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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. Mechanisms of cytoskeleton-mediated mechanical signal transmission in cells

    PubMed Central

    Hwang, Yongyun; Gouget, Cecile L.M.; Barakat, Abdul I.

    2012-01-01

    Recent experiments have demonstrated very rapid long-distance transmission of mechanical forces within cells. Because the speed of this transmission greatly exceeds that of reaction-diffusion signaling, it has been conjectured that it occurs via the propagation of elastic waves through the actin stress fiber network. To explore the plausibility of this conjecture, we recently developed a model of small amplitude stress fiber deformations in prestressed viscoelastic stress fibers subjected to external forces. The model results demonstrated that rapid mechanical signal transmission is only possible when the external force is applied orthogonal to the stress fiber axis and that the dynamics of this transmission are governed by a balance between the prestress in the stress fiber and the stress fiber's material viscosity. The present study, which is a follow-up on our previous model, uses dimensional analysis to: (1) further evaluate the plausibility of the elastic wave conjecture and (2) obtain insight into mechanical signal transmission dynamics in simple stress fiber networks. We show that the elastic wave scenario is likely not the mechanism of rapid mechanical signal transmission in actin stress fibers due to the highly viscoelastic character of these fibers. Our analysis also demonstrates that the time constant characterizing mechanical stimulus transmission is strongly dependent on the topology of the stress fiber network, implying that network organization plays an important role in determining the dynamics of cellular responsiveness to mechanical stimulation. PMID:23336020

  3. Molecular Mechanisms of Treg-Mediated T Cell Suppression

    PubMed Central

    Schmidt, Angelika; Oberle, Nina; Krammer, Peter H.

    2012-01-01

    CD4+CD25highFoxp3+ regulatory T cells (Tregs) can suppress other immune cells and, thus, are critical mediators of peripheral self-tolerance. On the one hand, Tregs avert autoimmune disease and allergies. On the other hand, Tregs can prevent immune reactions against tumors and pathogens. Despite the importance of Tregs, the molecular mechanisms of suppression remain incompletely understood and controversial. Proliferation and cytokine production of CD4+CD25− conventional T cells (Tcons) can be inhibited directly by Tregs. In addition, Tregs can indirectly suppress Tcon activation via inhibition of the stimulatory capacity of antigen presenting cells. Direct suppression of Tcons by Tregs can involve immunosuppressive soluble factors or cell contact. Different mechanisms of suppression have been described, so far with no consensus on one universal mechanism. Controversies might be explained by the fact that different mechanisms may operate depending on the site of the immune reaction, on the type and activation state of the suppressed target cell as well as on the Treg activation status. Further, inhibition of T cell effector function can occur independently of suppression of proliferation. In this review, we summarize the described molecular mechanisms of suppression with a particular focus on suppression of Tcons and rapid suppression of T cell receptor-induced calcium (Ca2+), NFAT, and NF-κB signaling in Tcons by Tregs. PMID:22566933

  4. Mechanisms of gold nanoparticle mediated ultrashort laser cell membrane perforation

    NASA Astrophysics Data System (ADS)

    Schomaker, M.; Baumgart, J.; Motekaitis, D.; Heinemann, D.; Krawinkel, J.; Pangalos, M.; Bintig, W.; Boulais, E.; Lachaine, R.; St.-Louis Lalonde, B.; Ngezahayo, A.; Meunier, M.; Heisterkamp, A.

    2011-03-01

    The gold nanoparticle (AuNP) mediated ultrashort laser cell membrane perforation has been proven as an efficient delivery method to bring membrane impermeable molecules into the cytoplasm. Nevertheless, the underlying mechanisms have not been fully determined yet. Different effects may occur when irradiating a AuNP with ultrashort laser pulses and finally enable the molecule to transfer. Depending on the parameters (pulse length, laser fluence and wavelength, particle size and shape, etc.) light absorption or an enhanced near field scattering can lead to perforation of the cell membrane when the particle is in close vicinity. Here we present our experimental results to clarify the perforation initiating mechanisms. The generation of cavitation and gas bubbles due to the laser induced effects were observed via time resolved imaging. Additionally, pump-probe experiments for bubble detection was performed. Furthermore, in our patch clamp studies a depolarization of the membrane potential and the current through the membrane of AuNP loaded cell during laser treatment was detected. This indicates an exchange of extra- and intra cellular ions trough the perforated cell membrane for some milliseconds. Additionally investigations by ESEM imaging were applied to study the interaction of cells and AuNP after co incubation. The images show an attachment of AuNP at the cell membrane after several hours of incubation. Moreover, images of irradiated and AuNP loaded cells were taken to visualize the laser induced effects.

  5. Controlling Stem Cell-mediated Bone Regeneration through Tailored Mechanical Properties of Collagen Scaffolds

    PubMed Central

    Sun, Hongli; Zhu, Feng; Hu, Qingang; Krebsbach, Paul H.

    2014-01-01

    Mechanical properties of the extracellular matrix (ECM) play an essential role in cell fate determination. To study the role of mechanical properties of ECM in stem cell-mediated bone regeneration, we used a 3D in vivo ossicle model that recapitulates endochondral bone formation. Three-dimensional gelatin scaffolds with distinct stiffness were developed using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) mediated zero-length crosslinking. The mechanical strength of the scaffolds was significantly increased by EDC treatment, while the microstructure of the scaffold was preserved. Cell behavior on the scaffolds with different mechanical properties was evaluated in vitro and in vivo. EDC-treated scaffolds promoted early chondrogenic differentiation, while it promoted both chondrogenic and osteogenic differentiation at later time points. Both micro-computed tomography and histologic data demonstrated that EDC-treatment significantly increased trabecular bone formation by transplanted cells transduced with AdBMP. Moreover, significantly increased chondrogenesis was observed in the EDC-treated scaffolds. Based on both in vitro and in vivo data, we conclude that the high mechanical strength of 3D scaffolds promoted stem cell mediated bone regeneration by promoting endochondral ossification. These data suggest a new method for harnessing stem cells for bone regeneration in vivo by tailoring the mechanical properties of 3D scaffolds. PMID:24211076

  6. Molecular mechanisms mediating metastasis of hypoxic breast cancer cells.

    PubMed

    Semenza, Gregg L

    2012-09-01

    Breast cancers contain regions of intratumoral hypoxia in which reduced O(2) availability activates the hypoxia-inducible factors HIF-1 and HIF-2, which increase the transcription of genes encoding proteins that are required for many important steps in cancer progression. Recently, HIFs have been shown to play critical roles in the metastasis of breast cancer to the lungs through the transcriptional activation of genes encoding angiopoietin-like 4 and L1 cell adhesion molecule, which promote the extravasation of circulating cancer cells from the lung vasculature, and the lysyl oxidase family members LOX, LOXL2, and LOXL4, which promote invasion and metastatic niche formation. Digoxin, a drug that inhibits HIF-1 activity, blocks primary tumor growth, vascularization, invasion, and metastasis in ex vivo and in vivo assays. PMID:22921864

  7. 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. PMID:26947806

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

    PubMed

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

    2016-01-18

    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

  9. 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

  10. Epigenetic mechanisms of cell adhesion-mediated drug resistance in multiple myeloma.

    PubMed

    Furukawa, Yusuke; Kikuchi, Jiro

    2016-09-01

    Multiple myeloma cells acquire the resistance to anti-cancer drugs through physical and functional interactions with the bone marrow microenvironment via two overlapping mechanisms. First, bone marrow stromal cells (BMSCs) produce soluble factors, such as interleukin-6 and insulin-like growth factor-1, to activate signal transduction pathways leading to drug resistance (soluble factor-mediated drug resistance). Second, BMSCs up-regulate the expression of cell cycle inhibitors, anti-apoptotic members of the Bcl-2 family and ABC drug transporters in myeloma cells upon direct adhesion [cell adhesion-mediated drug resistance (CAM-DR)]. Elucidation of the mechanisms underlying drug resistance may greatly contribute to the advancement of cancer therapies. Recent investigations, including ours, have revealed the involvement of epigenetic alterations in drug resistance especially CAM-DR. For example, we found that class I histone deacetylases (HDACs) determine the sensitivity of proteasome inhibitors and the histone methyltransferase EZH2 regulates the transcription of anti-apoptotic genes during the acquisition of CAM-DR by myeloma cells. In addition, another histone methyltransferase MMSET was shown to confer drug resistance to myeloma cells by facilitating DNA repair. These findings provide a rationale for the inclusion of epigenetic drugs, such as HDAC inhibitors and histone methylation modifiers, in combination chemotherapy for MM patients to increase the therapeutic index. PMID:27411688

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. A Dystroglycan/Plectin Scaffold Mediates Mechanical Pathway Bifurcation in Lung Epithelial Cells*

    PubMed Central

    Takawira, Desire; Budinger, G. R. Scott; Hopkinson, Susan B.; Jones, Jonathan C. R.

    2011-01-01

    In alveolar epithelial cells (AECs), the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the ERK1/2 and the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling cascades in a process called pathway bifurcation. We tested the hypothesis that the cytoskeleton cross-linker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. We demonstrate that plectin and DG form a complex in AECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in AECs deficient in plectin. Thus, DG has the dual role of mechanical receptor and scaffold for ERK1/2, whereas plectin acts as a scaffold for AMPK signaling but is also required for DG-mediated ERK1/2 activation. We conclude that the DG-plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix to the cytoplasm. PMID:21149456

  17. 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...

  18. Cancer resistance in the blind mole rat is mediated by concerted necrotic cell death mechanism.

    PubMed

    Gorbunova, Vera; Hine, Christopher; Tian, Xiao; Ablaeva, Julia; Gudkov, Andrei V; Nevo, Eviatar; Seluanov, Andrei

    2012-11-20

    Blind mole rats Spalax (BMR) are small subterranean rodents common in the Middle East. BMR is distinguished by its adaptations to life underground, remarkable longevity (with a maximum documented lifespan of 21 y), and resistance to cancer. Spontaneous tumors have never been observed in spalacids. To understand the mechanisms responsible for this resistance, we examined the growth of BMR fibroblasts in vitro of the species Spalax judaei and Spalax golani. BMR cells proliferated actively for 7-20 population doublings, after which the cells began secreting IFN-β, and the cultures underwent massive necrotic cell death within 3 d. The necrotic cell death phenomenon was independent of culture conditions or telomere shortening. Interestingly, this cell behavior was distinct from that observed in another long-lived and cancer-resistant African mole rat, Heterocephalus glaber, the naked mole rat in which cells display hypersensitivity to contact inhibition. Sequestration of p53 and Rb proteins using SV40 large T antigen completely rescued necrotic cell death. Our results suggest that cancer resistance of BMR is conferred by massive necrotic response to overproliferation mediated by p53 and Rb pathways, and triggered by the release of IFN-β. Thus, we have identified a unique mechanism that contributes to cancer resistance of this subterranean mammal extremely adapted to life underground. PMID:23129611

  19. 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

  20. Ubiquitin-proteasome-mediated degradation of keratin intermediate filaments in mechanically stimulated A549 cells.

    PubMed

    Jaitovich, Ariel; Mehta, Semil; Na, Ni; Ciechanover, Aaron; Goldman, Robert D; Ridge, Karen M

    2008-09-12

    We previously reported that shear stress induces phosphorylation and disassembly of keratin intermediate filaments (IFs). Shear stress also induces a time- and strain-dependent degradation of keratin IFs, and the current study examines the mechanisms involved in degradation of keratin proteins in human A549 cells exposed to 0-24 h of shear stress (7.5-30 dynes/cm(2)). Ubiquitin was found to be covalently associated with keratin proteins immunoprecipitated from shear-stressed cells, and pretreatment with the proteasomal inhibitor MG132 prevented the degradation of the keratin IF network. Importantly, phosphorylation of K8 Ser-73 is required for the shear stress-mediated ubiquitination, disassembly, and degradation of the keratin IF network. Immunofluorescence microscopy revealed that shear stress caused the thin array of keratin fibrils observed in control cells to be reorganized into a perinuclear aggregate, known as an aggresome, and that ubiquitin was also associated with this structure. Finally, the E2 enzymes, UbcH5b, -c, and Ubc3, but not E2-25K are required for the shear stress-mediated ubiquitin-proteasomal degradation of keratin proteins. These data suggest that shear stress promotes the disassembly and degradation of the keratin IF network via phosphorylation and the ubiquitin-proteasome pathway. PMID:18617517

  1. 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

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

    PubMed

    Ruhe, Zachary C; Nguyen, Josephine Y; Chen, Annette J; Leung, Nicole Y; Hayes, Christopher S; Low, David A

    2016-06-01

    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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Mechanism of lymphocyte-mediated cytolysis: functional cytolytic T cells lacking perforin and granzymes.

    PubMed Central

    Berke, G; Rosen, D; Ronen, D

    1993-01-01

    Involvement of the lytic protein perforin (c. 65,000 MW) and of granule proteases (granzymes) in cell lysis induced by cytolytic T lymphocytes (CTL) has been suggested, but is still controversial. For example, in vivo-primed peritoneal exudate CTL (PEL) have been found to express perforin and granzyme activity in amounts comparable to those found in non-lytic lymphocytes, although PEL are the most potent of all CTL. Exploiting several cloned CTL hybridomas developed in this laboratory and newly available molecular probes for detecting perforin, granzymes, protein and mRNA, we now directly demonstrate killer T lymphocytes which kill effectively and specifically, but are free from perforin, lytic granules and granzymes, all three of which have been postulated to be involved in lymphocyte-mediated killing. The CTL hybridomas are completely devoid of perforin and granzymes prior to, during, and after activation by antigen, mitogen or interleukin-2 (IL-2). The induction of lytic granules, perforin, and granzymes in the in vivo-primed PEL, but not in the cloned CTL hybridomas, upon cultivation in IL-2, further suggests the involvement of these constituents in antigen/lymphokine-induced CTL activation and differentiation rather than directly in their cytocidal activity. Together, these findings support a perforin- and granzyme-independent CTL lytic mechanism. Images Figure 1 Figure 2 PMID:8436395

  11. 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

  12. Mechanisms of Contact-Mediated Killing of Yeast Cells on Dry Metallic Copper Surfaces▿

    PubMed Central

    Quaranta, Davide; Krans, Travis; Santo, Christophe Espírito; Elowsky, Christian G.; Domaille, Dylan W.; Chang, Christopher J.; Grass, Gregor

    2011-01-01

    Surfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivate Candida albicans and Saccharomyces cerevisiae within minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay with Saccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC2(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated. PMID:21097600

  13. 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

  14. 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

  15. 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

  16. CD8+ T Cell-Independent Immune-Mediated Mechanisms of Anti-Tumor Activity

    PubMed Central

    Pluhar, G. Elizabeth; Pennell, Christopher A.; Olin, Michael R.

    2016-01-01

    Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Cancer immunotherapy seeks to recruit an effective immune response to eliminate tumor cells. To date, cancer vaccines have shown only limited effectiveness because of our incomplete understanding of the necessary effector cells and mechanisms that yield efficient tumor clearance. CD8+ T cell cytotoxic activity has long been proposed as the primary effector function necessary for tumor regression. However, there is increasing evidence that indicates that components of the immune system other than CD8+ T cells play important roles in tumor eradication and control. The following review should provide an understanding of the mechanisms involved in an effective antitumor response to guide future therapeutic designs. The information provided suggests an alternate means of effective tumor clearance in malignant glioma to the canonical CD8+ cytotoxic T cell mechanism. PMID:26351148

  17. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells

    PubMed Central

    Geisen, Ulf; Zenthoefer, Marion; Peipp, Matthias; Kerber, Jannik; Plenge, Johannes; Managò, Antonella; Fuhrmann, Markus; Geyer, Roland; Hennig, Steffen; Adam, Dieter; Piker, Levent; Rimbach, Gerald; Kalthoff, Holger

    2015-01-01

    Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application. PMID:26204945

  18. Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells.

    PubMed

    Geisen, Ulf; Zenthoefer, Marion; Peipp, Matthias; Kerber, Jannik; Plenge, Johannes; Managò, Antonella; Fuhrmann, Markus; Geyer, Roland; Hennig, Steffen; Adam, Dieter; Piker, Levent; Rimbach, Gerald; Kalthoff, Holger

    2015-07-01

    Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application. PMID:26204945

  19. The role of T-cell-mediated mechanisms in virus infections of the nervous system.

    PubMed

    Dörries, R

    2001-01-01

    during or shortly after exerting their effector functions. The clinical consequences and the influence of the effector phase on the further course of the infection depends on the balance and fine-tuning of the contributing lymphoid cell populations. Generally, any delay in the recruitment of effector lymphocytes to the tissue or an unbalanced combination of lymphocyte subsets allows the virus to spread in the CNS, which in turn will cause severe immune-mediated tissue effects as well as disease. If either too late or partially deficient, the immune system response may contribute to a lethal outcome or cause autosensitization to brain-specific antigens by epitope spreading to the antigen-presenting system in peripheral lymphoid tissue. This could form the basis for subsequent booster reactions of autosensitized CD4+ T cells--a process that finally will end in an inflammatory autoimmune reaction, which in humans we call multiple sclerosis. In contrast, a rapid and specific local response in the brain tissue will result in efficient limitation of viral spread and thereby a subclinical immune system-mediated termination of the infection. After clearance of virus-infected cells, downsizing of the local response probably occurs via self-elimination of the contributing T cell populations and/or by so far unidentified signal pathways. However, much of this is highly speculative, and more data have to be collected to make decisive conclusions regarding this matter. Several strategies have been developed by viruses to escape T cell-mediated eradication, including interference with the MHC class I presentation pathway of the host cell or "hiding" in cells which lack MHC class I expression. This may result in life-long persistence of the virus in the brain, a state which probably is actively controlled by T lymphocytes. Under severe immunosuppression, however, reactivation of viral replication can occur, which is a lethal threat to the host. PMID:11417137

  20. iTRAQ-based proteomic analysis reveals the mechanisms of silicon-mediated cadmium tolerance in rice (Oryza sativa) cells.

