mutant cell line responsive to IFN-gamma but deficient in IDO activity was shown to support C. trachomatis growth, but aberrant organisms were not induced in response to IFN-gamma treatment. Analyses of infected cells cultured in medium with incremental levels of exogenous tryptophan indicated that persistent growth was induced by reducing the amount of this essential amino acid. These studies confirmed that nutrient deprivation by IDO-mediated tryptophan catabolism was the mechanism by which IFN-gamma mediates persistent growth of C. trachomatis. Images PMID:8063385

  11. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  12. CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

    PubMed Central

    Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

    2016-01-01

    Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate

  13. A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

    PubMed Central

    Muehlbauer, Stefan M.; Chandran, Kartik; Diaz-Griffero, Felipe

    2014-01-01

    Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants. PMID:24893007

  14. Drug-induced hyperploidy stimulates an antitumor NK cell response mediated by NKG2D and DNAM-1 receptors

    PubMed Central

    Acebes-Huerta, Andrea; Lorenzo-Herrero, Seila; Folgueras, Alicia R.; Huergo-Zapico, Leticia; Lopez-Larrea, Carlos; López-Soto, Alejandro; Gonzalez, Segundo

    2016-01-01

    ABSTRACT Formation of polyploid or aneuploid cells is a pathological hallmark of malignant tumors. Cell cycle checkpoint mechanisms play a crucial role in ensuring genomic integrity during mitosis, avoiding the generation of aneuploid cells. Additionally, cancer cell DNA ploidy is subjected to extrinsic controls operated by activation of adaptive immune responses mediated by T cells. NK cells exert a central role in the innate anticancer immunity; however, the mechanisms involved in the recognition of tumor cells by NK cells have not been fully elucidated. Herein, we report that drug-induced polyploidy in cancer cells activates antitumor responses mediated by NK cells. Thus, hyperploidy-inducing chemotherapeutic agents strongly upregulate the tumor expression of ligands for the NK cell activating receptors NKG2D and DNAM-1. Drug-induced hyperploidy modulated the repertoire of activating receptors and the cytokine profile of NK cells, rendering tumor cells more susceptible to NK cell-mediated lysis through the activation of NKG2D and DNAM-1 receptors. In addition, hyperploidization stimulated the production of IL-2 by CD4 T cells, which induced NK cell proliferation and activity. The stimulation of MICA, a key NKG2D ligand, in hyperploid cells was mainly mediated by ATM protein kinase. Likewise, pharmacological inhibition of key regulators of endoplasmic reticulum stress in certain cell models supports a role for this pathway in NKG2D ligand upregulation. Overall, our findings indicate that, besides the cytotoxic effect on tumor cells, the therapeutic activity of anti-mitotic drugs may be mediated by the induction of a coordinated antitumor immune response involving NK and T cells. PMID:27057443

  15. Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1.

    PubMed

    Speyer, Cecilia L; Nassar, Mahdy A; Hachem, Ali H; Bukhsh, Miriam A; Jafry, Waris S; Khansa, Rafa M; Gorski, David H

    2016-06-01

    Riluzole, the only drug approved by the FDA for treating amyotrophic lateral sclerosis, inhibits melanoma proliferation through its inhibitory effect on glutamatergic signaling. We demonstrated that riluzole also inhibits the growth of triple-negative breast cancer (TNBC) and described a role for metabotropic glutamate receptor-1 (GRM1) in regulating TNBC cell growth and progression. However, the role of GRM1 in mediating riluzole's effects in breast cancer has not been fully elucidated. In this study, we seek to determine how much of riluzole's action in breast cancer is mediated through GRM1. We investigated anti-tumor properties of riluzole in TNBC and ER+ cells using cell growth, invasion, and soft-agar assays and compared riluzole activity with GRM1 levels. Using Lentiviral vectors expressing GRM1 or shGRM1, these studies were repeated in cells expressing high or low GRM1 levels where the gene was either silenced or overexpressed. Riluzole inhibited proliferation, invasion, and colony formation in both TNBC and ER+ cells. There was a trend between GRM1 expression in TNBC cells and their response to riluzole in both cell proliferation and invasion assays. However, silencing and overexpression studies had no effect on cell sensitivity to riluzole. Our results clearly suggest a GRM1-independent mechanism through which riluzole mediates its effects on breast cancer cells. Understanding the mechanism by which riluzole mediates breast cancer progression will be useful in identifying new therapeutic targets for treating TNBC and in facilitating stratification of patients in clinical trials using riluzole in conjunction with conventional therapy. PMID:27146584

  16. Biomarkers of CD4+ CTL cell Mediated Immunity to Tuberculosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immune responses mediated by interactions between T-lymphocyte subsets and mycobacteria-infected macrophages are critical for control of tuberculosis. In these studies, the bovine model was used to characterize the cytolytic and mycobactericidal CD4+ T cell response induced by BCG vaccination. ...