    PubMed

    Ma, Jie; Sheng, Huachun; Li, Xiuli; Wang, Lijun

    2016-07-01

    Silicon (Si) can alleviate cadmium (Cd) stress in rice (Oryza sativa) plants, however, the understanding of the molecular mechanisms at the single-cell level remains limited. To address these questions, we investigated suspension cells of rice cultured in the dark environment in the absence and presence of Si with either short- (12 h) or long-term (5 d) Cd treatments using a combination of isobaric tags for relative and absolute quantitation (iTRAQ), fluorescent staining, and inductively coupled plasma mass spectroscopy (ICP-MS). We identified 100 proteins differentially regulated by Si under the short- or long-term Cd stress. 70% of these proteins were down-regulated, suggesting that Si may improve protein use efficiency by maintaining cells in the normal physiological status. Furthermore, we showed two different mechanisms for Si-mediated Cd tolerance. Under the short-term Cd stress, the Si-modified cell walls inhibited the uptake of Cd ions into cells and consequently reduced the expressions of glycosidase, cell surface non-specific lipid-transfer proteins (nsLTPs), and several stress-related proteins. Under the long-term Cd stress, the amount of Cd in the cytoplasm in Si-accumulating (+Si) cells was decreased by compartmentation of Cd into vacuoles, thus leading to a lower expression of glutathione S-transferases (GST). These results provide protein-level insights into the Si-mediated Cd detoxification in rice single cells. PMID:27017433

  1. 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

  2. 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

  3. 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

  4. 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

  5. Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms.

    PubMed

    Chang, H C; Lee, T H; Chuang, L Y; Yen, M H; Hung, W C

    1999-10-18

    The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21-/- cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine specifically inhibited cyclin D1 expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21CIP1 expression in H226 and H358 cells, while it activated p27KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because significant changes in cyclin D1 or CDKI expression were observed at 12-24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control. PMID:10530763

  6. 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

  7. Signalling mechanism for somatostatin receptor 5-mediated suppression of AMPA responses in rat retinal ganglion cells.

    PubMed

    Deng, Qin-Qin; Sheng, Wen-Long; Zhang, Gong; Weng, Shi-Jun; Yang, Xiong-Li; Zhong, Yong-Mei

    2016-08-01

    Somatostatin (SRIF) is involved in a variety of physiological functions via the activation of five subtypes of specific receptors (sst1-5). Here, we investigated the effects of SRIF on AMPA receptor (AMPAR)-mediated currents (AMPA currents) in isolated rat retinal ganglion cells (GCs) using patch-clamp techniques. Immunofluorescence double labelling demonstrated the expression of sst5 in rat GCs. Consistent to this, whole cell AMPA currents of GCs were dose-dependently suppressed by SRIF, and the effect was reversed by the sst5 antagonist BIM-23056. Intracellular dialysis of GDP-β-S or pre-incubation with the Gi/o inhibitor pertussis toxin (PTX) abolished the SRIF effect. The SRIF effect was mimicked by the administration of either 8-Br-cAMP or forskolin, but was eliminated by the protein kinase A (PKA) antagonists H-89/KT5720/Rp-cAMP. Moreover, SRIF increased intracellular Ca(2+) levels and did not suppress the AMPA currents when GCs were infused with an intracellular Ca(2+)-free solution or in the presence of ryanodine receptor modulators caffeine/ryanodine. Furthermore, the SRIF effect was eliminated when the activity of calmodulin (CaM), calcineurin and protein phosphatase 1 (PP1) was blocked with W-7, FK-506 and okadaic acid, respectively. SRIF persisted to suppress the AMPA currents when cGMP-protein kinase G (PKG) and phosphatidylinositol (PI)-/phosphatidylcholine (PC)-phospholipase C (PLC) signalling pathways were blocked. In rat flat-mount retinas, SRIF suppressed AMPAR-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) in GCs. We conclude that a distinct Gi/o/cAMP-PKA/ryanodine/Ca(2+)/CaM/calcineurin/PP1 signalling pathway comes into play due to the activation of sst5 to mediate the SRIF effect on GCs. PMID:26969240

  8. 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

  9. 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

  10. Cytotoxicity mechanism of α-MMC in normal liver cells through LRP1 mediated endocytosis and JNK activation.

    PubMed

    Wang, Ling; Shen, Fubing; Zhang, Min; He, Qianchuan; Zhao, Hui; Yu, Xiaoping; Yang, Shuxia; Liu, Yang; Deng, Nianhua; Zheng, Juecun; Zhu, Lixia; Liu, Xiaolan

    2016-05-16

    Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein isolated from Momordica charantia, is a potential drug candidate with strong anti-tumor activity. However, α-MMC has a severe hepatotoxicity when applied in vivo, which may greatly hinders its use in clinic in the future. The biological mechanism of hepatotoxicity induced by α-MMC is largely unknown, especially the mechanism by which α-MMC enters the hepatocytes. In this study, we investigated α-MMC-induced cytotoxicity in normal liver L02 cell line as well as the mechanism underlying it. As expected, α-MMC is more toxic in L02 cells than in various normal cells from other organs. The cytotoxic effect of α-MMC on L02 cells is found to be mediated through cell apoptosis as detected by flow cytometry and fluorescence microscopy. Importantly, α-MMC was shown to bind to a specific receptor on cell membrane, as the density of the cell membrane receptor is closely related to both the amount of α-MMC endocytosed and the cytotoxicity in different cell lines. By using LRP1 competitive inhibitor α2-M or siRNA targeting LRP1, we further identified that LRP1 protein served as the membrane receptor for α-MMC. Both α2-M and siRNA targeting LRP1 can significantly inhibit α-MMC's endocytosis as well as its cytotoxicity in L02 cells. In addition, it was found that α-MMC can activate the JNK signalling pathways via LRP1 in L02 cells. As JNK activation often leads to cell apoptosis, the activation of JNK may play an important role in α-MMC-induced cytotoxicity. To our knowledge, this is the first report showing that LRP1 mediates the cytotoxicity of α-MMC through (1) endocytosis and induced apoptosis and (2) the activation of the JNK pathway. Our findings shed light on the fundamental mechanism of hepatotoxicity of α-MMC and offer reference to understand its mechanism of lymphocytotoxicity and neurotoxicity. PMID:27262837

  11. Notch signaling enhances FcεRI-mediated cytokine production by mast cells through direct and indirect mechanisms.

    PubMed

    Nakano, Nobuhiro; Nishiyama, Chiharu; Yagita, Hideo; Hara, Mutsuko; Motomura, Yasutaka; Kubo, Masato; Okumura, Ko; Ogawa, Hideoki

    2015-05-01

    Th2-type cytokines and TNF-α secreted by activated mast cells upon cross-linking of FcεRI contribute to the development and maintenance of Th2 immunity to parasites and allergens. We have previously shown that cytokine secretion by mouse mast cells is enhanced by signaling through Notch receptors. In this study, we investigated the molecular mechanisms by which Notch signaling enhances mast cell cytokine production induced by FcεRI cross-linking. FcεRI-mediated production of cytokines, particularly IL-4, was significantly enhanced in mouse bone marrow-derived mast cells by priming with Notch ligands. Western blot analysis showed that Notch signaling augmented and prolonged FcεRI-mediated phosphorylation of MAPKs, mainly JNK and p38 MAPK, through suppression of the expression of SHIP-1, a master negative regulator of FcεRI signaling, resulting in the enhanced production of multiple cytokines. The enhancing effect of Notch ligand priming on multiple cytokine production was abolished by knockdown of Notch2, but not Notch1, and FcεRI-mediated production of multiple cytokines was enhanced by retroviral transduction with the intracellular domain of Notch2. However, only IL-4 production was enhanced by both Notch1 and Notch2. The enhancing effect of Notch signaling on IL-4 production was lost in bone marrow-derived mast cells from mice lacking conserved noncoding sequence 2, which is located at the distal 3' element of the Il4 gene locus and contains Notch effector RBP-J binding sites. These results indicate that Notch2 signaling indirectly enhances the FcεRI-mediated production of multiple cytokines, and both Notch1 and Notch2 signaling directly enhances IL-4 production through the noncoding sequence 2 enhancer of the Il4 gene. PMID:25821223

  12. Hic-5 mediates TGFβ-induced adhesion in vascular smooth muscle cells by a Nox4-dependent mechanism

    PubMed Central

    Fernandez, Isabel; Martin-Garrido, Abel; Zhou, Dennis W.; Clempus, Roza E.; Seidel-Rogol, Bonnie; Valdivia, Alejandra; Lassègue, Bernard; García, Andrés J.; Griendling, Kathy K.; Martin, Alejandra San

    2015-01-01

    Objective Focal adhesions (FAs) link the cytoskeleton to the extracellular matrix and as such play important roles in growth, migration and contractile properties of vascular smooth muscle cells (VSMCs). Recently, it has been shown that downregulation of Nox4, a transforming growth factor beta (TGFβ)-inducible, H2O2-producing enzyme, affects the number of FAs. However, the effectors downstream of Nox4 that mediate FA regulation are unknown. The FA resident protein hydrogen peroxide-inducible clone-5 (Hic-5) is H2O2- and TGFβ-inducible, and a binding partner of the heat shock protein (Hsp)27. The objective of this study was to elucidate the mechanism by which Hic-5 and Hsp27 participate in TGFβ-induced, Nox4-mediated VSMC adhesion and migration. Approach and Results Through a combination of molecular biology and biochemistry techniques, we found that TGFβ, by a Nox4-dependent mechanism, induces the expression and interaction of Hic-5 and Hsp27, which is essential for Hic-5 localization to FAs. Importantly, we found that Hic-5 expression is required for the TGFβ-mediated increase in FA number, and adhesive forces and migration. Mechanistically, Nox4 downregulation impedes Smad signaling by TGFβ, and Hsp27 and Hic-5 upregulation by TGFβ is blocked in Smad4-deficient cells. Conclusions Hic-5 and Hsp27 are effectors of Nox4 required for TGFβ-stimulated FA formation and adhesion strength and migration in VSMC. PMID:25814672

  13. CD11b+Ly6G- myeloid cells mediate mechanical inflammatory pain hypersensitivity.

    PubMed

    Ghasemlou, Nader; Chiu, Isaac M; Julien, Jean-Pierre; Woolf, Clifford J

    2015-12-01

    Pain hypersensitivity at the site of inflammation as a result of chronic immune diseases, pathogenic infection, and tissue injury is a common medical condition. However, the specific contributions of the innate and adaptive immune system to the generation of pain during inflammation have not been systematically elucidated. We therefore set out to characterize the cellular and molecular immune response in two widely used preclinical models of inflammatory pain: (i) intraplantar injection of complete Freund's adjuvant (CFA) as a model of adjuvant- and pathogen-based inflammation and (ii) a plantar incisional wound as a model of tissue injury-based inflammation. Our findings reveal differences in temporal patterns of immune cell recruitment and activation states, cytokine production, and pain in these two models, with CFA causing a nonresolving granulomatous inflammatory response whereas tissue incision induced resolving immune and pain responses. These findings highlight the significant differences and potential clinical relevance of the incisional wound model compared with the CFA model. By using various cell-depletion strategies, we find that, whereas lymphocyte antigen 6 complex locus G (Ly)6G(+)CD11b(+) neutrophils and T-cell receptor (TCR) β(+) T cells do not contribute to the development of thermal or mechanical pain hypersensitivity in either model, proliferating CD11b(+)Ly6G(-) myeloid cells were necessary for mechanical hypersensitivity during incisional pain, and, to a lesser extent, CFA-induced inflammation. However, inflammatory (CCR2(+)Ly6C(hi)) monocytes were not responsible for these effects. The finding that a population of proliferating CD11b(+)Ly6G(-) myeloid cells contribute to mechanical inflammatory pain provides a potential cellular target for its treatment in wound inflammation. PMID:26598697

  14. CD11b+Ly6G− myeloid cells mediate mechanical inflammatory pain hypersensitivity

    PubMed Central

    Ghasemlou, Nader; Chiu, Isaac M.; Julien, Jean-Pierre; Woolf, Clifford J.

    2015-01-01

    Pain hypersensitivity at the site of inflammation as a result of chronic immune diseases, pathogenic infection, and tissue injury is a common medical condition. However, the specific contributions of the innate and adaptive immune system to the generation of pain during inflammation have not been systematically elucidated. We therefore set out to characterize the cellular and molecular immune response in two widely used preclinical models of inflammatory pain: (i) intraplantar injection of complete Freund’s adjuvant (CFA) as a model of adjuvant- and pathogen-based inflammation and (ii) a plantar incisional wound as a model of tissue injury-based inflammation. Our findings reveal differences in temporal patterns of immune cell recruitment and activation states, cytokine production, and pain in these two models, with CFA causing a nonresolving granulomatous inflammatory response whereas tissue incision induced resolving immune and pain responses. These findings highlight the significant differences and potential clinical relevance of the incisional wound model compared with the CFA model. By using various cell-depletion strategies, we find that, whereas lymphocyte antigen 6 complex locus G (Ly)6G+CD11b+ neutrophils and T-cell receptor (TCR) β+ T cells do not contribute to the development of thermal or mechanical pain hypersensitivity in either model, proliferating CD11b+Ly6G− myeloid cells were necessary for mechanical hypersensitivity during incisional pain, and, to a lesser extent, CFA-induced inflammation. However, inflammatory (CCR2+Ly6Chi) monocytes were not responsible for these effects. The finding that a population of proliferating CD11b+Ly6G− myeloid cells contribute to mechanical inflammatory pain provides a potential cellular target for its treatment in wound inflammation. PMID:26598697

  15. 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

  16. Different immune cells mediate mechanical pain hypersensitivity in male and female mice

    PubMed Central

    Sorge, Robert E.; Mapplebeck, Josiane C.S.; Rosen, Sarah; Beggs, Simon; Taves, Sarah; Alexander, Jessica K.; Martin, Loren J.; Austin, Jean-Sebastien; Sotocinal, Susana G.; Chen, Di; Yang, Mu; Shi, Xiang Qun; Huang, Hao; Pillon, Nicolas J.; Bilan, Philip J.; Tu, Yu Shan; Klip, Amira; Ji, Ru-Rong; Zhang, Ji; Salter, Michael W.; Mogil, Jeffrey S.

    2016-01-01

    A large and rapidly increasing body of evidence indicates that microglia-neuron signaling is essential for chronic pain hypersensitivity. Here we show using multiple approaches that microglia are not required for mechanical pain hypersensitivity in female mice; female mice achieve similar levels of pain hypersensitivity using adaptive immune cells, likely T-lymphocytes. This sexual dimorphism suggests that male mice cannot be used as proxies for females in pain research. PMID:26120961

  17. 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

  18. ATP release mechanisms of endothelial cell-mediated stimulus-dependent hyperalgesia

    PubMed Central

    Joseph, Elizabeth K.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTRinh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes. PMID:24793242

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

    PubMed Central

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

    2015-01-01

    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. PMID:26446836

  20. Inter-cellular nanovesicle mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth

    PubMed Central

    Kogure, Takayuki; Lin, Wen-Lang; Yan, Irene K.; Braconi, Chiara; Patel, Tushar

    2011-01-01

    Hepatocellular carcinoma (HCC) is characterized by a propensity for multifocality, growth by local spread, and dysregulation of multiple signaling pathways. These features may be determined by the tumoral microenvironment. The potential of tumor cells to modulate HCC growth and behavior by secreted proteins has been extensively studied. In contrast the potential for genetic modulation is poorly understood. We investigated the role and involvement of tumor derived nanovesicles capable of altering gene expression, and characterized their ability to modulate cell signaling and biological effects in other cells. We show that HCC cells can produce nanovesicles, exosomes, that differ in both RNA and protein content from their cells of origin. These can be taken up and internalized by other cells, and can transmit a functional transgene. The microRNA content of these exosomes was examined, and a subset that is highly enriched within exosomes was identified. A combinatorial approach to identify potential targets identified transforming growth factor β activated kinase-1 (TAK1) as the most likely candidate pathway that could be modulated by these miRNA. Loss of TAK1 has been implicated in hepatocarcinogenesis and is a biologically plausible target for inter-cellular modulation. We showed that HCC cell derived exosomes can modulate TAK1 expression and associated signaling and enhance transformed cell growth in recipient cells. Conclusion: Exosome mediated miRNA transfer is an important mechanism of inter-cellular communication in HCC cells. These observations identify a unique inter-cellular mechanism that could potentially contribute to local spread, intrahepatic metastases or multifocal growth in HCC. PMID:21721029

  1. 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

  2. Telaprevir may induce adverse cutaneous reactions by a T cell immune-mediated mechanism.

    PubMed

    Federico, Alessandro; Aitella, Ernesto; Sgambato, Dolores; Savoia, Alfonso; De Bartolomeis, Fabio; Dallio, Marcello; Ruocco, Eleonora; Pezone, Luciano; Abbondanza, Ciro; Loguercio, Carmela; Astarita, Corrado

    2015-01-01

    The HCV protease inhibitor telaprevir associated with peginterferon-alpha and ribavirin, was widely used in the recent past as standard treatment in HCV genotype-1 infected patients. Telaprevir improves the sustained virology response rates, but at the same time increases the frequency of adverse cutaneous reactions. However, mechanisms through which telaprevir induces cutaneous lesions are not yet defined. A 50-year-old woman, affected by HCV genotype 1b, was admitted to our Department for a telaprevir-related severe cutaneous eruptions, eight weeks after starting a triple therapy (telaprevir associated with Peginterferon-alpha and ribavirin). Mechanisms of cutaneous reactions were investigated by skin tests with non-irritating concentrations of telaprevir and by activating in vitro T lymphocyte with different concentrations. Immediate and delayed responses to skin testing were negative, but the drug-induced lymphocytes activation was significantly higher as compared to patient's baseline values and to parallel results obtained in three healthy subjects (p < 0.05). In conclusion, adverse cutaneous reactions of our patient were caused by a telaprevir-induced T-cell dependent immune mechanism. PMID:25864225

  3. 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

  4. 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

  5. Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells.