  17. Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

    NASA Astrophysics Data System (ADS)

    Khetan, Sudhir; Guvendiren, Murat; Legant, Wesley R.; Cohen, Daniel M.; Chen, Christopher S.; Burdick, Jason A.

    2013-05-01

    Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

  18. TRPC1 and TRPC6 channels cooperate with TRPV4 to mediate mechanical hyperalgesia and nociceptor sensitization

    PubMed Central

    Alessandri-Haber, Nicole; Dina, Olayinka A.; Chen, Xiaoje; Levine, Jon D.

    2009-01-01

    The transient receptor potential vanilloid 4 (TRPV4) contributes to mechanical hyperalgesia of diverse etiologies, presumably as part of a mechanoreceptor signaling complex (Alessandri-Haber et al., 2008). To investigate the hypothesis that a functional interaction between TRPV4 and stretch-activated ion channels (SACs) is involved in this mechanical transduction mechanism, we used a selective SACs inhibitor, GsMTx-4. Intradermal injection of GsMTx-4 in the rat hind paw reversed the mechanical hyperalgesia induced by intradermal injection of inflammatory mediators. In vivo single fiber recordings showed that GsMTx-4 reversed inflammatory mediator-induced decrease in mechanical threshold in half of sensitized C-fibers. Furthermore, GsMTx-4 reduced hyperalgesia to both mechanical and hypotonic stimuli in different models of inflammatory and neuropathic pain while it had no effect on baseline mechanical nociceptive thresholds. TRPC1 and TRPC6, two GsMTx-4-sensitive SACs are expressed in dorsal root ganglion neurons (DRG). Single-cell RT-PCR showed that messenger RNAs for TRPV4, TRPC1 and TRPC6 are frequently co-expressed in DRG neurons. Spinal intrathecal administration of oligodeoxynucleotides antisense to TRPC1 and TRPC6, like that to TRPV4, reversed the hyperalgesia to mechanical and hypotonic stimuli induced by inflammatory mediators without affecting baseline mechanical nociceptive threshold. However, antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4 selective agonist 4α-PDD. We conclude that TRPC1 and TRPC6 channels cooperate with TRPV4 channels to mediate mechanical hyperalgesia and primary afferent nociceptor sensitization although they may have distinctive roles. PMID:19439599

  19. Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

    PubMed Central

    Brown, Gabriella K.; Kreiss, Alexandre; Lyons, A. Bruce; Woods, Gregory M.

    2011-01-01

    The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research. PMID:21957452

  20. Ptch2 mediates the Shh response in Ptch1-/- cells.

    PubMed

    Alfaro, Astrid C; Roberts, Brock; Kwong, Lina; Bijlsma, Maarten F; Roelink, Henk

    2014-09-01

    The Hedgehog (Hh) signaling response is regulated by the interaction of three key components that include the sonic hedgehog (Shh) ligand, its receptor patched 1 (Ptch1) and the pathway activator smoothened (Smo). Under the prevailing model of Shh pathway activation, the binding of Shh to Ptch1 (the key Shh receptor) results in the release of Ptch1-mediated inhibition of Smo, leading to Smo activation and subsequent cell-autonomous activation of the Shh response. Consistent with this model, Ptch1(-/-) cells show a strong upregulation of the Shh response. Our finding that this response can be inhibited by the Shh-blocking antibody 5E1 indicates that the Shh response in Ptch1(-/-) cells remains ligand dependent. Furthermore, we find that Shh induces a strong response in Ptch1(-/-);Shh(-/-) cells, and that Ptch1(-/-) fibroblasts retain their ability to migrate towards Shh, demonstrating that Ptch1(-/-) cells remain sensitive to Shh. Expression of a dominant-negative Ptch1 mutant in the developing chick neural tube had no effect on Shh-mediated patterning, but expression of a dominant-negative form of patched 2 (Ptch2) caused an activation of the Shh response. This indicates that, at early developmental stages, Ptch2 functions to suppress Shh signaling. We found that Ptch1(-/-);Ptch2(-/-) cells cannot further activate the Shh response, demonstrating that Ptch2 mediates the response to Shh in the absence of Ptch1. PMID:25085974

  1. Cell mechanics and human disease states

    NASA Astrophysics Data System (ADS)

    Suresh, Subra

    2006-03-01

    This presentation will provide summary of our very recent studies exploring the effects of biochemical factors, influenced by foreign organisms or in vivo processes, on intracellular structural reorganization, single-cell mechanical response and motility of a population of cells in the context of two human diseases: malaria induced by Plasmodium falciparum merozoites that invade red blood cells, and gastrointestinal cancer metastasis involving epithelial cells. In both cases, particular attention will be devoted to systematic changes induced in specific molecular species in response to controlled alterations in disease state. The role of critical proteins in influencing the mechanical response of human red bloods during the intra-erythrocytic development of P. falciparum merozoites has also been assessed quantitatively using specific protein knock-out experiments by recourse to gene inactivation methods. Single-cell mechanical response characterization entails such tools as optical tweezers and mechanical plate stretchers whereas cell motility assays and cell-population biorheology characterization involves microfluidic channels. The experimental studies are accompanied by three-dimensional computational simulations at the continuum and mesoscopic scales of cell deformation. An outcome of such combined experimental and computational biophysical studies is the realization of how chemical factors influence single-cell mechanical response, cytoadherence, the biorheology of a large population of cells through microchannels representative of in vivo conditions, and the onset and progression of disease states.

  2. Environmental Estrogens Induce Mast Cell Degranulation and Enhance IgE-Mediated Release of Allergic Mediators

    PubMed Central

    Narita, Shin-ichiro; Goldblum, Randall M.; Watson, Cheryl S.; Brooks, Edward G.; Estes, D. Mark; Curran, Edward M.; Midoro-Horiuti, Terumi

    2007-01-01

    Background Prevalence and morbidity of allergic diseases have increased over the last decades. Based on the recently recognized differences in asthma prevalence between the sexes, we have examined the effect of endogenous estrogens on a key element of the allergic response. Some lipophilic pollutants have estrogen-like activities and are termed environmental estrogens. These pollutants tend to degrade slowly in the environment and to bioaccumulate and bioconcentrate in the food chain; they also have long biological half-lives. Objectives Our goal in this study was to identify possible pathogenic roles for environmental estrogens in the development of allergic diseases. Methods We screened a number of environmental estrogens for their ability to modulate the release of allergic mediators from mast cells. We incubated a human mast cell line and primary mast cell cultures derived from bone marrow of wild type and estrogen receptor α (ER-α )–deficient mice with environmental estrogens with and without estradiol or IgE and allergens. We assessed degranulation of mast cells by quantifying the release of β -hexosaminidase. Results All of the environmental estrogens tested caused rapid, dose-related release of β -hexosaminidase from mast cells and enhanced IgE-mediated release. The combination of physiologic concentrations of 17β -estradiol and several concentrations of environmental estrogens had additive effects on mast cell degranulation. Comparison of bone marrow mast cells from ER-α –sufficient and ER-α –deficient mice indicated that much of the effect of environmental estrogens was mediated by ER-α . Conclusions Our findings suggest that estrogenic environmental pollutants might promote allergic diseases by inducing and enhancing mast cell degranulation by physiologic estrogens and exposure to allergens. PMID:17366818

  3. Retinoic acid receptor alpha mediates growth inhibition by retinoids in human colon carcinoma HT29 cells.

    PubMed

    Nicke, B; Kaiser, A; Wiedenmann, B; Riecken, E O; Rosewicz, S

    1999-08-11

    Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.