    PubMed

    Jain, Aklank; Magistri, Marco; Napoli, Sara; Carbone, Giuseppina M; Catapano, Carlo V

    2010-03-01

    Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells. PMID:20045441

  6. 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.

  7. Induction of Osteogenic Differentiation of Adipose Derived Stem Cells by Microstructured Nitinol Actuator-Mediated Mechanical Stress

    PubMed Central

    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. PMID:23236461

  8. Vascular patterning regulates interdigital cell death by a ROS-mediated mechanism.

    PubMed

    Eshkar-Oren, Idit; Krief, Sharon; Ferrara, Napoleone; Elliott, Alison M; Zelzer, Elazar

    2015-02-15

    Blood vessels serve as key regulators of organogenesis by providing oxygen, nutrients and molecular signals. During limb development, programmed cell death (PCD) contributes to separation of the digits. Interestingly, prior to the onset of PCD, the autopod vasculature undergoes extensive patterning that results in high interdigital vascularity. Here, we show that in mice, the limb vasculature positively regulates interdigital PCD. In vivo, reduction in interdigital vessel number inhibited PCD, resulting in syndactyly, whereas an increment in vessel number and distribution resulted in elevation and expansion of PCD. Production of reactive oxygen species (ROS), toxic compounds that have been implicated in PCD, also depended on interdigital vascular patterning. Finally, ex vivo incubation of limbs in gradually decreasing oxygen levels led to a correlated reduction in both ROS production and interdigital PCD. The results support a role for oxygen in these processes and provide a mechanistic explanation for the counterintuitive positive role of the vasculature in PCD. In conclusion, we suggest a new role for vascular patterning during limb development in regulating interdigital PCD by ROS production. More broadly, we propose a double safety mechanism that restricts PCD to interdigital areas, as the genetic program of PCD provides the first layer and vascular patterning serves as the second. PMID:25617432

  9. 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

  10. MDM2-Mediated Degradation of p14ARF: A Novel Mechanism to Control ARF Levels in Cancer Cells

    PubMed Central

    Vivo, Maria; Matarese, Maria; Sepe, Maria; Di Martino, Rosaria; Festa, Luisa; Calabrò, Viola; Mantia, Girolama La; Pollice, Alessandra

    2015-01-01

    We here show a new relationship between the human p14ARF oncosuppressor and the MDM2 oncoprotein. MDM2 overexpression in various cancer cell lines causes p14ARF reduction inducing its degradation through the proteasome. The effect does not require the ubiquitin ligase activity of MDM2 and preferentially occurs in the cytoplasm. Interestingly, treatment with inhibitors of the PKC (Protein Kinase C) pathway and use of p14ARF phosphorylation mutants indicate that ARF phosphorylation could play a role in MDM2 mediated ARF degradation reinforcing our previous observations that ARF phosphorylation influences its stability and biological activity. Our study uncovers a new potentially important mechanism through which ARF and MDM2 can counterbalance each other during the tumorigenic process. PMID:25723571

  11. 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.

  12. 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

  13. High-mobility group boxes mediate cell proliferation and radiosensitivity via retinoblastoma-interaction-dependent and -independent mechanisms.

    PubMed

    Wang, Li-Li; Meng, Qing-Hui; Jiao, Yang; Xu, Jia-Ying; Ge, Chun-Min; Zhou, Ju-Ying; Rosen, Eliot M; Wang, Hai-Chao; Fan, Sai-Jun

    2012-06-01

    Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1-3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2-3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation-Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1-4 interactions. Enforced expression of HMGB1-3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1-4 association and modulation of cancer cell growth, but not radiosensitivity. PMID:22655796

  14. A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1.

    PubMed Central

    Wesseling, J; van der Valk, S W; Hilkens, J

    1996-01-01

    Episialin (MUC1, PEM, EMA, CA15-3 antigen) is a sialylated, membrane-associated glycoprotein with an extended mucin-like ectodomain. This domain mainly consists of 30-90 homologous 20-amino acid repeats that are rich in O-glycosylation sites (serines and threonines). It is likely that this part forms a polyproline beta-turn helix. As a result, the ectodomain can protrude more than 200 nm above the cell surface, whereas most cell surface molecules do not exceed a length of 35 nm. Normally, episialin is present at the apical side of glandular epithelial cells. On carcinoma cells, however, it can be strongly overexpressed and it is often present over the entire cell surface. We have previously shown that episialin, if it is interspersed between adhesion molecules, nonspecifically reduces cell-cell and cell-extracellular matrix interactions in vitro and in vivo, presumably by steric hindrance caused by the extreme length and high density of the episialin molecules at the cell surface. To analyze the molecular mechanism for this anti-adhesion effect in more detail, we have now deleted an increasing number of repeats in the episialin cDNA and transfected the resulting mutants into murine L929 cells expressing the homophilic adhesion molecule E-cadherin. Here we show that the length of episialin is the dominant factor that determines the inhibition of E-cadherin-mediated cell-cell interactions. For the anti-adhesive effect mediated by the full length episialin, charge repulsion by negatively charged sialylated O-linked glycans is far less important. Images PMID:8730100

  15. GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation

    PubMed Central

    Nolan-Stevaux, Olivier; Lau, Janet; Truitt, Morgan L.; Chu, Gerald C.; Hebrok, Matthias; Fernández-Zapico, Martin E.; Hanahan, Douglas

    2009-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by the deregulation of the hedgehog signaling pathway. The Sonic Hedgehog ligand (Shh), absent in the normal pancreas, is highly expressed in pancreatic tumors and is sufficient to induce neoplastic precursor lesions in mouse models. We investigated the mechanism of Shh signaling in PDAC carcinogenesis by genetically ablating the canonical bottleneck of hedgehog signaling, the transmembrane protein Smoothened (Smo), in the pancreatic epithelium of PDAC-susceptible mice. We report that multistage development of PDAC tumors is not affected by the deletion of Smo in the pancreas, demonstrating that autocrine Shh–Ptch–Smo signaling is not required in pancreatic ductal cells for PDAC progression. However, the expression of Gli target genes is maintained in Smo-negative ducts, implicating alternative means of regulating Gli transcription in the neoplastic ductal epithelium. In PDAC tumor cells, we find that Gli transcription is decoupled from upstream Shh–Ptch–Smo signaling and is regulated by TGF-β and KRAS, and we show that Gli1 is required both for survival and for the KRAS-mediated transformed phenotype of cultured PDAC cancer cells. PMID:19136624

  16. 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

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

    PubMed

    Almatouq, Abdullah; Babatunde, Akintunde O

    2016-04-01

    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, 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. PMID:27043584

  18. Cocaine mediated apoptosis of vascular cells as a mechanism for carotid artery dissection leading to ischemic stroke.

    PubMed

    Dabbouseh, Noura M; Ardelt, Agnieszka

    2011-08-01

    In arterial dissection, blood may enter the arterial wall through an intimal tear, splitting the arterial wall and activating the coagulation cascade at the site of endothelial damage. Dissection of extracranial and intracranial vessels may lead to ischemic stroke through thromboembolic or hemodynamic mechanisms. Major blunt trauma or rapid acceleration-deceleration may cause dissection, but in patients with inherent arterial wall weakness, dissection can occur spontaneously or as a result of minor neck movement. Cocaine use has been associated with dissection of the aortic arch and coronary and renal arteries through cocaine-mediated hypertension. Recent preclinical studies have suggested, however, that cocaine may cause apoptosis of cells in the vascular wall. In this article, we postulate that cocaine may cause apoptosis of vascular endothelial and/or smooth muscle cells, thus weakening the vascular wall and resulting in a dissection-prone state. We review the literature and propose a biological basis for vasculopathy, vascular dissection, and ischemic stroke in the setting of cocaine use. Further research studies on vascular cells, as well as focused analysis of human pathological material, will be important in providing evidence for or against our hypotheses. PMID:21546166

  19. 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

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

    PubMed Central

    Sartorius, Carol A.; Hanna, Colton T.; Gril, Brunilde; Cruz, Hazel; Serkova, Natalie J.; Huber, Kendra M.; Kabos, Peter; Schedin, Troy B.; Borges, Virginia F.; Steeg, Patricia S.; Cittelly, Diana M.

    2015-01-01

    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 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 MRI detectable lesions by 56% as compared to 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 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 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 to vehicle-control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. ShRNA-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 studies provide a novel mechanism by which estrogens, acting through ER

  1. Mechanisms of genetic resistance to Friend virus leukemia. III. Susceptibility of mitogen-responsive lymphocytes mediated by T cells.

    PubMed

    Kumar, V; Caruso, T; Bennett, M

    1976-04-01

    Friend leukemia virus (FV) suppressed the proliferative responses of spleen, lymph node, marrow, and thymus cell populations to various T- and B-cell mitogens. Cells taken from mice, e.g. BALB/c genetically susceptible to leukemogenesis in vivo were much more susceptible to suppression of mitogenesis in vitro than similar cells from genetically resistant mice, e.g., C57BL/6. Nylon wool-purified splenic T cells from BALB/c and C3H mice lost susceptibility to FV-induced suppression of mitogenesis but became suppressible by addition of 10% unfiltered spleen cell. Thus, FV mediates in vitro suppression of lymphocyte proliferation indirectly by "activating" a suppressor cell. The suppressor cell adhered to nylon wool but not to glass wool or rayon wool columns. Pretreatment of spleen cells with carbonyl iron and a magnet did not abrogate the suppressor cell function. Suppressor cells were not eliminated by treatment with rabbit antimouse immunoglobulin (7S) and complement (C). However, high concentrations of anti-Thy-1 plus C destroyed suppressor cells of the spleen; thymic suppressor cells were much more susceptible to anti-Thy-1 serum. Nude athymic mice were devoid of suppressor cells and their B-cell proliferation was relatively resistant to FV-induced suppression in vitro. The suppressor cells in the thymus (but not in the spleen) were eliminated by treatment of mice with cortisol. Thus, FV appears to mediate its suppressive effect on mitogen-responsive lymphocytes by affecting "T-suppressor cells." Spleen cells from C57BL/6 mice treated with 89Sr to destroy marrow-dependent (M) cells were much more suppressible by FV in virto than normal C57BL/6 spleen cells. However, nylon-filtered spleen cells of 89Sr-treated C57BL/6 mice were resistant to FV-induced suppression in vitro, indicating that the susceptibility of spleen cells from 89Sr-treated B6 mice is also mediated by suppressor cells. Normal B6 splenic T cells were rendered susceptible to FV-induced suppression

  2. 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

  3. 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

  4. Human lung cancer cell lines express cell membrane complement inhibitory proteins and are extremely resistant to complement-mediated lysis; a comparison with normal human respiratory epithelium in vitro, and an insight into mechanism(s) of resistance.

    PubMed

    Varsano, S; Rashkovsky, L; Shapiro, H; Ophir, D; Mark-Bentankur, T

    1998-08-01

    Human lung cancer expresses cell membrane complement inhibitory proteins (CIP). We investigated whether human lung cancer cell lines also express cell-membrane CIP molecules and whether the biology of CIP molecules in these cell lines differs from that of CIP in normal human respiratory epithelium in culture. The cell lines ChaGo K-1 and NCI-H596 were compared with normal human nasal epithelium in primary cultures in respect to the level of cell membrane CIP expression of membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and CD59, in respect to the level of cell resistance to complement-mediated lysis, and in respect to the contribution of cell membrane CIP to cell resistance against complement-mediated lysis. We found, using flow cytometry, that both human lung cancer cell lines expressed MCP, DAF and CD59, as did normal nasal epithelial cells. However, normal cells showed a large subpopulation of low DAF-expressing cells (60% of all cells) and a smaller subpopulation of high DAF-expressing cells (40%), while the lung cancer cell lines showed only one cell population, of high DAF expression. In addition, both lung cancer cell lines expressed higher MCP levels, and NCI-H596 cells showed higher levels of CD59. Cell resistance to complement-mediated lysis of both lung cancer cell lines was much higher than that of normal cells. Fifty percent normal human serum, under the same concentrations of complement activators, induced lysis of less than a mean of 10% of lung cancer cells, while lysing up to a mean of 50% of nasal epithelial cells. Lung cancer cell resistance to complement was due to its ability to prevent significant activation of complement upon its cell membrane, as manifested by a failure of complement activators to increase cell membrane deposition of C3-related fragments. The exact mechanism for this resistance remains obscure. Unexpectedly, neutralizing antibodies, anti-MCP and anti-DAF were entirely ineffective and anti-CD59

  5. Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells

    SciTech Connect

    Li, Xin-Yu; Zhan, Xiao-Rong; Lu, Chong; Liu, Xiao-Min; Wang, Xiao-Chen

    2010-07-30

    Research highlights: {yields} A hypothesis that the differentiation of PDEC is through MAPKs or PI3K/AKT pathways. {yields} Determine if kinases (ERK1/2, p38, JNK, and AKT) are activated in these pathways. {yields} Determine signal pathway(s) that may effect on HGF-induced differentiation of PDEC. {yields} PI3K-AKT pathway is involved in the differentiation of PDECs induced by HGF. {yields} MEK-ERK pathway effect on the proliferation of PDECs but not the differentiation. -- Abstract: Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28 days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors

  6. Inhibition of Elastin Peptide-Mediated Angiogenic Signaling Mechanism(s) in Choroidal Endothelial Cells by the α6(IV)NC1 Collagen Fragment

    PubMed Central

    Gunda, Venugopal; Verma, Raj Kumar; Sudhakar, Yakkanti Akul

    2013-01-01

    Purpose. The inhibitory effects and mechanism(s) of type IV collagen α-6 chain–derived noncollagenous domain (α6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)–activated choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloproteinase (MT1-MMP) activation are explored. Methods. Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for different time intervals with or without α6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and α6(IV)NC1 treatments. The MCECs also were subjected to EDPs, and specific inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) phosphorylation. Results. Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting the proliferation of MCECs. The α6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by α6(IV)NC1. Further, EDP-activated MT1-MMP expression and FAK/phosphoinositide-3-kinase (PI-3K)/mammalian target of rapamycin (mToR)/Akt phosphorylation in MCECs, were reduced by α6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK- and Akt-specific inhibitors. Conclusions. The EDPs and α6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The α6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates α6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye. PMID:24194191

  7. 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. PMID:21833088

  8. 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

  9. Sequestration of inhaled particulate antigens by lung phagocytes. A mechanism for the effective inhibition of pulmonary cell-mediated immunity.

    PubMed Central

    MacLean, J. A.; Xia, W.; Pinto, C. E.; Zhao, L.; Liu, H. W.; Kradin, R. L.

    1996-01-01

    Dendritic cells (DCs) have emerged as the dominant antigen-presenting cells (APCs) of the lung, playing a vital role in the induction of cell-mediated immunity to inhaled antigens. We have previously demonstrated that an airway challenge with the soluble antigen hen egg lysozyme yields rapid acquisition of specific antigen-presenting cell activity by purified pulmonary DCs and a cell-mediated immune response in the lung upon secondary challenge. To examine how a particulate antigen leads to a cell-mediated response in vivo, graded concentrations of heat-killed Listeria (HKL) were injected intratracheally into Lewis rats. The bacteria were rapidly ingested by lung macrophages and polymorphonuclear leukocytes. The ability of purified pulmonary DCs pulsed in vivo by an airway challenge with HKL to subsequently stimulate HKL-specific responses ex vivo showed a threshold response, requiring a dose in excess of 10(9) organisms/rat. By contrast, all dosages of HKL yielded specific sensitization of lymphocytes in the draining bilar nodes. Pulmonary DCs purified from rats after a secondary in vivo airway challenge with HKL at day 14 were ineffective antigen-presenting cells except at high dosages of antigen. The generation of cell-mediated pulmonary inflammation paralleled the antigen-presenting cell activity of pulmonary DCs and was observed only at high antigen dosages. Hen egg lysozyme immobilized onto polystyrene beads and injected intratracheally yielded comparable results to those observed with HKL. We suggest that a pulmonary cellular immune response is generated to an inhaled particulate antigen when the protective phagocytic capacities of the lung are exceeded and antigen is able to interact directly with interstitial DCs. The diversion of particulate antigens by pulmonary phagocytes may help to limit undesirable pulmonary inflammation while allowing the generation of antigen-specific immune lymphocytes in vivo. Images Figure 2 Figure 4 Figure 5 Figure 7 Figure 9

  10. 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. PMID:25504014

  11. Mechanisms of Immune-Mediated Liver Injury

    PubMed Central

    Adams, David H.; Ju, Cynthia; Ramaiah, Shashi K.; Uetrecht, Jack; Jaeschke, Hartmut

    2010-01-01

    Hepatic inflammation is a common finding during a variety of liver diseases including drug-induced liver toxicity. The inflammatory phenotype can be attributed to the innate immune response generated by Kupffer cells, monocytes, neutrophils, and lymphocytes. The adaptive immune system is also influenced by the innate immune response leading to liver damage. This review summarizes recent advances in specific mechanisms of immune-mediated hepatotoxicity and its application to drug-induced liver injury. Basic mechanisms of activation of lymphocytes, macrophages, and neutrophils and their unique mechanisms of recruitment into the liver vasculature are discussed. In particular, the role of adhesion molecules and various inflammatory mediators in this process are explored. In addition, the authors describe mechanisms of liver cell damage by these inflammatory cells and critically evaluate the functional significance of each cell type for predictive and idiosyncratic drug-induced liver injury. It is expected that continued advances in our understanding of immune mechanisms of liver injury will lead to an earlier detection of the hepatotoxic potential of drugs under development and to an earlier identification of susceptible individuals at risk for predictive and idiosyncratic drug toxicities. PMID:20071422

  12. Investigation of Biophysical Mechanisms in Gold Nanoparticle Mediated Laser Manipulation of Cells Using a Multimodal Holographic and Fluorescence Imaging Setup

    PubMed Central

    Rakoski, Mirko S.; Heinemann, Dag; Schomaker, Markus; Ripken, Tammo; Meyer, Heiko

    2015-01-01

    Laser based cell manipulation has proven to be a versatile tool in biomedical applications. In this context, combining weakly focused laser pulses and nanostructures, e.g. gold nanoparticles, promises to be useful for high throughput cell manipulation, such as transfection and photothermal therapy. Interactions between laser pulses and gold nanoparticles are well understood. However, it is still necessary to study cell behavior in gold nanoparticle mediated laser manipulation. While parameters like cell viability or perforation efficiency are commonly addressed, the influence of the manipulation process on other essential cell parameters is not sufficiently investigated yet. Thus, we set out to study four relevant cell properties: cell volume and area, ion exchange and cytoskeleton structure after gold nanoparticle based laser manipulation. For this, we designed a multimodal imaging and manipulation setup. 200 nm gold nanoparticles were attached unspecifically to canine cells and irradiated by weakly focused 850 ps laser pulses. Volume and area change in the first minute post laser manipulation was monitored using digital holography. Calcium imaging and cells expressing a marker for filamentous actin (F-actin) served to analyze the ion exchange and the cytoskeleton, respectively. High radiant exposures led to cells exhibiting a tendency to shrink in volume and area, possibly due to outflow of cytoplasm. An intracellular raise in calcium was observed and accompanied by an intercellular calcium wave. This multimodal approach enabled for the first time a comprehensive analysis of the cell behavior in gold nanoparticle mediated cell manipulation. Additionally, this work can pave the way for a better understanding and the evaluation of new applications in the context of cell transfection or photothermal therapy. PMID:25909631

  13. Clearance of Herpes Simplex Virus Type 2 by CD8+ T Cells Requires Gamma Interferon and either Perforin- or Fas-Mediated Cytolytic Mechanisms

    PubMed Central

    Dobbs, Melanie E.; Strasser, Jane E.; Chu, Chin-Fun; Chalk, Claudia; Milligan, Gregg N.