  4. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

    PubMed Central

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  5. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

    PubMed

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular

  6. Methoxychlor enhances degranulation of murine mast cells by regulating FcεRI-mediated signal transduction.

    PubMed

    Yasunaga, Sho; Nishi, Kosuke; Nishimoto, Sogo; Sugahara, Takuya

    2015-01-01

    Methoxychlor, an organochlorine insecticide developed to replace DDT (dichlorodiphenyltrichloroethane), has been reported to induce mast cell degranulation and to enhance IgE-mediated allergic responses. However, the mechanisms underlying these effects are not clear. To clarify potential mechanisms, the effects of methoxychlor on degranulation of mast cells were examined. Degranulation responses were evaluated using RBL-2H3 cells and mouse bone marrow-derived mast cells with either the antigen-induced or calcium ionophore-induced stimulation. Phosphorylation of enzymes related to signaling events associated with mast cell degranulation was analyzed by immunoblotting. Effects on vascular permeability in the passive cutaneous anaphylaxis reaction were evaluated following oral administration of methoxychlor to BALB/c mice. The results indicated that methoxychlor caused increased mast cell degranulation in the presence of antigen, whereas it had no effect on calcium ionophore-induced degranulation of RBL-2H3 cells. Immunoblot analyses demonstrated that the phosphorylation level of phosphoinositide 3-kinase (which plays a central role in mast cell signaling) was increased by methoxychlor during antigen-induced degranulation. In addition, methoxychlor activated the signaling pathway via the high-affinity IgE receptor by inducing phosphorylation of Syk and PLCγ1/2, which transfer the signal for degranulation downstream. Lastly, oral administration of methoxychlor exhibited a tendency to promote vascular permeability in passive cutaneous anaphylaxis model mice. Taken together, the results here suggested that methoxychlor enhanced degranulation through FcεRI-mediated signaling and promoted allergenic symptoms involved in mast cell degranulation.

  7. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation.

    PubMed

    Wen, Li-Li; Lin, Chien-Yu; Chou, Hsiu-Chu; Chang, Chih-Cheng; Lo, Hau-Yin; Juan, Shu-Hui

    2016-01-01

    Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation. PMID:27171144

  8. Perfluorooctanesulfonate Mediates Renal Tubular Cell Apoptosis through PPARgamma Inactivation

    PubMed Central

    Chou, Hsiu-Chu; Chang, Chih-Cheng; Lo, Hau-Yin; Juan, Shu-Hui

    2016-01-01

    Perfluorinated chemicals (PFCs) are ubiquitously distributed in the environments including stainless pan-coating, raincoat, fire extinguisher, and semiconductor products. The PPAR family has been shown to contribute to the toxic effects of PFCs in thymus, immune and excretory systems. Herein, we demonstrated that perfluorooctanesulfonate (PFOS) caused cell apoptosis through increasing ratio of Bcl-xS/xL, cytosolic cytochrome C, and caspase 3 activation in renal tubular cells (RTCs). In addition, PFOS increased transcription of inflammatory cytokines (i.e., TNFα, ICAM1, and MCP1) by NFκB activation. Conversely, PFOS reduced the mRNA levels of antioxidative enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase, as a result of reduced PPARγ transactivational activity by using reporter and chromatin immuoprecipitation (ChIP) assays. PFOS reduced the protein interaction between PPARγ and PPARγ coactivator-1 alpha (PGC1α) by PPARγ deacetylation through Sirt1 upregulation, of which the binding of PPARγ and PGC1α to a peroxisome proliferator response element (PPRE) in the promoter regions of these antioxidative enzymes was alleviated in the ChIP assay. Furthermore, Sirt1 also deacetylated p53 and then increased the binding of p53 to Bax, resulting in increased cytosolic cytochrome C. The effect of PPARγ inactivation by PFOS was validated using the PPARγ antagonist GW9662, whereas the adverse effects of PFOS were prevented by PPARγ overexpression and activators, rosiglitozone and L-carnitine, in RTCs. The in vitro finding of protective effect of L-carnitine was substantiated in vivo using Balb/c mice model subjected to PFOS challenge. Altogether, we provide in vivo and in vitro evidence for the protective mechanism of L-carnitine in eliminating PFOS-mediated renal injury, at least partially, through PPARγ activation. PMID:27171144

  9. Dormancy activation mechanism of tracheal stem cells

    PubMed Central

    Li, Xin; Xu, Jing-xian; Jia, Xin-Shan; Li, Wen-ya; Han, Yi-chen; Wang, En-hua; Li, Fang

    2016-01-01

    Accurate markers and molecular mechanisms of stem cell dormancy and activation are poorly understood. In this study, the anti-cancer drug, 5-fluorouracil, was used to selectively kill proliferating cells of human bronchial epithelial (HBE) cell line. This method can enrich and purify stem cell population. The dormant versus active status of stem cells was determined by phosphorylation of RNAp II Ser2. The surviving stem cells were cultured to form stem cell spheres expressing stem cell markers and transplanted into nude mice to form a teratoma. The results demonstrated the properties of stem cells and potential for multi-directional differentiation. Bisulfite sequencing polymerase chain reaction showed that demethylation of the Sox2 promoter by 5-FU resulted in Sox2 expression in the dormant stem cells. This study shows that the dormancy and activation of HBE stem cells is closely related to epigenetic modification. PMID:27009861

  10. Release and Elementary Mechanisms of Nitric Oxide in Hair Cells

    PubMed Central

    Lv, Ping; Rodriguez-Contreras, Adrian; Kim, Hyo Jeong; Zhu, Jun; Wei, Dongguang; Choong-Ryoul, Sihn; Eastwood, Emily; Mu, Karen; Levic, Snezana; Song, Haitao; Yevgeniy, Petrov Y.; Smith, Peter J. S.

    2010-01-01

    The enzyme nitric oxide (NO) synthase, that produces the signaling molecule NO, has been identified in several cell types in the inner ear. However, it is unclear whether a measurable quantity of NO is released in the inner ear to confer specific functions. Indeed, the functional significance of NO and the elementary cellular mechanism thereof are most uncertain. Here, we demonstrate that the sensory epithelia of the frog saccule release NO and explore its release mechanisms by using self-referencing NO-selective electrodes. Additionally, we investigated the functional effects of NO on electrical properties of hair cells and determined their underlying cellular mechanism. We show detectable amounts of NO are released by hair cells (>50 nM). Furthermore, a hair-cell efferent modulator acetylcholine produces at least a threefold increase in NO release. NO not only attenuated the baseline membrane oscillations but it also increased the magnitude of current required to generate the characteristic membrane potential oscillations. This resulted in a rightward shift in the frequency–current relationship and altered the excitability of hair cells. Our data suggest that these effects ensue because NO reduces whole cell Ca2+ current and drastically decreases the open probability of single-channel events of the L-type and non L-type Ca2+ channels in hair cells, an effect that is mediated through direct nitrosylation of the channel and activation of protein kinase G. Finally, NO increases the magnitude of Ca2+-activated K+ currents via direct NO nitrosylation. We conclude that NO-mediated inhibition serves as a component of efferent nerve modulation of hair cells. PMID:20220083

  11. Mesenchymal stromal cells protect against caspase 3-mediated apoptosis of CD19(+) peripheral B cells through contact-dependent upregulation of VEGF.

    PubMed

    Healy, Marc E; Bergin, Ronan; Mahon, Bernard P; English, Karen

    2015-10-15

    The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease. PMID:26076727

  12. Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

    PubMed

    Brojatsch, Jürgen; Lima, Heriberto; Palliser, Deborah; Jacobson, Lee S; Muehlbauer, Stefan M; Furtado, Raquel; Goldman, David L; Lisanti, Michael P; Chandran, Kartik

    2015-01-01

    Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis. PMID:25830414

  13. A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells

    PubMed Central

    Luo, Judong; Li, Zengpeng; Zhu, Hong; Wang, Chenying; Zheng, Weibin; He, Yan; Song, Jianyuan; Wang, Wenjie; Zhou, Xifa; Lu, Xujing; Zhang, Shuyu; Chen, Jianming

    2016-01-01

    Ca2+-binding protein of 45 kDa (Cab45), a CREC family member, is reported to be associated with Ca2+-dependent secretory pathways and involved in multiple diseases including cancers. Cab45-G, a Cab45 isoform protein, plays an important role in protein sorting and secretion at Golgi complex. However, its role in cancer cell migration remains elusive. In this study, we demonstrate that Cab45-G exhibited an increased expression in cell lines with higher metastatic potential and promoted cell migration in multiple types of cancer cells. Overexpression of Cab45-G resulted in an altered expression of the molecular mediators of epithelial-mesenchymal transition (EMT), which is a critical step in the tumor metastasis. Quantitative real-time PCR showed that overexpression of Cab45-G increased the expression of matrix metalloproteinase-2 and -7 (MMP-2 and MMP-7). Conversely, knock-down of Cab45-G reduced the expression of the above MMPs. Moreover, forced expression of Cab45-G upregulated the level of phosphorylated ERK and modulated the secretion of extracellular proteins fibronectin and fibulin. Furthermore, in human cervical and esophageal cancer tissues, the expression of Cab45-G was found to be significantly correlated with that of MMP-2, further supporting the importance of Cab45-G on regulating cancer metastasis. Taken together, these results suggest that Cab45-G could regulate cancer cell migration through various molecular mechanisms, which may serve as a therapeutic target for the treatment of cancers. PMID:27194945