    2005-01-01

    The T-cell-mediated resolution of herpes simplex virus type 2 (HSV-2) genital infections is not fully understood. In these studies, the mechanisms by which CD8+ T cells clear virus from the genital epithelium were examined. Ovalbumin (OVA)-specific CD8+ T cells from OT-I transgenic mice cleared a thymidine kinase-deficient, ovalbumin-expressing HSV-2 virus (HSV-2 tk− OVA) from the genital epithelium of recipient mice, and clearance was abrogated by in vivo neutralization of gamma interferon (IFN-γ). Further, CD8+ OT-I T cells deficient in IFN-γ were unable to clear HSV-2 tk− OVA from the vaginal epithelium. The requirement for cytolytic mechanisms in HSV-2 tk− OVA clearance was tested in radiation chimeras by adoptive transfer of wild-type or perforin-deficient OT-I T cells to irradiated Fas-defective or wild-type recipients. Although a dramatic decrease in viral load was observed early after challenge with HSV-2 tk− OVA, full resolution of the infection was not achieved in recipients lacking both perforin- and Fas-mediated cytolytic pathways. These results suggest that IFN-γ was responsible for an early rapid decrease in HSV-2 virus titer. However, either perforin- or Fas-mediated cytolytic mechanisms were required to achieve complete clearance of HSV-2 from the genital epithelium. PMID:16282454

  14. Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain.

    PubMed

    Inoue, Shigeki; Murata, Kaoru; Tanaka, Aiko; Kakuta, Eri; Tanemura, Saori; Hatakeyama, Shiori; Nakamura, Atsunao; Yamamoto, Chihiro; Hasebe, Masaharu; Kosakai, Kumiko; Yoshino, Masami

    2014-09-01

    Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 μM) potently blocked INaP with little effect on INaf. Riluzole (>20 μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 μM riluzole in the bath solution. These membrane oscillations disappeared with 1 μM TTX, 50 μM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in

  15. Loss of E-Cadherin–mediated Cell–Cell Contacts Activates a Novel Mechanism for Up-Regulation of the Proto-Oncogene c-Jun

    PubMed Central

    Knirsh, Revital; Ben-Dror, Iris; Spangler, Barbara; Matthews, Gideon D.; Kuphal, Silke; Bosserhoff, Anja K.

    2009-01-01

    Loss of E-cadherin–mediated cell–cell contacts can elicit a signaling pathway that leads to acquisition of an invasive phenotype. Here, we show that at the receiving end of this pathway is the proto-oncogene c-Jun, a member of the activator protein-1 family of transcription factors that play a key role in stimulation of cell proliferation and tumor promotion. Cell separation or abrogation of E-cadherin–mediated cell–cell contacts both cause a dramatic increase in accumulation of the c-Jun protein. Unlike growth factors that enhance the expression of c-Jun by activating the transcription of the c-jun gene, the cell contact-dependent increase in c-Jun accumulation is not accompanied by a corresponding increase in c-Jun mRNA or c-Jun protein stability but rather in the translatability of the c-Jun transcript. Consistently, the increase in c-Jun accumulation is not dependent on activation of the mitogen-activated protein kinase or β-catenin pathways but is mediated by signals triggered by the restructured cytoskeleton. Depolymerization of the cytoskeleton can mimic the effect of cell separation and cause a dramatic increase in c-Jun accumulation, whereas Taxol inhibits the cell contact-dependent increase. This novel mechanism of c-Jun regulation seems to underlie the robust overexpression of c-Jun in tumor cells of patients with colon carcinoma. PMID:19193763

  16. 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

  17. 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.

  18. 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.

  19. 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

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

    PubMed

    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

  1. Signal strength regulates antigen-mediated T-cell deceleration by distinct mechanisms to promote local exploration or arrest

    PubMed Central

    Moreau, Hélène D.; Lemaître, Fabrice; Garrod, Kym R.; Garcia, Zacarias; Lennon-Duménil, Ana-Maria; Bousso, Philippe

    2015-01-01

    T lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca2+ signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells. PMID:26371316

  2. 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

  3. 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

  4. Notch/HES1-mediated PARP1 activation: a cell type–specific mechanism for tumor suppression

    PubMed Central

    Kannan, Sankaranarayanan; Fang, Wendy; Song, Guangchun; Mullighan, Charles G.; Hammitt, Richard; McMurray, John

    2011-01-01

    Notch signaling plays both oncogenic and tumor suppressor roles, depending on cell type. In contrast to T-cell acute lymphoblastic leukemia (ALL), where Notch activation promotes leukemogenesis, induction of Notch signaling in B-cell ALL (B-ALL) leads to growth arrest and apoptosis. The Notch target Hairy/Enhancer of Split1 (HES1) is sufficient to reproduce this tumor suppressor phenotype in B-ALL; however, the mechanism is not yet known. We report that HES1 regulates proapoptotic signals by the novel interacting protein Poly ADP-Ribose Polymerase1 (PARP1) in a cell type–specific manner. Interaction of HES1 with PARP1 inhibits HES1 function, induces PARP1 activation, and results in PARP1 cleavage in B-ALL. HES1-induced PARP1 activation leads to self-ADP ribosylation of PARP1, consumption of nicotinamide adenine dinucleotide+, diminished adenosine triphosphate levels, and translocation of apoptosis-inducing factor from mitochondria to the nucleus, resulting in apoptosis in B-ALL but not T-cell ALL. Importantly, induction of Notch signaling by the Notch agonist peptide Delta/Serrate/Lag-2 can reproduce these events and leads to B-ALL apoptosis. The novel interaction of HES1 and PARP1 in B-ALL modulates the function of the HES1 transcriptional complex and signals through PARP1 to induce apoptosis. This mechanism shows a cell type–specific proapoptotic pathway that may lead to Notch agonist–based cancer therapeutics. PMID:21224467

  5. 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

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

    PubMed

    Shpak, Guy; Zylbertal, Asaph; Wagner, Shlomo

    2014-01-01

    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

  7. Chemokine-mediated redirection of T cells constitutes a critical mechanism of glucocorticoid therapy in autoimmune CNS responses

    PubMed Central

    Schweingruber, Nils; Fischer, Henrike J.; Fischer, Lisa; van den Brandt, Jens; Karabinskaya, Anna; Labi, Verena; Villunger, Andreas; Kretzschmar, Benedikt; Huppke, Peter; Simons, Mikael; Tuckermann, Jan P.; Flügel, Alexander

    2016-01-01

    Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of gC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor® strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS. PMID:24488308

  8. Cardiotrophin-1 determines liver engraftment of syngenic colon carcinoma cells through an immune system-mediated mechanism.

    PubMed

    Bustos, Matilde; Dubrot, Juan; Martinez-Anso, Eduardo; Larequi, Eduardo; Castaño, David; Palazon, Asis; Belza, Idoia; Sanmamed, Miguel F; Perez-Gracia, Jose Luis; Ortiz de Solorzano, Carlos; Alfaro, Carlos; Melero, Ignacio

    2012-12-01

    Cardiotrophin-1 (CT-1/CTF1) is a member of the interleukin-6 (IL-6) family of cytokines that stimulates STAT-3 phosphorylation in cells bearing the cognate receptor. We report that Ctf1(-/-) mice (hereby referred to as CT-1(-/-) mice) are resistant to the hepatic engraftment of MC38 colon carcinoma cells, while these cells engraft normally in the mouse subcutaneous tissue. Tumor intake in the liver could be enhanced by the systemic delivery of a recombinant adenovirus encoding CT-1, which also partly rescued the resistance of CT-1(-/-) mice to the hepatic engraftment of MC38 cells. Moreover, systemic treatment of wild-type (WT) mice with a novel antibody-neutralizing mouse CT-1 also reduced engraftment of this model. Conversely, experiments with Panc02 pancreatic cancer and B16-OVA melanoma cells in CT-1(-/-) mice revealed rates of hepatic engraftment similar to those observed in WT mice. The mechanism whereby CT-1 renders the liver permissive for MC38 metastasis involves T lymphocytes and natural killer (NK) cells, as shown by selective depletion experiments and in genetically deficient mice. However, no obvious changes in the number or cell killing capacity of liver lymphocytes in CT-1(-/-) animals could be substantiated. These findings demonstrate that the seed and soil concept to understand metastasis can be locally influenced by cytokines as well as by the cellular immune system. PMID:23264899

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

    PubMed Central

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

    2013-01-01

    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

  10. Stress-mediated progression of solid tumors: effect of mechanical stress on tissue oxygenation, cancer cell proliferation, and drug delivery.

    PubMed

    Mpekris, Fotios; Angeli, Stelios; Pirentis, Athanassios P; Stylianopoulos, Triantafyllos

    2015-11-01

    Oxygen supply plays a central role in cancer cell proliferation. While vascular density increases at the early stages of carcinogenesis, mechanical solid stresses developed during growth compress tumor blood vessels and, thus, drastically reduce not only the supply of oxygen, but also the delivery of drugs at inner tumor regions. Among other effects, hypoxia and reduced drug delivery compromise the efficacy of radiation and chemo/nanotherapy, respectively. In the present study, we developed a mathematical model of tumor growth to investigate the interconnections among tumor oxygenation that supports cancer cell proliferation, the heterogeneous accumulation of mechanical stresses owing to tumor growth, the non-uniform compression of intratumoral blood vessels due to the mechanical stresses, and the insufficient delivery of oxygen and therapeutic agents because of vessel compression. We found that the high vascular density and increased cancer cell proliferation often observed in the periphery compared to the interior of a tumor can be attributed to heterogeneous solid stress accumulation. Highly vascularized peripheral regions are also associated with greater oxygenation compared with the compressed, less vascularized inner regions. We also modeled the delivery of drugs of two distinct sizes, namely chemotherapy and nanomedicine. Model predictions suggest that drug delivery is affected negatively by vessel compression independently of the size of the therapeutic agent. Finally, we demonstrated the applicability of our model to actual geometries, employing a breast tumor model derived from MR images. PMID:25968141

  11. Stress-mediated progression of solid tumors: effect of mechanical stress on tissue oxygenation, cancer cell proliferation and drug delivery

    PubMed Central

    Mpekris, Fotios; Angeli, Stelios; Pirentis, Athanassios P.; Stylianopoulos, Triantafyllos

    2015-01-01

    Oxygen supply plays a central role in cancer cell proliferation. While vascular density increases at the early stages of carcinogenesis, mechanical solid stresses developed during growth compress tumor blood vessels and, thus, drastically reduce the supply of oxygen, but also the delivery of drugs at inner tumor regions. Among other effects, hypoxia and reduced drug delivery compromise the efficacy of radiation and chemo/nano therapy, respectively. In the present study, we developed a mathematical model of tumor growth to investigate the interconnections among tumor oxygenation that supports cancer cell proliferation, the heterogeneous accumulation of mechanical stresses owing to tumor growth, the non-uniform compression of intratumoral blood vessels due to the mechanical stresses, and the insufficient delivery of oxygen and therapeutic agents because of vessel compression. We found that the high vascular density and increased cancer cell proliferation often observed in the periphery compared to the interior of a tumor can be attributed to heterogeneous solid stress accumulation. Highly vascularized peripheral regions are also associated with greater oxygenation compared with the compressed, less vascularized inner regions. We also modeled the delivery of drugs of two distinct sizes, namely chemotherapy and nanomedicine. Model predictions suggest that drug delivery is affected negatively by vessel compression independently of the size of the therapeutic agent. Finally, we demonstrated the applicability of our model to actual geometries, employing a breast tumor model derived from MR images. PMID:25968141

  12. Molecular mechanisms underlying β-adrenergic receptor-mediated cross-talk between sympathetic neurons and immune cells.

    PubMed

    Lorton, Dianne; Bellinger, Denise L

    2015-01-01

    Cross-talk between the sympathetic nervous system (SNS) and immune system is vital for health and well-being. Infection, tissue injury and inflammation raise firing rates of sympathetic nerves, increasing their release of norepinephrine (NE) in lymphoid organs and tissues. NE stimulation of β2-adrenergic receptors (ARs) in immune cells activates the cAMP-protein kinase A (PKA) intracellular signaling pathway, a pathway that interfaces with other signaling pathways that regulate proliferation, differentiation, maturation and effector functions in immune cells. Immune-SNS cross-talk is required to maintain homeostasis under normal conditions, to develop an immune response of appropriate magnitude after injury or immune challenge, and subsequently restore homeostasis. Typically, β2-AR-induced cAMP is immunosuppressive. However, many studies report actions of β2-AR stimulation in immune cells that are inconsistent with typical cAMP-PKA signal transduction. Research during the last decade in non-immune organs, has unveiled novel alternative signaling mechanisms induced by β2-AR activation, such as a signaling switch from cAMP-PKA to mitogen-activated protein kinase (MAPK) pathways. If alternative signaling occurs in immune cells, it may explain inconsistent findings of sympathetic regulation of immune function. Here, we review β2-AR signaling, assess the available evidence for alternative signaling in immune cells, and provide insight into the circumstances necessary for "signal switching" in immune cells. PMID:25768345

  13. Palmitic acid in chicken granulosa cell death-lipotoxic mechanisms mediate reproductive inefficacy of broiler breeder hens.

    PubMed

    Xie, Y-L; Pan, Y-E; Chang, C-J; Tang, P-C; Huang, Y-F; Walzem, R L; Chen, S-E

    2012-12-01

    In vivo and in vitro approaches were used to elucidate mechanisms of palmitate-induced cytotoxicity of follicle granulosa cells in fuel-overloaded broiler hens. In contrast to their energy-restricted counterparts, broiler breeder hens fed ad libitum for 2 wk had dyslipidemia, atresia within hierarchical ovarian follicles, and a 34% reduction in egg production (P < 0.05). Based on vital staining of freshly isolated granulosa cells with annexin V/propidium iodide, there were increases in apoptosis consistent with suppressed Akt activation (P < 0.05). Supplementing primary granulosa cell cultures with 0.5 mM palmitate for 48 or 96 h increased apoptosis (P < 0.05). Palmitate-induced cell death was accompanied by increased acyl-CoA oxidase, carnitine palmitoyl transferase-1, serine palmitoyl transferase, and sphingomyelinase transcripts and increased concentrations of proinflammatory interleukin-1β (P < 0.05). Triacsin-C inhibition of fatty acyl-CoA synthesis blunted interleukin-1β production and rescued granulosa cultures from palmitate-induced cell death. That there was partial to complete prevention of cell death with addition of the free radical scavenger pyrrolidine dithiocarbamate, the sphingomyelinase inhibitor imipramine, or the de novo ceramide synthesis inhibitor fumonisin B1, supported the notion that palmitate-induced granulosa cell cytotoxicity operated through a palmitate-derived metabolite. Palmitoyl-CoA may be channeled into β-oxidation and/or into bioactive metabolites that increase free radical generation, an inflammatory response, and ceramide production. In conclusion, palmitate-derived metabolites activated apoptotic machinery in avian granulosa cells, which caused ovarian follicular atresia and reduced egg production in fuel-overloaded broiler breeder hens. PMID:23058789

  14. 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

  15. 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

  16. Direct and indirect mechanisms mediating apoptosis during HIV infection: contribution to in vivo CD4 T cell depletion.