  14. A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells.

    PubMed

    Luo, Judong; Li, Zengpeng; Zhu, Hong; Wang, Chenying; Zheng, Weibin; He, Yan; Song, Jianyuan; Wang, Wenjie; Zhou, Xifa; Lu, Xujing; Zhang, Shuyu; Chen, Jianming

    2016-01-01

    Ca(2+)-binding protein of 45 kDa (Cab45), a CREC family member, is reported to be associated with Ca(2+)-dependent secretory pathways and involved in multiple diseases including cancers. Cab45-G, a Cab45 isoform protein, plays an important role in protein sorting and secretion at Golgi complex. However, its role in cancer cell migration remains elusive. In this study, we demonstrate that Cab45-G exhibited an increased expression in cell lines with higher metastatic potential and promoted cell migration in multiple types of cancer cells. Overexpression of Cab45-G resulted in an altered expression of the molecular mediators of epithelial-mesenchymal transition (EMT), which is a critical step in the tumor metastasis. Quantitative real-time PCR showed that overexpression of Cab45-G increased the expression of matrix metalloproteinase-2 and -7 (MMP-2 and MMP-7). Conversely, knock-down of Cab45-G reduced the expression of the above MMPs. Moreover, forced expression of Cab45-G upregulated the level of phosphorylated ERK and modulated the secretion of extracellular proteins fibronectin and fibulin. Furthermore, in human cervical and esophageal cancer tissues, the expression of Cab45-G was found to be significantly correlated with that of MMP-2, further supporting the importance of Cab45-G on regulating cancer metastasis. Taken together, these results suggest that Cab45-G could regulate cancer cell migration through various molecular mechanisms, which may serve as a therapeutic target for the treatment of cancers. PMID:27194945

  15. Single cell migration dynamics mediated by geometric confinement.

    PubMed

    Zhang, Hua; Hou, Ruixia; Xiao, Peng; Xing, Rubo; Chen, Tao; Han, Yanchun; Ren, Penggang; Fu, Jun

    2016-09-01

    The migration dynamics of cells plays a key role in tissue engineering and regenerative medicine. Previous studies mostly focus on regulating stem cell fate and phenotype by biophysical cues. In contrast, less is known about how the geometric cues mediate the migration dynamics of cells. Here, we fabricate graphene oxide (GO) microstripes on cell non-adhesive PEG substrate by using micromolding in capillary (MIMIC) method. Such micropatterns with alternating cell adhesion and cell resistance enable an effective control of selective adhesion and migration of single cells. The sharp contrast in cell adhesion minimizes the invasion of cells into the PEG patterns, and thereby strongly confines the cells on GO microstripes. As a result, the cells are forced to adapt highly polarized, elongated, and oriented geometry to fit the patterns. A series of pattern widths have been fabricated to modulate the extent of cell deformation and polarization. Under strong confinement, the cytoskeleton contractility, intracellular traction, and actin filament elongation are highly promoted, which result in enhanced cell migration along the patterns. This work provides an important insight into developing combinatorial graphene-based patterns for the control of cell migration dynamics, which is of great significance for tissue engineering and regenerative medicine. PMID:27137805

  16. Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in Hep G2 Hepatocelluar Carcinoma Cells

    PubMed Central

    Han, Hee Jeong; Kwon, Hee Young; Sohn, Eun Jung; Ko, Hyunsuk; Kim, Bogeun; Jung, Kwon; Lew, Jae Hwan; Kim, Sung-Hoon

    2014-01-01

    Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells. PMID:24795530

  17. Nostalgia-Evoked Inspiration: Mediating Mechanisms and Motivational Implications.

    PubMed

    Stephan, Elena; Sedikides, Constantine; Wildschut, Tim; Cheung, Wing-Yee; Routledge, Clay; Arndt, Jamie

    2015-10-01

    Six studies examined the nostalgia-inspiration link and its motivational implications. In Study 1, nostalgia proneness was positively associated with inspiration frequency and intensity. In Studies 2 and 3, the recollection of nostalgic (vs. ordinary) experiences increased both general inspiration and specific inspiration to engage in exploratory activities. In Study 4, serial mediational analyses supported a model in which nostalgia increases social connectedness, which subsequently fosters self-esteem, which then boosts inspiration. In Study 5, a rigorous evaluation of this serial mediational model (with a novel nostalgia induction controlling for positive affect) reinforced the idea that nostalgia-elicited social connectedness increases self-esteem, which then heightens inspiration. Study 6 extended the serial mediational model by demonstrating that nostalgia-evoked inspiration predicts goal pursuit (intentions to pursue an important goal). Nostalgia spawns inspiration via social connectedness and attendant self-esteem. In turn, nostalgia-evoked inspiration bolsters motivation. PMID:26228477

  18. Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake.