    PubMed

    Gougeon, M L; Laurent-Crawford, A G; Hovanessian, A G; Montagnier, L

    1993-06-01

    The gradual depletion of CD4+ T lymphocytes during the development of AIDS may be due, at least in part, to a process referred to as apoptosis. This process involves a Ca2+ dependent nuclear endonuclease that cleaves the chromatin at internucleosomal junctions. In addition, we have recently provided evidence that apoptosis may be responsible not only for the progressive loss of CD4+ T lymphocytes but may be operative in CD8+ T lymphocytes as well. Here, we describe mechanisms which by direct and indirect pathways may induce apoptosis during HIV infection and thus leading to elimination of T cells. PMID:8102263

  17. Mechanism of Siglec-8-mediated Cell Death in IL-5-activated Eosinophils: Role for ROS-enhanced MEK/ERK Activation

    PubMed Central

    Kano, Gen; Almanan, Maha; Bochner, Bruce S.; Zimmermann, Nives

    2014-01-01

    Background Siglec-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity. Objective In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils. Methods Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by flow cytometry and morphology. Phosphorylation of MAPK was determined by phospho-luminex, flow cytometry, and Western blotting. ROS accumulation was determined by dihydrorhodamine (DHR) fluorescence. Results Co-stimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cells dying by necrosis accompanied by granule release as compared to stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in co-stimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 co-stimulation. Phosphorylation of ERK1/2 and MEK1 was significantly enhanced and sustained in co42 stimulated cells compared to cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation. Conclusions In activated eosinophils, ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK

  18. 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

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

    PubMed

    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

  20. Short-chain fatty acid (SCFA) uptake into Caco-2 cells by a pH-dependent and carrier mediated transport mechanism.

    PubMed

    Stein, J; Zores, M; Schröder, O

    2000-06-01

    The short-chain fatty acids, acetate, propionate, and butyrate, are the most abundant organic anions in the human colon. SCFA play a pivotal role in maintaining homeostasis in the colon. Particularly butyrate induces cell differentiation and regulates growth and proliferation of colonic mucosal epithelial cells, whereas it reduces the growth rate of colorectal cancer cell. Previous studies by several groups, including our own, using isolated membrane vesicles have demonstrated that the uptake of butyrate is at least in part mediated by a non-electrogenic SCFA-/HCO3- antiporter. The purpose of the present study was to determine (1) whether Caco-2 cells could serve as an experimental model to assess the mechanisms of SCFA transport, and (2) whether monocarboxlate transporters could play a role in SCFA transport in these cells. Caco-2 cells were found to transport 14C-butyrate in a concentration and time dependent manner. The uptake was sodium independent, but was stimulated by lowering extracellular pH. The uptake of 500 microM butyrate was reduced by 49.6% +/- 3.3% in the presence of propionate and by 57.2% +/- 4.8% in the presence of 10 mM L-lactate. The addition of 1 mM alpha-cyano-4-hydroxycinnamate and phloretin, both known to be potent inhibitors of MCT1, decreased the uptake of 500 microM 14C-butyrate by 59.4% +/- 4.1% and 48.9% +/- 3.3%, respectively, whereas similar concentrations of DIDS did not have any effect. These data suggest that the uptake of butyrate in Caco-2 cells occurs via a carrier mediated transport system specific for monocarboxylic acids, which is in accordance with characteristics of the MCT 1. PMID:10918994

  1. Macrophage-Associated Osteoactivin/GPNMB Mediates Mesenchymal Stem Cell Survival, Proliferation, and Migration Via a CD44-Dependent Mechanism.

    PubMed

    Yu, Bing; Sondag, Gregory R; Malcuit, Christopher; Kim, Min-Ho; Safadi, Fayez F

    2016-07-01

    Although MSCs have been widely recognized to have therapeutic potential in the repair of injured or diseased tissues, it remains unclear how functional activities of mesenchymal stem cells (MSCs) are influenced by the surrounding inflammatory milieu at the site of tissue injury. Macrophages constitute an essential component of innate immunity and have been shown to exhibit a phenotypic plasticity in response to various stimuli, which play a central role in both acute inflammation and wound repair. Osteoactivin (OA)/Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein that plays a role in cell differentiation, survival, and angiogenesis. The objective of this study was to investigate the potential role of OA/GPNMB in macrophage-induced MSC function. We found that reparative M2 macrophages express significantly greater levels of OA/GPNMB than pro-inflammatory M1 macrophages. Furthermore, using loss of function and rescue studies, we demonstrated that M2 macrophages-secreted OA/GPNMB positively regulates the viability, proliferation, and migration of MSCs. More importantly, we demonstrated that OA/GPNMB acts through ERK and AKT signaling pathways in MSCs via CD44, to induce these effects. Taken together, our results provide pivotal insight into the mechanism by which OA/GPNMB contributes to the tissue reparative phenotype of M2 macrophages and positively regulates functional activities of MSCs. J. Cell. Biochem. 117: 1511-1521, 2016. © 2015 Wiley Periodicals, Inc. PMID:26442636

  2. Anti-inflammatory mechanism of α-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells.

    PubMed

    Dilshara, Matharage Gayani; Lee, Kyoung-Tae; Kim, Hee Ju; Lee, Hak-Ju; Choi, Yung Hyun; Lee, Chang-Min; Kim, Lark Kyun; Kim, Gi-Young

    2014-07-01

    α-Viniferin is an oligostilbene of trimeric resveratrol and has anticancer activity; however, the molecular mechanism underlying the anti-inflammatory effects of α-viniferin has not been completely elucidated thus far. Therefore, we determined the mechanism by which α-viniferin regulates lipopolysaccharide (LPS)-induced expression of proinflammatory mediators in BV2 microglial cells. Treatment with α-viniferin isolated from Clematis mandshurica decreased LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). α-Viniferin also downregulated the LPS-induced expression of proinflammatory genes such as iNOS and COX-2 by suppressing the activity of nuclear factor kappa B (NF-κB) via dephosphorylation of Akt/PI3K. Treatment with a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), indirectly showed that NF-κB is a crucial transcription factor for expression of these genes in the early stage of inflammation. Additionally, our results indicated that α-viniferin suppresses NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1) regulated by nuclear factor erythroid 2-related factor (Nrf2). Taken together, our data indicate that α-viniferin suppresses the expression of proinflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/PI3K-dependent NF-κB activation and inhibits the production of proinflammatory mediators NO and PGE2 in the late stage by stimulating Nrf2-mediated HO-1 signaling pathway in LPS-stimulated BV2 microglial cells. These results suggest that α-viniferin may be a potential candidate to regulate LPS-induced inflammation. PMID:24859013

  3. Notch-Mediated Cell Adhesion.

    PubMed

    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

  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. Transcriptional mechanisms and protein kinase signaling mediate organic dust induction of IL-8 expression in lung epithelial and THP-1 cells

    PubMed Central

    Gottipati, Koteswara R.; Bandari, Shiva Kumar; Nonnenmann, Matthew W.; Levin, Jeffrey L.; Dooley, Gregory P.; Reynolds, Stephen J.

    2014-01-01

    Exposure to the agricultural work environment is a risk factor for the development of respiratory symptoms and chronic lung diseases. Inflammation is an important contributor to the pathogenesis of tissue injury and disease. Cellular and molecular mechanisms mediating lung inflammatory responses to agricultural dust are not yet fully understood. We studied the effects of poultry dust extract on molecular regulation of interleukin-8 (IL-8), a proinflammatory cytokine, in A549 and Beas2B lung epithelial and THP-1 monocytic cells. Our findings indicate that poultry dust extract potently induces IL-8 levels by increasing IL-8 gene transcription without altering IL-8 mRNA stability. Increase in IL-8 promoter activity was due to enhanced binding of activator protein 1 and NF-κB. IL-8 induction was associated with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation and inhibited by PKC and MAPK inhibitors. IL-8 increase was not inhibited by polymyxin B or l-nitroarginine methyl ester, indicating lack of involvement of lipopolysaccharide and nitric oxide in the induction. Lung epithelial and THP-1 cells share common mechanisms for induction of IL-8 levels. Our findings identify key roles for transcriptional mechanisms and protein kinase signaling pathways for IL-8 induction and provide insights into the mechanisms regulating lung inflammatory responses to organic dust exposure. PMID:25398986

  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. Arsenic Trioxide Induces Apoptosis and Incapacitates Proliferation and Invasive Properties of U87MG Glioblastoma Cells through a Possible NF-κB-Mediated Mechanism.

    PubMed

    Ghaffari, Seyed H; Yousefi, Meysam; Dizaji, Majid Zaki; Momeny, Majid; Bashash, Davood; Zekri, Ali; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir

    2016-01-01

    Identification of novel therapeutics in glioblastoma remains crucial due to the devastating and infiltrative capacity of this malignancy. The current study was aimed to appraise effect of arsenic trioxide (ATO) in U87MG cells. The results demonstrated that ATO induced apoptosis and impeded proliferation of U87MG cells in a dosedependent manner and also inhibited classical NF-κB signaling pathway. ATO further upregulated expression of Bax as an important proapoptotic target of NF-κB and also inhibited mRNA expression of survivin, c-Myc and hTERT and suppressed telomerase activity. Moreover, ATO significantly increased adhesion of U87MG cells and also diminished transcription of NF-κB down-stream targets involved in cell migration and invasion, including cathepsin B, uPA, MMP-2, MMP-9 and MMP-14 and suppressed proteolytic activity of cathepsin B, MMP-2 and MMP-9, demonstrating a possible mechanism of ATO effect on a well-known signaling in glioblastoma dissemination. Taken together, here we suggest that ATO inhibits survival and invasion of U87MG cells possibly through NF-κB-mediated inhibition of survivin and telomerase activity and NF-κB-dependent suppression of cathepsin B, MMP-2 and MMP-9. PMID:27039805

  10. 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

  11. γδT Cell-derived interleukin-17A via an interleukin-1β-dependent mechanism mediates cardiac injury and fibrosis in hypertension.

    PubMed

    Li, Yulin; Wu, Yina; Zhang, Congcong; Li, Ping; Cui, Wei; Hao, Jianlei; Ma, Xinliang; Yin, Zhinan; Du, Jie

    2014-08-01

    Inflammation is implicated in the initiation of hypertension and end-organ injury. Interleukin-17A (IL-17A) is a key pathogenic factor in a variety of inflammatory diseases and hypertension. However, the mechanisms underlying IL-17A production, and its role in mediating inflammation and early cardiovascular injury in hypertensive heart, remain unknown. Angiotensin II (Ang II) infusion increased cardiac IL-17A mRNA expression and IL-17A+CD3+ cell infiltration in a time-dependent manner. IL-17A in the hypertensive heart was derived mostly from infiltrating γδT cells rather than from CD4 T cells. Genetic knockdown of γδT cells or specific anti-γδT antibody abolished IL-17A production in Ang II–infused heart. Moreover, monocyte-secreted IL-1β, not cardiac fibroblast–secreted IL-6 or transforming growth factor-β, was required for IL-17A production from γδT cell. IL-17A accelerated differentiation of myofibroblast through promoting IL-6 production from cardiac fibroblast. Finally, inflammatory cell infiltration, proinflammatory or profibrotic cytokine expression, and fibrotic lesion induced by Ang II were attenuated in IL-17A–deficient mice. Moreover, the deletion of γδT cell was protected from Ang II–induced cardiac injury. Thus, a triangular positive feedback loop exists among monocytic-secreted IL-1β, γδT-cell–derived IL-17A, and cardiac fibroblast–produced IL-6, which triggers the cardiac injury in hypertension. PMID:24866139

  12. Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

    PubMed

    Mushtaq, Gohar; Greig, Nigel H; Anwar, Firoz; Al-Abbasi, Fahad A; Zamzami, Mazin A; Al-Talhi, Hasan A; Kamal, Mohammad A

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  13. Neuroprotective Mechanisms Mediated by CDK5 Inhibition

    PubMed Central

    Mushtaq, Gohar; Greig, Nigel H.; Anwar, Firoz; Al-Abbasi, Fahad A.; Zamzami, Mazin A.; Al-Talhi, Hasan A.; Kamal, Mohammad A.

    2016-01-01

    Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not well-understood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine-induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer’s disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke. PMID:26601962

  14. Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

    SciTech Connect

    Pearce, N.W.; Spinelli, A.; Gurley, K.E.; Dorsch, S.E.; Hall, B.M.

    1989-07-15

    In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth (x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.

  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. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

    PubMed Central

    Saile, B.; Knittel, T.; Matthes, N.; Schott, P.; Ramadori, G.

    1997-01-01

    During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable

  17. Defect in insulin-like growth factor-1 survival mechanism in atherosclerotic plaque-derived vascular smooth muscle cells is mediated by reduced surface binding and signaling.

    PubMed

    Patel, V A; Zhang, Q J; Siddle, K; Soos, M A; Goddard, M; Weissberg, P L; Bennett, M R

    2001-05-11

    Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis. PMID:11348998

  18. 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

  19. Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.

    PubMed

    Rohringer, Sabrina; Hofbauer, Pablo; Schneider, Karl H; Husa, Anna-Maria; Feichtinger, Georg; Peterbauer-Scherb, Anja; Redl, Heinz; Holnthoner, Wolfgang

    2014-10-01

    Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering. PMID:25086616

  20. 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

  1. Cell mediated immune regulation in autoimmunity.

    PubMed

    Gillissen, G; Pusztai-Markos, Z

    1979-01-01

    Autoimmunity is the term for the immune conditions characterized by a specific humoral or cell mediated response to the body's own tissues. The termination of the natural state of self tolerance may lead to immunopathological manifestations with clinical consequences, i.e. autoimmune diseases. In a very general sense, one may classify autoimmune diseases into two groups with respect to the underlying mechanism: 1. There are autoimmune diseases which develop in the presence of a normal intact regulation mechanism. 2. Another group whose development must be understood on the basis of a cellular dysfunction. In the first case, dequestered or semi-sequestered autoantigens are liberated as a consequence of exogenic influences inducing the sensitization of immunocompetent cells. The immune system then reacts with these autoantigens in the same way as with foreign substances. This kind of autoimmune disease will, however, not be dealt with here. In the second case, autoantigens are normally, i.e. in healthy individuals, accessible to the immunocompetent cells. To understand the reason for the development of an autoimmune reaction one must first clarify the mechanism of self tolerance. Then one must examine the way in which a break of this physiological state takes place. One of the major unanswered questions is the relative importance of antibody-mediated and cell-mediated immune mechanisms in the onset and further development of autoimmune diseases. Recently it has been suggested that a dysfunction at the cellular level might represent the basic cause which induces the termination of selftolerance. Most of the conceptions about the mechanism by which autoimmune diseases are triggered were gained through experiments with animals. It is, however, difficult to use these experimental results to explain human diseases; in humans many questions are still open. Undoubtedly, the mechanisms of induction and maintenance of self tolerance and also the ways in which autoimmune

  2. Cell and tissue mechanics in cell migration

    PubMed Central

    Lange, Janina R.; Fabry, Ben

    2013-01-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  3. Cell and tissue mechanics in cell migration.

    PubMed

    Lange, Janina R; Fabry, Ben

    2013-10-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  4. 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

  5. Nonthermal-plasma-mediated animal cell death

    NASA Astrophysics Data System (ADS)

    Kim, Wanil; Woo, Kyung-Chul; Kim, Gyoo-Cheon; Kim, Kyong-Tai

    2011-01-01

    Animal cell death comprising necrosis and apoptosis occurred in a well-regulated manner upon specific stimuli. The physiological meanings and detailed molecular mechanisms of cell death have been continuously investigated over several decades. Necrotic cell death has typical morphological changes, such as cell swelling and cell lysis followed by DNA degradation, whereas apoptosis shows blebbing formation and regular DNA fragmentation. Cell death is usually adopted to terminate cancer cells in vivo. The current strategies against tumour are based on the induction of cell death by adopting various methods, including radiotherapy and chemotherapeutics. Among these, radiotherapy is the most frequently used treatment method, but it still has obvious limitations. Recent studies have suggested that the use of nonthermal air plasma can be a prominent method for inducing cancer cell death. Plasma-irradiated cells showed the loss of genomic integrity, mitochondrial dysfunction, plasma membrane damage, etc. Tumour elimination with plasma irradiation is an emerging concept in cancer therapy and can be accelerated by targeting certain tumour-specific proteins with gold nanoparticles. Here, some recent developments are described so that the mechanisms related to plasma-mediated cell death and its perspectives in cancer treatment can be understood.

  6. 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

  7. 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

  8. 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

  9. Mechanisms of platelet-mediated liver regeneration.

    PubMed

    Lisman, Ton; Porte, Robert J

    2016-08-01

    Platelets have multiple functions beyond their roles in thrombosis and hemostasis. Platelets support liver regeneration, which is required after partial hepatectomy and acute or chronic liver injury. Although it is widely assumed that platelets stimulate liver regeneration by local excretion of mitogens stored within platelet granules, definitive evidence for this is lacking, and alternative mechanisms deserve consideration. In-depth knowledge of mechanisms of platelet-mediated liver regeneration may lead to new therapeutic strategies to treat patients with failing regenerative responses. PMID:27297793

  10. 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.

  11. 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

  12. Molecular mechanisms of IgE mediated food allergy.

    PubMed

    Kumar, Sandeep; Verma, Alok Kumar; Das, Mukul; Dwivedi, Premendra D

    2012-08-01

    The purpose of this review is to collate current knowledge and recent advances in molecular mechanism behind the immediate type hypersensitivity of foods. Food allergy is a growing concern of human health in developed as well as developing countries now days. Food allergic reactions are mostly IgE mediated and also known as immediate type hypersensitivity or type I reaction. This review encompasses a wide range of molecular events during IgE mediated reactions like primary exposure of allergens, processing of allergens by antigen presenting cells, role of transcription factors like GATA-3, STAT-6, NF-AT, c-maf, c-kit and NF-κB, Treg cells, toll like receptors, cytokines and chemokines, class switch to IgE, FcεR1 receptor, priming of IgE on mast cells or basophils, signaling events followed by secondary exposure of allergens, degranulation and release of mediators like leukotrienes, histamines, prostaglandins, β-hexosaminidase and ultimately anaphylaxis. This review may be helpful to beginners as well as experts working in the field of allergy and immunology because of the stepwise explanations of molecular mechanisms involved in IgE mediated reactions. PMID:22668720

  13. Molecular pathways mediating mechanical signaling in bone

    PubMed Central

    Rubin, Janet; Rubin, Clinton; Jacobs, Christopher Rae

    2013-01-01

    Bone tissue has the capacity to adapt to its functional environment such that its morphology is “optimized” for the mechanical demand. The adaptive nature of the skeleton poses an interesting set of biological questions (e.g., how does bone sense mechanical signals, what cells are the sensing system, what are the mechanical signals that drive the system, what receptors are responsible for transducing the mechanical signal, what are the molecular responses to the mechanical stimuli). Studies of the characteristics of the mechanical environment at the cellular level, the forces that bone cells recognize, and the integrated cellular responses are providing new information at an accelerating speed. This review first considers the mechanical factors that are generated by loading in the skeleton, including strain, stress and pressure. Mechanosensitive cells placed to recognize these forces in the skeleton, osteoblasts, osteoclasts, osteocytes and cells of the vasculature are reviewed. The identity of the mechanoreceptor(s) is approached, with consideration of ion channels, integrins, connexins, the lipid membrane including caveolar and noncaveolar lipid rafts and the possibility that altering cell shape at the membrane or cytoskeleton alters integral signaling protein associations. The distal intracellular signaling systems on-line after the mechanoreceptor is activated are reviewed, including those emanating from G-proteins (e.g., intracellular calcium shifts), MAPKs, and nitric oxide. The ability to harness mechanical signals to improve bone health through devices and exercise is broached. Increased appreciation of the importance of the mechanical environment in regulating and determining the structural efficacy of the skeleton makes this an exciting time for further exploration of this area. PMID:16361069

  14. NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

    PubMed Central

    Elmetwali, T; Salman, A; Palmer, D H

    2016-01-01

    Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies. PMID:26986513

  15. NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism.

    PubMed

    Elmetwali, T; Salman, A; Palmer, D H

    2016-01-01

    Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies. PMID:26986513

  16. An antioxidant agent prevents NO sub 2 -induced inhibition of mast cell mediator release: Evidence that the mechanism involves free radicals

    SciTech Connect

    Fujimaki, Hidekazu; Shiraishi, Fujio; Wakamori, Kazuo Jikei Univ. School of Medicine, Tokyo )

    1990-12-01

    Previously we established that in vitro NO{sub 2} exposure induced inhibition of histamine release from rat peritoneal mast cells (PMC) stimulated with secretagogues such as compound 48/80 or substance P. To further explore the effects of NO{sub 2} exposure on mast cells, we investigated whether the addition of an antioxidant agent, 2-mercaptoethanol (2-ME), can prevent NO{sub 2}-induced inhibition of mediator release from PMC. Histamine release from 5 ppm NO{sub 2}-exposed PMC stimulated with 20 {mu}M substance P was also significantly inhibited compared with that from the controls. {beta}-Hexosaminidase release from 5 ppm NO{sub 2}-exposed PMC stimulated with 20 {mu}M substance P was also significantly inhibited. However, the inhibition of both histamine and {beta}-hexosaminidase release from exposed PMC was diminished by the addition of 5 mM 2-ME during NO{sub 2} exposure. Although IgE-mediated histamine release from NO{sub 2} exposed PMC was markedly inhibited, the addition of 5 mM 2-ME during NO{sub 2} exposure induced no inhibition of histamine release. These results suggest a possible relationship between NO{sub 2}-induced inhibition of mast cell mediator release and production of free radicals by the action of NO{sub 2}.

  17. Mechanical Forces Mediate Localized Topological Change in Epithelia

    PubMed Central

    Li, Yingzi; Naveed, Hammad; Kachalo, Sema; Xu, Lisa X.; Liang, Jie

    2013-01-01

    Regulation of cell growth and proliferation has a fundamental role in tissue development, organogenesis, and disease progression. Conserved distribution of the number of sides of cells with a mean value of six was found in a variety of proliferating epithelia. Previous studies have shown that clones of proliferating cells bounded by quiescent cells have fewer sides than normal epithelia. However, the mechanisms for mediating such localized topological change remain poorly understood. In this study, we use a two-dimensional vertex model with consideration of mechanical forces to investigate how differential proliferation and forces can influence cell shape and tissue morphogenesis, and how they may lead to distorted topological change. We find that differential proliferation alone is insufficient to affect the topology of boundary proliferating cells. Rather, increased surface tension on the boundary, in addition to differential proliferation, can significantly decrease the average number of cell sides. Our results are consistent with experimental observations. We conclude that mechanical forces in addition to localized differential proliferation are required to produce the distorted topological change which significantly impacts the overall cell shape and tissue morphogenesis. PMID:22254279

  18. Pathogenesis and mechanisms of antibody-mediated hemolysis

    PubMed Central

    Flegel, Willy A

    2015-01-01

    Background The clinical consequences of antibodies to red blood cells (RBC) have been studied for a century. Most clinically relevant antibodies can be detected by sensitive in vitro assays. Several mechanisms of antibody-mediated hemolysis are well understood. Such hemolysis following transfusion is reliably avoided in a donor/recipient pair, if one individual is negative for the cognate antigen to which the other has the antibody. Study design and results Mechanisms of antibody-mediated hemolysis were reviewed based on a presentation at the Strategies to Address Hemolytic Complications of Immune Globulin Infusions Workshop addressing intravenous immunoglobulin (IVIG) and ABO antibodies. The presented topics included the rates of intravascular and extravascular hemolysis; IgM and IgG isoagglutinins; auto- and alloantibodies; antibody specificity; A, B, A,B and A1 antigens; A1 versus A2 phenotypes; monocytes/macrophages, other immune cells and complement; monocyte monolayer assay (MMA); antibody-dependent cell-mediated cytotoxicity (ADCC); and transfusion reactions due to ABO and other antibodies. Conclusion Several clinically relevant questions remained unresolved, and diagnostic tools were lacking to routinely and reliably predict the clinical consequences of RBC antibodies. Most hemolytic transfusion reactions associated with IVIG were due to ABO antibodies. Reducing the titers of such antibodies in IVIG may lower the frequency of this kind of adverse event. The only way to stop these events is to have no anti-A or anti-B antibodies in the IVIG products. PMID:26174897

  19. 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

  20. The Spliceosomal Phosphopeptide P140 Controls the Lupus Disease by Interacting with the HSC70 Protein and via a Mechanism Mediated by γδ T Cells

    PubMed Central

    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 γδ 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. PMID:19390596

  1. 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.

  2. Propionibacterium acnes induces an adjuvant effect in B-1 cells and affects their phagocyte differentiation via a TLR2-mediated mechanism.

    PubMed

    Gambero, Monica; Teixeira, Daniela; Butin, Liane; Ishimura, Mayari Eika; Mariano, Mario; Popi, Ana Flavia; Longo-Maugéri, Ieda Maria

    2016-09-01

    B-1 lymphocytes are present in large numbers in the mouse peritoneal cavity, as are macrophages, and are responsible for natural IgM production. These lymphocytes migrate to inflammatory foci and are also involved in innate immunity. It was also demonstrated that B-1 cells are able to differentiated into phagocytes (B-1CDP), which is characterized by expression of F4/80 and increased phagocytic activity. B-1 cell responses to antigens and adjuvants are poorly characterized. It has been shown that Propionibacterium acnes suspensions induce immunomodulatory effects in both macrophages and B-2 lymphocytes. We recently demonstrated that this bacterium has the ability to increase B-1 cell populations both in vitro and in vivo. P. acnes induces B-1CDP differentiation, increases the expression of TLR2, TLR4 and TLR9 and augments the expression of CD80, CD86 and CD40 in B-1 and B-1CDP cells. Because P. acnes has been shown to modulate TLR expression, in this study, we investigated the role of TLR2 and TLR4 in B-1 cell population, including B-1CDP differentiation and phagocytic activity in vitro and in vivo. Interestingly, we have demonstrated that TLR2 signaling could be involved in the increase in the B-1 cell population induced by P. acnes. Furthermore, the early differentiation of B-1CDP is also dependent of TLR2. It was also observed that TLR signals also interfere in the phagocytic ability of B-1 cells and their phagocytes. According to these data, it is clear that P. acnes promotes an important adjuvant effect in B-1 cells by inducing them to differentiate into B-1CDP cells and modulates their phagocytic functions both in vivo and in vitro. Moreover, most of these effects are mediated primarily via TLR2. These data reinforce the findings that such bacterial suspensions have powerful adjuvant properties. The responses of B-1 cells to exogenous stimulation indicate that these cells are important to the innate immune response. PMID:27233619

  3. 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

  4. 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

  5. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism.

    PubMed

    Chen, Julia C; Hoey, David A; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R

    2016-04-01

    It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitmentin vitro In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cellsin vivo Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitmentin vivoand that the primary cilium contributes to this process.-Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

  6. 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

  7. The Mechanics of Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Mani, Madhav; Shraiman, Boris; Lecuit, Thomas

    2014-03-01

    Cell-intercalation involves the cytoskeleton-driven exchange of cellular neighbors. Developmental cues produce directional biases in the pattern of neighbor-exchanges, resulting in the alteration of tissue shape - morphogenesis. Focusing on cell-intercalation during early fly development, I will address both static and dynamical aspects. A quantitative correspondence is drawn between cytoskeletal levels, stresses and geometry. This construction of a constitutive law, relies on a novel image analysis tool that infers mechanical features of the cellular lattice from live imaging (from the Lecuit Lab, Marseilles). Building on our understanding of these static aspects, we construct a phenomenological, and physically-motivated, model for cytoskeletal remodeling based on temporal correlation analyses. This model predicts the qualitative phases of junctional states, insights into the T1 event that mediates intercalation, and several of the collective properties of cell-intercalation that have remained unaddressed so far - we go on to validate these predictions. We conclude with introducing the idea that tissue-wide anisotropies, central to morphogenesis and patterning in the embryo, can emerge as a consequences of the collective aspects of mechanical interactions.

  8. 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

  9. 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

  10. Mouse embryonic stem cells have underdeveloped antiviral mechanisms that can be exploited for the development of mRNA-mediated gene expression strategy.

    PubMed

    Wang, Ruoxing; Teng, Chengwen; Spangler, Joseph; Wang, Jundi; Huang, Faqing; Guo, Yan-Lin

    2014-03-15

    We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFN) when exposed to viral infection and double-stranded RNA. In this study, we extended our investigation and demonstrated that single-stranded RNA and protein-encoding mRNA can induce strong IFN expression and cytotoxicity in fibroblasts and epithelial cells, but none of the effects associated with these antiviral responses were observed in mESCs. Our results provided additional data to support the conclusion that mESCs are intrinsically deficient in antiviral responses. While our findings represent a novel feature of mESCs that in itself is important for understanding innate immunity development, we exploited this property to develop a novel mRNA-mediated gene expression cell model. Direct introduction of synthetic mRNA to express desired genes has been shown as an effective alternative to DNA/viral vector-based gene expression. However, a major biological challenge is that a synthetic mRNA is detected as a viral RNA analog by the host cell, resulting in a series of adverse effects associated with antiviral responses. We demonstrate that the lack of antiviral responses in mESCs effectively avoids this problem. mESCs can tolerate repeated transfection and effectively express proteins from their synthetic mRNA with expected biological functions, as demonstrated by the expression of green fluorescent protein and the transcription factor Etv2. Therefore, mRNA-based gene expression could be developed into a novel ESC differentiation strategy that avoids safety concerns associated with viral/DNA-based vectors in regenerative medicine. PMID:24219369

  11. 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

  12. Identifying immune mechanisms mediating the hypertension during preeclampsia.

    PubMed

    LaMarca, Babbette; Cornelius, Denise C; Harmon, Ashlyn C; Amaral, Lorena M; Cunningham, Mark W; Faulkner, Jessica L; Wallace, Kedra

    2016-07-01

    Preeclampsia (PE) is a pregnancy-associated disorder that affects 5-8% of pregnancies and is a major cause of maternal, fetal, and neonatal morbidity and mortality. Hallmark characteristics of PE are new onset hypertension after 20 wk gestation with or without proteinuria, chronic immune activation, fetal growth restriction, and maternal endothelial dysfunction. However, the pathophysiological mechanisms that lead to the development of PE are poorly understood. Recent data from studies of both clinical and animal models demonstrate an imbalance in the subpopulations of CD4+ T cells and a role for these cells as mediators of inflammation and hypertension during pregnancy. Specifically, it has been proposed that the imbalance between two CD4+ T cell subtypes, regulatory T cells (Tregs) and T-helper 17 cells (Th17s), is involved in the pathophysiology of PE. Studies from our laboratory highlighting how this imbalance contributes to vasoactive factors, endothelial dysfunction, and hypertension during pregnancy will be discussed in this review. Therefore, the purpose of this review is to highlight hypertensive mechanisms stimulated by inflammatory factors in response to placental ischemia, thereby elucidating a role. PMID:27097659

  13. 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. PMID:25682075

  14. 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.

  15. Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component

    SciTech Connect

    Sykes, M.; Eisenthal, A.; Sachs, D.H.

    1988-05-01

    Splenocyte populations from whole body-irradiated recipients of mixed T cell-depleted (TCD) syngeneic and allogeneic (complete H-2 disparity) bone marrow, or of TCD syngeneic marrow alone, contain cells with the ability to suppress the generation of cell-mediated lympholysis responses in vitro. This activity, which is present by 8 days after bone marrow transplantation and persists for several weeks, has been analyzed for possible veto-like or other specificity. Although reproducible patterns of suppression were observed, depending both on host strain and on the genetic combination of the response examined, the overall suppression in vitro most closely resembles that which has been ascribed to natural suppressor cells in other systems. The suppression appears to be mediated by a non-T cell, non-B cell, nonadherent, asialo GM1-negative population. Cold target inhibition and CTL activity of chimeric cells have been ruled out as factors contributing to the observed suppression. Significantly, in mixed chimeras, suppression was found to be mediated exclusively by cells which were syngeneic to the recipient in both recipient strains tested. The rapid development of this suppressive activity may explain the resistance to graft-vs-host disease conferred on whole body-irradiated mice by the addition of TCD syngeneic marrow to an allogeneic graft-vs-host disease-producing inoculum.

  16. S. aureus haemolysin A-induced IL-8 and IL-6 release from human airway epithelial cells is mediated by activation of p38- and Erk-MAP kinases and additional, cell type-specific signalling mechanisms.

    PubMed

    Räth, Susann; Ziesemer, Sabine; Witte, Amelie; Konkel, Anne; Müller, Christian; Hildebrandt, Petra; Völker, Uwe; Hildebrandt, Jan-Peter

    2013-07-01

    Soluble virulence-associated factors of Staphylococcus aureus like haemolysin A (Hla) induce secretion of chemo/cytokines from airway epithelial cells. To elucidate the potential roles of specific signalling pathways in this response, we treated 16HBE14o-, S9 or A549 cells with recombinant Hla (rHla). In a dose-dependent manner, rHla induced secretion of IL-8 in all three cell types, but IL-6 release only in 16HBE14o- and S9 cells. rHla-mediated secretion of IL-8 and IL-6 was suppressed by pre-incubation of cells with inhibitors of Erk type or p38 MAP kinases, indicating that activation of these signalling pathways is essential for IL-8 release in all three cell types and for IL-6 release in 16HBE14o- and S9 cells. The rHla-mediated phosphorylation and activation of p38 MAP kinase seem to depend on elevations in [Ca(2+)]i, an early response in rHla-treated cells. Inhibitors of calmodulin or calcium/calmodulin-dependent kinase II attenuated rHla-mediated release of IL-8 in 16HBE14o- and A549 cells and of IL-6 in 16HBE14o- cells. This indicates that rHla may mediate simultaneous activation of calmodulin-dependent processes as additional prerequisites for chemo/cytokine secretion.However, the inhibitors of calmodulin-dependent signalling did not affect rHla-induced p38 MAP kinase phosphorylation, indicating that this pathway works in parallel with p38 MAP kinase. PMID:23347173

  17. 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. PMID:12782208

  18. Down Regulation of the TCR Complex CD3ζ-Chain on CD3+ T Cells: A Potential Mechanism for Helminth-Mediated Immune Modulation

    PubMed Central

    Appleby, Laura J.; Nausch, Norman; Heard, Francesca; Erskine, Louise; Bourke, Claire D.; Midzi, Nicholas; Mduluza, Takafira; Allen, Judith E.; Mutapi, Francisca

    2015-01-01

    The CD3ζ forms part of the T cell receptor (TCR) where it plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways leading to T cell effector functions. Down regulation of CD3ζ leads to impairment of immune responses including reduced cell proliferation and cytokine production. In experimental models, helminth parasites have been shown to modulate immune responses directed against them and unrelated antigens, so called bystander antigens, but there is a lack of studies validating these observations in humans. This study investigated the relationship between expression levels of the TCR CD3ζ chain with lymphocyte cell proliferation during human infection with the helminth parasite, Schistosoma haematobium, which causes uro-genital schistosomiasis. Using flow cytometry, peripheral blood mononuclear cells (PBMCs) from individuals naturally exposed to S. haematobium in rural Zimbabwe were phenotyped, and expression levels of CD3ζ on T cells were related to intensity of infection. In this population, parasite infection intensity was inversely related to CD3ζ expression levels (p < 0.05), consistent with downregulation of CD3ζ expression during helminth infection. Furthermore, PBMC proliferation was positively related to expression levels of CD3ζ (p < 0.05) after allowing for confounding variables (host age, sex, and infection level). CD3ζ expression levels had a differing relationship between immune correlates of susceptibility and immunity, measured by antibody responses, indicating a complex relationship between immune activation status and immunity. The relationships between the CD3ζ chain of the TCR and schistosome infection, PBMC proliferation and schistosome-specific antibody responses have not previously been reported, and these results may indicate a mechanism for the impaired T cell proliferative responses observed during human schistosome infection. PMID:25741337

  19. 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

  20. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25644501

  2. B-cell-activating factor inhibits CD20-mediated and B-cell receptor-mediated apoptosis in human B cells

    PubMed Central

    Saito, Yohei; Miyagawa, Yoshitaka; Onda, Keiko; Nakajima, Hideki; Sato, Ban; Horiuchi, Yasuomi; Okita, Hajime; Katagiri, Yohko U; Saito, Masahiro; Shimizu, Toshiaki; Fujimoto, Junichiro; Kiyokawa, Nobutaka