    PubMed

    Firsching, R; Buchholz, C J; Schneider, U; Cattaneo, R; ter Meulen, V; Schneider-Schaulies, J

    1999-07-01

    CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell. PMID:10364272

  19. Cancer Cell Fusion: Mechanisms Slowly Unravel

    PubMed Central

    Noubissi, Felicite K.; Ogle, Brenda M.

    2016-01-01

    Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion was a mechanism of cancer metastasis. This hypothesis gained some support over the years, and recently became the focus of many studies that revealed increasing evidence pointing to the possibility that cancer cell fusion probably gives rise to the metastatic phenotype by generating widespread genetic and epigenetic diversity, leading to the emergence of critical populations needed to evolve resistance to the treatment and development of metastasis. In this review, we will discuss the clinical relevance of cancer cell fusion, describe emerging mechanisms of cancer cell fusion, address why inhibiting cancer cell fusion could represent a critical line of attack to limit drug resistance and to prevent metastasis, and suggest one new modality for doing so. PMID:27657058

  20. Mechanisms of mesenchymal stem/stromal cell function.

    PubMed

    Spees, Jeffrey L; Lee, Ryang Hwa; Gregory, Carl A

    2016-01-01

    The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell-cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics. PMID:27581859

  1. MAR characteristic motifs mediate episomal vector in CHO cells.

    PubMed

    Lin, Yan; Li, Zhaoxi; Wang, Tianyun; Wang, Xiaoyin; Wang, Li; Dong, Weihua; Jing, Changqin; Yang, Xianjun

    2015-04-01

    An ideal gene therapy vector should enable persistent transgene expression without limitations in safety and reproducibility. Recent researches' insight into the ability of chromosomal matrix attachment regions (MARs) to mediate episomal maintenance of genetic elements allowed the development of a circular episomal vector. Although a MAR-mediated engineered vector has been developed, little is known on which motifs of MAR confer this function during interaction with the host genome. Here, we report an artificially synthesized DNA fragment containing only characteristic motif sequences that served as an alternative to human beta-interferon matrix attachment region sequence. The potential of the vector to mediate gene transfer in CHO cells was investigated. The short synthetic MAR motifs were found to mediate episomal vector at a low copy number for many generations without integration into the host genome. Higher transgene expression was maintained for at least 4 months. In addition, MAR was maintained episomally and conferred sustained EGFP expression even in nonselective CHO cells. All the results demonstrated that MAR characteristic sequence-based vector can function as stable episomes in CHO cells, supporting long-term and effective transgene expression.

  2. Phenotypically distinct helper NK cells are required for gp96-mediated anti-tumor immunity

    PubMed Central

    Sedlacek, Abigail L.; Kinner-Bibeau, Lauren B.; Binder, Robert J.

    2016-01-01

    A number of Heat Shock Proteins (HSPs), in the extracellular environment, are immunogenic. Following cross-presentation of HSP-chaperoned peptides by CD91+ antigen presenting cells (APCs), T cells are primed with specificity for the derivative antigen-bearing cell. Accordingly, tumor-derived HSPs are in clinical trials for cancer immunotherapy. We investigate the role of NK cells in gp96-mediated anti-tumor immune responses given their propensity to lyse tumor cells. We show that gp96-mediated rejection of tumors requires a unique and necessary helper role in NK cells. This helper role occurs during the effector phase of the anti-tumor immune response and is required for T cell and APC function. Gp96 activates NK cells indirectly via APCs to a phenotype distinct from NK cells activated by other mechanisms such as IL-2. While NK cells have both lytic and cytokine producing properties, we show that gp96 selectively activates cytokine production in NK cells, which is important in the HSP anti-tumor immune response, and leaves their cytotoxic capacity unchanged. PMID:27431727