    2008-01-01

    B-cell-activating factor (BAFF) is a survival and maturation factor for B cells belonging to the tumour necrosis factor superfamily. Among three identified functional receptors, the BAFF receptor (BAFF-R) is thought to be responsible for the effect of BAFF on B cells though details of how remain unclear. We determined that a hairy-cell leukaemia line, MLMA, expressed a relatively high level of BAFF-R and was susceptible to apoptosis mediated by either CD20 or B-cell antigen receptor (BCR). Using MLMA cells as an in vitro model of mature B cells, we found that treatment with BAFF could inhibit apoptosis mediated by both CD20 and BCR. We also observed, using immunoblot analysis and microarray analysis, that BAFF treatment induced activation of nuclear factor-κB2 following elevation of the expression level of Bcl-2, which may be involved in the molecular mechanism of BAFF-mediated inhibition of apoptosis. Interestingly, BAFF treatment was also found to induce the expression of a series of genes, such as that for CD40, related to cell survival, suggesting the involvement of a multiple mechanism in the BAFF-mediated anti-apoptotic effect. MLMA cells should provide a model for investigating the molecular basis of the effect of BAFF on B cells in vitro and will help to elucidate how B cells survive in the immune system in which BAFF-mediated signalling is involved. PMID:18540961

  3. HIV-1 Tat Protein Induces PD-L1 (B7-H1) Expression on Dendritic Cells through Tumor Necrosis Factor Alpha- and Toll-Like Receptor 4-Mediated Mechanisms

    PubMed Central

    Planès, Rémi; BenMohamed, Lbachir; Leghmari, Kaoutar; Delobel, Pierre; Izopet, Jacques

    2014-01-01

    ABSTRACT Chronic human immunodeficiency virus type 1 (HIV-1) infection is associated with induction of T-cell coinhibitory pathways. However, the mechanisms by which HIV-1 induces upregulation of coinhibitory molecules remain to be fully elucidated. The aim of the present study was to determine whether and how HIV-1 Tat protein, an immunosuppressive viral factor, induces the PD-1/PD-L1 coinhibitory pathway on human dendritic cells (DCs). We found that treatment of DCs with whole HIV-1 Tat protein significantly upregulated the level of expression of PD-L1. This PD-L1 upregulation was observed in monocyte-derived dendritic cells (MoDCs) obtained from either uninfected or HIV-1-infected patients as well as in primary myeloid DCs from HIV-negative donors. In contrast, no effect on the expression of PD-L2 or PD-1 molecules was detected. The induction of PD-L1 on MoDCs by HIV-1 Tat (i) occurred in dose- and time-dependent manners, (ii) was mediated by the N-terminal 1–45 fragment of Tat, (iii) did not require direct cell-cell contact but appeared rather to be mediated by soluble factor(s), (iv) was abrogated following neutralization of tumor necrosis factor alpha (TNF-α) or blocking of Toll-like receptor 4 (TLR4), (v) was absent in TLR4-knockoout (KO) mice but could be restored following incubation with Tat-conditioned medium from wild-type DCs, (vi) impaired the capacity of MoDCs to functionally stimulate T cells, and (vii) was not reversed functionally following PD-1/PD-L1 pathway blockade, suggesting the implication of other Tat-mediated coinhibitory pathways. Our results demonstrate that HIV-1 Tat protein upregulates PD-L1 expression on MoDCs through TNF-α- and TLR4-mediated mechanisms, functionally compromising the ability of DCs to stimulate T cells. The findings offer a novel potential molecular target for the development of an anti-HIV-1 treatment. IMPORTANCE The objective of this study was to investigate the effect of human immunodeficiency virus type 1 (HIV

  4. 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

  5. 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

  6. Heat stroke: opioid-mediated mechanisms.

    PubMed

    Romanovsky, A A; Blatteis, C M

    1996-12-01

    In our previous study in guinea pigs, intensive and prolonged intraperitoneal heating (IPH) caused heat stroke characterized by high mortality and accompanied by two paradoxical phenomena: ear skin vasoconstriction at a high body temperature (Tb) (hyperthermia-induced vasoconstriction) and a post-IPH Tb fall at an ambient temperature (Ta) below thermoneutrality (hyperthermia-induced hypothermia). In this study, we tested the hypothesis that the mechanisms of the two phenomena involve endogenous opioid agonists. Experiments were conducted in 24 unanesthetized, lightly restrained guinea pigs, each chronically implanted with an intraperitoneal thermode and intrahypothalamic thermocouple. The thermoregulatory effects of a wide-spectrum opioid-receptor antagonist, naltrexone (NTX; 50 or 0 mumol/kg sc), were studied in IPH-induced heat stroke and under normal conditions. IPH was accomplished by perfusing (50 ml/min; 80 min) water (45 degrees C) through the thermode. Ta was maintained at approximately 24 degrees C. Skin vasodilation occurred at the onset of IPH but later changed to vasoconstriction despite high Tb and continuing IPH. IPH-induced hyperthermia (1.8 +/- 0.1 degrees C) was followed by a post-IPH Tb fall (-5.1 +/- 0.7 degree C; calculated for the survivors only). The 48-h mortality rate was 50%. NTX prevented the hyperthermia-induced vasoconstriction and attenuated the hyperthermia-induced hypothermia (-1.8 +/- 0.4 degree C). None of the NTX-treated animals died. The effects of NTX on Tb regulation under normal conditions were minor. These results indicate that the phenomena of both hyperthermia-induced vasoconstriction and hyperthermia-induced hypothermia are opioid dependent. The latter is speculated to reflect opioid-mediated inhibition of metabolism; the former is thought to result from opioid-induced hemodynamic alterations. Because both phenomena did not occur in the NTX-treated survivors, the skin vasoconstriction at high Tb and the posthyperthermia Tb

  7. Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

    PubMed

    Boutillier, A L; Barthel, F; Roberts, J L; Loeffler, J P

    1992-11-25

    Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants. PMID:1331087

  8. Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism.

    PubMed

    Jiang, N; Dreher, K L; Dye, J A; Li, Y; Richards, J H; Martin, L D; Adler, K B

    2000-03-15

    Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 microg/cm(2) ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular

  9. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. )

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  10. 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

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

    PubMed

    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

  12. 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

  13. 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

  14. Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

    PubMed Central

    Lopez-Haber, Cynthia

    2013-01-01

    The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl l-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac. PMID:23478800

  15. 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.

  16. 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

  17. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.

    PubMed

    Antognelli, Cinzia; Gambelunghe, Angela; Muzi, Giacomo; Talesa, Vincenzo Nicola

    2015-07-01

    Glyoxalase I (Glo1) is a cellular defense enzyme involved in the detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis, and MG-derived advanced glycation end products (AGEs). Argpyrimidine (AP), one of the major AGEs coming from MG modification of protein arginines, is a proapoptotic agent. Crystalline silica is a well-known occupational health hazard, responsible for a relevant number of pulmonary diseases. Exposure of cells to crystalline silica results in a number of complex biological responses, including apoptosis. The present study was aimed at investigating whether, and through which mechanism, Glo1 was involved in Min-U-Sil 5 crystalline silica-induced apoptosis. Apoptosis, by TdT-mediated dUTP nick-end labeling assay, and transcript and protein levels or enzymatic activity, by quantitative real-time PCR, Western blot, and spectrophotometric methods, respectively, were evaluated in human bronchial BEAS-2B cells exposed or not (control) to crystalline silica and also in experiments with appropriate inhibitors. Reactive oxygen species were evaluated by coumarin-7-boronic acid or Amplex red hydrogen peroxide/peroxidase methods for peroxynitrite (ONOO(-)) or hydrogen peroxide (H2O2) measurements, respectively. Our results showed that Min-U-Sil 5 crystalline silica induced a dramatic ONOO(-)-mediated inhibition of Glo1, leading to AP-modified Hsp70 protein accumulation that, in a mechanism involving JNK and NF-κB, triggered an apoptotic mitochondrial pathway. Inhibition of Glo1 occurred at both functional and transcriptional levels, the latter occurring via ERK1/2 MAPK and miRNA 101 involvement. Taken together, our data demonstrate that Glo1 is involved in the Min-U-Sil 5 crystalline silica-induced BEAS-2B cell mitochondrial apoptotic pathway via a novel mechanism involving Hsp70, JNK, and NF-κB. Because maintenance of an intact respiratory epithelium is a critically important determinant of normal respiratory function, the knowledge of

  18. Structural mechanisms of chaperone mediated protein disaggregation

    PubMed Central

    Sousa, Rui

    2014-01-01

    The ClpB/Hsp104 and Hsp70 classes of molecular chaperones use ATP hydrolysis to dissociate protein aggregates and complexes, and to move proteins through membranes. ClpB/Hsp104 are members of the AAA+ family of proteins which form ring-shaped hexamers. Loops lining the pore in the ring engage substrate proteins as extended polypeptides. Interdomain rotations and conformational changes in these loops coupled to ATP hydrolysis unfold and pull proteins through the pore. This provides a mechanism that progressively disrupts local secondary and tertiary structure in substrates, allowing these chaperones to dissociate stable aggregates such as β-sheet rich prions or coiled coil SNARE complexes. While the ClpB/Hsp104 mechanism appears to embody a true power-stroke in which an ATP powered conformational change in one protein is directly coupled to movement or structural change in another, the mechanism of force generation by Hsp70s is distinct and less well understood. Both active power-stroke and purely passive mechanisms in which Hsp70 captures spontaneous fluctuations in a substrate have been proposed, while a third proposed mechanism—entropic pulling—may be able to generate forces larger than seen in ATP-driven molecular motors without the conformational coupling required for a power-stroke. The disaggregase activity of these chaperones is required for thermotolerance, but unrestrained protein complex/aggregate dissociation is potentially detrimental. Disaggregating chaperones are strongly auto-repressed, and are regulated by co-chaperones which recruit them to protein substrates and activate the disaggregases via mechanisms involving either sequential transfer of substrate from one chaperone to another and/or simultaneous interaction of substrate with multiple chaperones. By effectively subjecting substrates to multiple levels of selection by multiple chaperones, this may insure that these potent disaggregases are only activated in the appropriate context. PMID

  19. 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

  20. Mechanics of the Cell

    NASA Astrophysics Data System (ADS)

    Boal, David

    2012-01-01

    Preface; List of symbols; 1. Introduction to the cell; 2. Soft materials and fluids; Part I. Rods and Ropes: 3. Polymers; 4. Complex filaments; 5. Two-dimensional networks; 6. Three-dimensional networks; Part II. Membranes: 7. Biomembranes; 8. Membrane undulations; 9. Intermembrane and electrostatic forces; Part III. The Whole Cell: 10. Structure of the simplest cells; 11. Dynamic filaments; 12. Growth and division; 13. Signals and switches; Appendixes; Glossary; References; Index.

  1. 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

  2. Thermally mediated mechanism to enhance magnetoelectric coupling in multiferroics.

    PubMed

    Chang, C-M; Mani, B K; Lisenkov, S; Ponomareva, I

    2015-05-01

    The main roadblock on the way to practical realization of magnetoelectric devices is the lack of multiferroics with strong magnetoelectric coupling. We propose an unusual route to dramatically enhance this coupling through a thermally mediated mechanism. Such a thermally mediated magnetoelectric effect is quantified by an isentropic rather than isothermal magnetoelectric response and is computed here from first principles. A robust enhancement of the magnetoelectric coupling is predicted for both naturally occurring and heterostructured materials. PMID:25978260

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

    PubMed

    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

  4. 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

  5. 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

  6. Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions

    PubMed Central

    Chatterjee, Moumita; Sequeira, Linda; Jenkins-Kabaila, Mashariki; Dubyk, Cara W.; Pathak, Surabhi; van Golen, Kenneth L.

    2011-01-01

    The Rho GTPases organize the actin cytoskeleton and are involved in cancer metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3 prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained activation of Rac1 GTPase and morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition. We also reported that Rac1 is required for PC-3 cell diapedesis across a bone marrow endothelial cell layer. In the current study, we queried whether Rac3 and RhoG GTPases also have a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation. Here we find that RhoG partially contributes to CCL2-induced tumor cell diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate cancer cells to bone marrow endothelial cells and promotes retraction of endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1 integrin activation, suggesting a mechanism that Rac1 can mediate tight binding with endothelial cells. Together, our data suggest that Rac1 GTPase is key mediator of prostate cancer cell-bone marrow endothelial cell interactions. PMID:21776386

  7. 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

  8. 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

  9. 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).

  10. 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

  11. 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

  12. 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

  13. Interferons Mediate Terminal Differentiation of Human Cortical Thymic Epithelial Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Laine, David; Zaffran, Yona; Azocar, Olga; Servet-Delprat, Christine; Wild, T. Fabian; Rabourdin-Combe, Chantal; Valentin, Hélène

    2002-01-01

    In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-β) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-β induction. Finally, we demonstrated that recombinant IFN-α, IFN-β, or IFN-γ was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections. PMID:12050353

  14. Hypoxia-mediated regulation of stem cell fate.

    PubMed

    Singh, Rashim Pal; Franke, Kristin; Wielockx, Ben

    2012-09-01

    Hypoxia-mediated regulation of stem cell fate, or reduced oxygen availability, is a prominent feature during mammalian development and under physiological and pathological conditions in adults. Oxygen-sensing is therefore indispensable as it enables the cells to adapt instantaneously to an inappropriate pO(2). This machinery relies primarily on hypoxia inducible factor (HIF). Moreover, a growing body of evidence proposes that different types of stem cells exist in a very hypoxic microenvironment, which may be beneficial for the maintenance of these cells and ensures continuous replenishment of dead or damaged cells in virtually all tissues of the body. Recent reports have shown that HIF is a critical player in these responses. However, a better understanding of the different HIF-related mechanisms is of utmost importance for the improvement of therapeutic strategies for tissue regeneration as well as hematological malignancies. PMID:22994515

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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…

  20. Cell-mediated immunity to schistosomes. Evaluation of mechanisms operating against lung-stage parasites which might be exploited in a vaccine.

    PubMed

    Pemberton, R M; Malaquias, L C; Falcao, P L; Silveira, A M; Rabello, A L; Katz, N; Amorim, M; Mountford, A P; Coffman, R L; Correa-Oliveira, R

    1994-01-01

    This report describes parallel studies examining T cell and cytokine responses to Schistosoma mansoni in mice and man. The prevalence of IFNg production amongst murine (C57BL/6) T cell lines and clones, plus good DTH reactivity by IFNg-secreting clones, highlights the predominance of the Th1 response in the pulmonary immunity characteristics of the murine irradiated vaccine model. In human studies, effects of anti-cytokine antibodies on the proliferation of PBMC from human patients to various soluble schistosome antigen preparations have been examined. Data suggest that both Th1 (against early antigens) and Th2 (against late antigens) responses are present. A role for IL-10 is highlighted in chronic intestinal, but not acute or chronic hepatosplenic patients, as a downregulator of responses which are associated with morbidity and are against late stage antigens. PMID:7825229

  1. 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

  2. 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. PMID:26450984

  3. B-Cell-Mediated Strategies to Fight Chronic Allograft Rejection

    PubMed Central

    Dalloul, Ali

    2013-01-01

    Solid organs have been transplanted for decades. Since the improvement in graft selection and in medical and surgical procedures, the likelihood of graft function after 1 year is now close to 90%. Nonetheless even well-matched recipients continue to need medications for the rest of their lives hence adverse side effects and enhanced morbidity. Understanding Immune rejection mechanisms, is of increasing importance since the greater use of living-unrelated donors and genetically unmatched individuals. Chronic rejection is devoted to T-cells, however the role of B-cells in rejection has been appreciated recently by the observation that B-cell depletion improve graft survival. By contrast however, B-cells can be beneficial to the grafted tissue. This protective effect is secondary to either the secretion of protective antibodies or the induction of B-cells that restrain excessive inflammatory responses, chiefly by local provision of IL-10, or inhibit effector T-cells by direct cellular interactions. As a proof of concept B-cell-mediated infectious transplantation tolerance could be achieved in animal models, and evidence emerged that the presence of such B-cells in transplanted patients correlate with a favorable outcome. Among these populations, regulatory B-cells constitute a recently described population. These cells may develop as a feedback mechanism to prevent uncontrolled reactivity to antigens and inflammatory stimuli. The difficult task for the clinician, is to quantify the respective ratios and functions of “tolerant” vs. effector B-cells within a transplanted organ, at a given time point in order to modulate B-cell-directed therapy. Several receptors at the B-cell membrane as well as signaling molecules, can now be targeted for this purpose. Understanding the temporal expansion of regulatory B-cells in grafted patients and the stimuli that activate them will help in the future to implement specific strategies aimed at fighting chronic allograft

  4. NKG2D ligands mediate immunosurveillance of senescent cells

    PubMed Central

    Moshayev, Zhana; Vadai, Ezra; Wensveen, Felix; Ben-Dor, Shifra; Golani, Ofra; Polic, Bojan; Krizhanovsky, Valery

    2016-01-01

    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

  5. 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

  6. The mechanics of cell crawling

    NASA Astrophysics Data System (ADS)

    Wolgemuth, Charles; Mogilner, Alex; Oster, George

    2004-03-01

    Crawling eukaryotic cells play many roles in biology. White blood cells chemotactically track down pathogens. Fibroblasts crawl and pull skin back together during wound healing. Cancer cells become metastatic and migrate to other points in the body. Therefore, understanding the physical mechanism driving crawling motility is very important. In crawling cells, a polymer meshwork, usually composed of actin filaments, provides both the structural integrity of the cell and is reponsible for the force production during migration. We present a theory that describes the dynamics of this actin gel and apply it to the crawling motility of nematode sperm cells. This model maintains the shape of the cell during crawling as occurs during the migration of Ascaris suum spermatozoa and also produces forces comparable to what has been measured in other crawling cells.