  3. CCCTC-binding Factor Mediates Effects of Glucose On Beta Cell Survival

    PubMed Central

    Tsui, Shanli; Dai, Wei; Lu, Luo

    2013-01-01

    Objectives Pancreatic islet β-cell survival is important in regulating insulin activities and maintaining glucose homeostasis. Recently, Pax6 has been shown to be essential for many vital functions in β-cells, though the molecular mechanisms of its regulation in β-cells remain unclear. The present study investigates the novel effects of glucose- and insulin-induced CTCF activity on Pax6 gene expression as well as the subsequent effects of insulin-activated signaling pathways on β-cell proliferation. Material and methods Pancreatic β-TC-1-6 cells were cultured in DMEM medium and stimulated with high concentrations of glucose (5 to 125 mM) and cell viability was assessed by MTT assays. The effect of CTCF on Pax6 was evaluated in high glucose-induced and CCCTC-binding Factor (CTCF)/Erk suppressed cells by promoter reporter and Western analyses. Results Increases in glucose and insulin concentrations up-regulated CTCF and consequently down-regulated Pax6 in β-cell survival and proliferation. Knocking-down CTCF directly affected Pax6 transcription through CTCF binding and blocked the response to glucose. Altered Erk activity mediated the effects of CTCF on controlling Pax6 expression, which partially regulates β-cell proliferation. Conclusions CTCF functions as a molecular mediator between insulin-induced upstream Erk signaling and Pax6 expression in pancreatic β-cells. This pathway may contribute to regulation of β-cell survival and proliferation. PMID:24354619

  4. Cutting edge: Human regulatory T cells require IL-35 to mediate suppression and infectious tolerance.

    PubMed

    Chaturvedi, Vandana; Collison, Lauren W; Guy, Clifford S; Workman, Creg J; Vignali, Dario A A

    2011-06-15

    Human regulatory T cells (T(reg)) are essential for the maintenance of immune tolerance. However, the mechanisms they use to mediate suppression remain controversial. Although IL-35 has been shown to play an important role in T(reg)-mediated suppression in mice, recent studies have questioned its relevance in human T(reg). In this study, we show that human T(reg) express and require IL-35 for maximal suppressive capacity. Substantial upregulation of EBI3 and IL12A, but not IL10 and TGFB, was observed in activated human T(reg) compared with conventional T cells (T(conv)). Contact-independent T(reg)-mediated suppression was IL-35 dependent and did not require IL-10 or TGF-β. Lastly, human T(reg)-mediated suppression led to the conversion of the suppressed T(conv) into iTr35 cells, an IL-35-induced T(reg) population, in an IL-35-dependent manner. Thus, IL-35 contributes to human T(reg)-mediated suppression, and its conversion of suppressed target T(conv) into IL-35-induced T(reg) may contribute to infectious tolerance.

  5. Differential mechanisms of memory CD8 T cell maintenance by individual myeloid cell types

    PubMed Central

    Frasca, Loredana; Stonier, Spencer W.; Overwijk, Willem W.; Schluns, Kimberly S.

    2010-01-01

    This study tested the hypothesis that individual myeloid subsets have a differential ability to maintain memory CD8 T cells via IL-15. Although DCs support IL-15-mediated homeostasis of memory CD8 T cells in vivo, whether various DC subsets and other myeloid cells similarly mediate homeostasis is unknown. Therefore, we studied the ability of different myeloid cells to maintain memory CD8 T cells in vitro. Using an in vitro cocoulture system that recapitulated known roles of DCs and IL-15 on memory CD8 T cells, all in vitro-derived or ex vivo-isolated DCs maintained CD8 T cells better than rIL-15 alone, and FLT-3L-DCs are the most efficient compared with GM-DCs, BM-derived macrophages, or freshly isolated DCs. Although FLT-3L-DCs were the least effective at inducing CD8 T cell proliferation, FLT-3L-DCs promoted better CD8 T cell survival and increased Bcl-2 and MCL-2 expression in CD8 T cells. T cell maintenance correlated only partially with DC expression of IL-15Rα and IL-15, suggesting that DCs provided additional support signals. Indeed, in the absence of IL-15 signals, CD70/CD27 further supported CD8 T cell maintenance. IFN-α enhanced CD70 expression by DCs, resulting in increased proliferation of CD8 T cells. Overall, this study supports our hypothesis by demonstrating that specific DC subtypes had a greater capacity to support memory CD8 T cell maintenance and did so through dif