  7. Regulatory T cells in immune-mediated renal disease.

    PubMed

    Ghali, Joanna R; Wang, Yuan Min; Holdsworth, Stephen R; Kitching, A Richard

    2016-02-01

    Regulatory T cells (Tregs) are CD4+ T cells that can suppress immune responses by effector T cells, B cells and innate immune cells. This review discusses the role that Tregs play in murine models of immune-mediated renal diseases and acute kidney injury and in human autoimmune kidney disease (such as systemic lupus erythematosus, anti-glomerular basement membrane disease, anti-neutrophil cytoplasmic antibody-associated vasculitis). Current research suggests that Tregs may be reduced in number and/or have impaired regulatory function in these diseases. Tregs possess several mechanisms by which they can limit renal and systemic inflammatory immune responses. Potential therapeutic applications involving Tregs include in vivo induction of Tregs or inducing Tregs from naïve CD4+ T cells or expanding natural Tregs ex vivo, to use as a cellular therapy. At present, the optimal method of generating a phenotypically stable pool of Tregs with long-lasting suppressive effects is not established, but human studies in renal transplantation are underway exploring the therapeutic potential of Tregs as a cellular therapy, and if successful may have a role as a novel therapy in immune-mediated renal diseases. PMID:26206106

  8. Minimal Synthetic Cells to Study Integrin-Mediated Adhesion

    PubMed Central

    Frohnmayer, Johannes P; Brüggemann, Dorothea; Eberhard, Christian; Neubauer, Stefanie; Mollenhauer, Christine; Boehm, Heike; Kessler, Horst; Geiger, Benjamin; Spatz, Joachim P

    2015-01-01

    To shed light on cell-adhesion-related molecular pathways, synthetic cells offer the unique advantage of a well-controlled model system with reduced molecular complexity. Herein, we show that liposomes with the reconstituted platelet integrin αIIbβ3 as the adhesion-mediating transmembrane protein are a functional minimal cell model for studying cellular adhesion mechanisms in a defined environment. The interaction of these synthetic cells with various extracellular matrix proteins was analyzed using a quartz crystal microbalance with dissipation monitoring. The data indicated that integrin was functionally incorporated into the lipid vesicles, thus enabling integrin-specific adhesion of the engineered liposomes to fibrinogen- and fibronectin-functionalized surfaces. Then, we were able to initiate the detachment of integrin liposomes from these surfaces in the presence of the peptide GRGDSP, a process that is even faster with our newly synthesized peptide mimetic SN529, which specifically inhibits the integrin αIIbβ3. PMID:26257266

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

    PubMed

    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. PMID:19932089

  10. 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.

  11. Myeloid-derived Suppressor Cells Adhere to Physiologic STAT3- vs STAT5-dependent Hematopoietic Programming, Establishing Diverse Tumor-Mediated Mechanisms of Immunologic Escape

    PubMed Central

    Cohen, Peter A.; Ko, Jennifer S.; Storkus, Walter J.; Spencer, Christopher D.; Bradley, Judy M.; Gorman, Jessica E.; McCurry, Dustin B.; Zorro-Manrique, Soroya; Dominguez, Anna Lucia; Pathangey, Latha B.; Rayman, Patricia A.; Rini, Brian I.; Gendler, Sandra J.; Finke, James H.

    2013-01-01

    The receptor tyrosine kinase inhibitor, sunitinib, is astonishingly effective in its capacity to reduce MDSCs in peripheral tissues such as blood (human) and spleen (mouse), restoring responsiveness of bystander T lymphocytes to TcR stimulation. Sunitinib blocks proliferation of undifferentiated MDSCs and decreases survival of more differentiated neutrophilic MDSC (n-MDSC) progeny. Ironically, sunitinib’s profound effects are observed even in a total absence of detectable anti-tumor therapeutic response. This is best explained by the presence of disparate MDSC-conditioning stimuli within individual body compartments, allowing sensitivity and resistance to sunitinib to coexist within the same mouse or patient. The presence or absence of GM-CSF is likely the major determinant in each compartment, given that GM-CSF’s capacity to preempt STAT3-dependent with dominant STAT5-dependent hematopoietic programming confers sunitinib resistance and redirects differentiation from the n-MDSC lineage to the more versatile monocytoid (m-MDSC) lineage. The clinical sunitinib experience underscores that strategies for MDSC and Treg depletions must be mindful of disparities among body compartments to avoid sanctuary effects. Ironically, m-MDSCs manifesting resistance to sunitinib also have the greatest potential to differentiate into tumoricidal accessory cells, by virtue of their capacity to respond to T cell-secreted IFN-γ or to TLR agonists with nitric oxide and peroxynitrate production. PMID:23017141

  12. Study of Molecular Mechanisms Involved in the Pathogenesis of Immune-Mediated Inflammatory Diseases, using Psoriasis As a Model

    PubMed Central

    Sobolev, V.V.; Abdeev, R.M.; Zolotarenko, A.D.; Nikolaev, A.A.; Sarkisova, M.K.; Sautin, M.E.; Ishkin, A.A.; Piruzyan, An.L.; Ilyina, S.A.; Korsunskaya, I.M.; Rahimova, O.Y.; Bruskin, S.A.

    2009-01-01

    Psoriasis was used as a model to analyze the pathogenetic pathways of immune-mediated inflammatory diseases, and the results of bioinformatic, molecular-genetic and proteomic studies are provided. Cell mechanisms, common for the pathogenesis of psoriasis, as well as Crohn's disease, are identified. New approaches for immune-mediated diseases are discussed. PMID:22649625

  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. TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin to integrin signaling

    PubMed Central

    Thodeti, Charles K.; Matthews, Benjamin; Ravi, Arvind; Mammoto, Akiko; Ghosh, Kaustabh; Bracha, Abigail L.; Ingber, Donald E.

    2009-01-01

    Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels, and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response, however, the molecular basis remains unknown. Here we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 ion channels that, in turn, stimulate phosphatidyl inositol-3-kinase-dependent activation and binding of additional ·1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knockdown of TRPV4 channels using specific siRNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent ‘integrin to integrin’ signaling mechanism mediated by force-induced activation of mechanically-gated TRPV4 ion channels on the cell surface. PMID:19359599

  15. ATR mediates a checkpoint at the nuclear envelope in response to mechanical stress.

    PubMed

    Kumar, Amit; Mazzanti, Michele; Mistrik, Martin; Kosar, Martin; Beznoussenko, Galina V; Mironov, Alexandre A; Garrè, Massimiliano; Parazzoli, Dario; Shivashankar, G V; Scita, Giorgio; Bartek, Jiri; Foiani, Marco

    2014-07-31

    ATR controls chromosome integrity and chromatin dynamics. We have previously shown that yeast Mec1/ATR promotes chromatin detachment from the nuclear envelope to counteract aberrant topological transitions during DNA replication. Here, we provide evidence that ATR activity at the nuclear envelope responds to mechanical stress. Human ATR associates with the nuclear envelope during S phase and prophase, and both osmotic stress and mechanical stretching relocalize ATR to nuclear membranes throughout the cell cycle. The ATR-mediated mechanical response occurs within the range of physiological forces, is reversible, and is independent of DNA damage signaling. ATR-defective cells exhibit aberrant chromatin condensation and nuclear envelope breakdown. We propose that mechanical forces derived from chromosome dynamics and torsional stress on nuclear membranes activate ATR to modulate nuclear envelope plasticity and chromatin association to the nuclear envelope, thus enabling cells to cope with the mechanical strain imposed by these molecular processes. PMID:25083873

  16. Novel mechanism of apoptosis resistance in cancer mediated by extracellular PAR-4.

    PubMed

    Burikhanov, Ravshan; Shrestha-Bhattarai, Tripti; Qiu, Shirley; Shukla, Nidhi; Hebbar, Nikhil; Lele, Subodh M; Horbinski, Craig; Rangnekar, Vivek M

    2013-01-15

    Tumor suppressor PAR-4 acts in part by modulating sensitivity to apoptosis, but the basis for its activity is not fully understood. In this study, we describe a novel mechanism of antiapoptosis by NF-κB, revealing that it can block PAR-4-mediated apoptosis by downregulating trafficking of the PAR-4 receptor GRP78 from the endoplasmic reticulum to the cell surface. Mechanistic investigations revealed that NF-κB mediated this antiapoptotic mechanism by upregulating expression of UACA, a proinflammatory protein in certain disease settings. In clinical specimens of cancer, a strong correlation existed between NF-κB activity and UACA expression, relative to normal tissues. UACA bound to intracellular PAR-4 in diverse cancer cells, where it prevented translocation of GRP78 from the endoplasmic reticulum to the cell surface. This pathway of antiapoptosis could be inhibited by suppressing levels of NF-κB or UACA expression, which enhanced endoplasmic reticulum stress and restored GRP78 trafficking to the cell surface, thereby sensitizing cancer cells to apoptosis by extracellular PAR-4 or GRP78 agonistic antibody. In summary, our results identify a novel intracellular pathway of apoptosis mediated by NF-κB through UACA elevation, which by attenuating endoplasmic reticulum stress and GRP78 translocation to the cell surface can blunt the sensitivity of cancer cells to apoptosis. PMID:23204231

  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. Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells.

    PubMed

    Schulz, Cordula; Wood, Cricket G; Jones, D Leanne; Tazuke, Salli I; Fuller, Margaret T

    2002-10-01

    Germ cells normally differentiate in the context of encapsulating somatic cells. However, the mechanisms that set up the special relationship between germ cells and somatic support cells and the signals that mediate the crucial communications between the two cell types are poorly understood. We show that interactions between germ cells and somatic support cells in Drosophila depend on wild-type function of the stet gene. In males, stet acts in germ cells to allow their encapsulation by somatic cyst cells and is required for germ cell differentiation. In females, stet function allows inner sheath cells to enclose early germ cells correctly at the tip of the germarium. stet encodes a homolog of rhomboid, a component of the epidermal growth factor receptor signaling pathway involved in ligand activation in the signaling cell. The stet mutant phenotype suggests that stet facilitates signaling from germ cells to the epidermal growth factor receptor on somatic cells, resulting in the encapsulation of germ cells by somatic support cells. The micro-environment provided by the surrounding somatic cells may, in turn, regulate differentiation of the germ cells they enclose. PMID:12223409

  19. Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism.

    PubMed

    Gegear, Robert J; Foley, Lauren E; Casselman, Amy; Reppert, Steven M

    2010-02-11

    Understanding the biophysical basis of animal magnetoreception has been one of the greatest challenges in sensory biology. Recently it was discovered that the light-dependent magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry). Here we show, using a transgenic approach, that the photoreceptive, Drosophila-like type 1 Cry and the transcriptionally repressive, vertebrate-like type 2 Cry of the monarch butterfly (Danaus plexippus) can both function in the magnetoreception system of Drosophila and require UV-A/blue light (wavelength below 420 nm) to do so. The lack of magnetic responses for both Cry types at wavelengths above 420 nm does not fit the widely held view that tryptophan triad-generated radical pairs mediate the ability of Cry to sense a magnetic field. We bolster this assessment by using a mutant form of Drosophila and monarch type 1 Cry and confirm that the tryptophan triad pathway is not crucial in magnetic transduction. Together, these results suggest that animal Crys mediate light-dependent magnetoreception through an unconventional photochemical mechanism. This work emphasizes the utility of Drosophila transgenesis for elucidating the precise mechanisms of Cry-mediated magnetosensitivity in insects and also in vertebrates such as migrating birds. PMID:20098414

  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. Overexpression of kinesins mediates docetaxel resistance in breast cancer cells.

    PubMed

    De, Sarmishtha; Cipriano, Rocky; Jackson, Mark W; Stark, George R

    2009-10-15

    Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumor-derived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes. PMID:19789344

  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. Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity.

    PubMed

    Colombo, E; Tentorio, P; Musio, S; Rajewsky, K; Pedotti, R; Casola, S; Farina, C

    2014-04-01

    B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP₁₈₅₋₂₀₆. However, ex-vivo T cell proliferation to PLP₁₈₅₋₂₀₆ peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses. PMID:24325711

  6. 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

  7. Immune-mediated mechanism for thrombocytopenia after Loxosceles spider bite.

    PubMed

    Levin, Carina; Bonstein, Lilach; Lauterbach, Roy; Mader, Rivka; Rozemman, Dganit; Koren, Ariel

    2014-08-01

    Loxoscelism, characterized by high fever, vomiting, malaise, a dermonecrotic lesion, and thrombocytopenia, was diagnosed in a 3-year-old female. Clinical laboratory and dermatological signs are described. Blood test showed a transient hypercoagulable state and the presence of IgG antibodies against platelets, suggesting an immune-mediated mechanism for platelet destruction, in addition to the direct toxic effect of the spider venom. The finding of platelet antibodies after a Loxosceles spider bite has not been previously reported. PMID:24497468

  8. Innate Immune Regulation by STAT-mediated Transcriptional Mechanisms

    PubMed Central

    Li, Haiyan S.; Watowich, Stephanie S.

    2014-01-01

    Summary The term innate immunity typically refers to a quick but nonspecific host defense response against invading pathogens. The innate immune system comprises particular immune cell populations, epithelial barriers, and numerous secretory mediators including cytokines, chemokines, and defense peptides. Innate immune cells are also now recognized to play important contributing roles in cancer and pathological inflammatory conditions. Innate immunity relies on rapid signal transduction elicited upon pathogen recognition via pattern recognition receptors (PRRs) and cell:cell communication conducted by soluble mediators, including cytokines. A majority of cytokines involved in innate immune signaling use a molecular cascade encompassing receptor-associated Jak protein tyrosine kinases and STAT (signal transducer and activator of transcription) transcriptional regulators. Here, we focus on roles for STAT proteins in three major innate immune subsets: neutrophils, macrophages, and dendritic cells (DCs). While knowledge in this area is only now emerging, understanding the molecular regulation of these cell types is necessary for developing new approaches to treat human disorders such as inflammatory conditions, autoimmunity, and cancer. PMID:25123278

  9. 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

  10. Separating monocular and binocular neural mechanisms mediating chromatic contextual interactions.

    PubMed

    D'Antona, Anthony D; Christiansen, Jens H; Shevell, Steven K

    2014-01-01

    When seen in isolation, a light that varies in chromaticity over time is perceived to oscillate in color. Perception of that same time-varying light may be altered by a surrounding light that is also temporally varying in chromaticity. The neural mechanisms that mediate these contextual interactions are the focus of this article. Observers viewed a central test stimulus that varied in chromaticity over time within a larger surround that also varied in chromaticity at the same temporal frequency. Center and surround were presented either to the same eye (monocular condition) or to opposite eyes (dichoptic condition) at the same frequency (3.125, 6.25, or 9.375 Hz). Relative phase between center and surround modulation was varied. In both the monocular and dichoptic conditions, the perceived modulation depth of the central light depended on the relative phase of the surround. A simple model implementing a linear combination of center and surround modulation fit the measurements well. At the lowest temporal frequency (3.125 Hz), the surround's influence was virtually identical for monocular and dichoptic conditions, suggesting that at this frequency, the surround's influence is mediated primarily by a binocular neural mechanism. At higher frequencies, the surround's influence was greater for the monocular condition than for the dichoptic condition, and this difference increased with temporal frequency. Our findings show that two separate neural mechanisms mediate chromatic contextual interactions: one binocular and dominant at lower temporal frequencies and the other monocular and dominant at higher frequencies (6-10 Hz). PMID:24744449

  11. Cell-mediated immunity in epidermodysplasia verruciformis.

    PubMed

    Gliński, W; Jablonska, S; Langner, A; Obalek, S; Haftek, M; Proniewska, M

    1976-01-01

    Investigations were performed in 6 cases of epidermodysplasia verruciformis and 2 healthy family members. Nonspecific cell-mediated immunity (CMI) was studied by measuring response to phytohemagglutinin (PHA) and concanavalin A (Con A), percentrages of E- and EAC-rosette-forming lymphocytes, bacterial skin tests, and allergic reactions to dinitrochloro-benzene (DNCB). Impairment of CMI was manifested by reduction in the percentage of E rosettes, and lowered response to PHA, and- to a lesser degree- to Con A. The immune response to DNCB sensitization was invariably negative. Impairment of CMI was greater in cases of long duration and with extensive lesions. The cases of similar duration and extent of lesions, which never showed tendency to tumor formation, were not different in CMI in comparison with cases with numerous tumors. Only in cases with very advanced tumors CMI was impaired parallel to the gravity of the patient's general condition. PMID:1017532

  12. 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

  13. Ethanol Mediates Cell Cycle Arrest and Apoptosis in SK-N-SH Neuroblastoma Cells

    PubMed Central

    Lee, Maria; Song, Byoung-Joon; Kwon, Yongil

    2014-01-01

    Background: The mechanisms of cell or organ damage by chronic alcohol consumption are still poorly understood. The present study aimed to investigate the role of the mitogen-activated protein kinases during ethanol-induced damage to SK-N-SH neuroblastoma cells. Methods: Cells were treated with ethanol and subsequently analyzed for cell morphology, viability, and DNA fragmentation. Immunoblot analysis was performed to assess various proteins levels associated with cell cycle arrest and apoptosis after ethanol exposure. Results: Ethanol induced time- and dose-dependent cell death in SK-N-SH cells and increased c-Jun N-terminal protein kinase (JNK) activity in a time- and concentration dependent manner. In contrast, p38 kinase activity increased transiently. After treatment with JNK or p38 kinase inhibitors, ethanol-induced cell death significantly reduced. Ethanol-induced cell death was accompanied by increased cytochrome c release and caspase 3 activity observed at 12 h. In contrast, the level of anti-apoptotic Bcl-2 protein did not change. Ethanol also increased the phosphorylation of p53 and p53 activation was followed by an increase in the p21 tumor suppressor protein accompanied by a gradual decrease in phospho-Rb protein. Conclusion: Our results suggest that ethanol mediates apoptosis of neuroblastoma cells by stimulating p53-related cell cycle arrest mediated through activation of the JNK-related pathway. PMID:25337571

  14. Shifting foundations: the mechanical cell wall and development.

    PubMed

    Braybrook, Siobhan A; Jönsson, Henrik

    2016-02-01

    The cell wall has long been acknowledged as an important physical mediator of growth in plants. Recent experimental and modelling work has brought the importance of cell wall mechanics into the forefront again. These data have challenged existing dogmas that relate cell wall structure to cell/organ growth, that uncouple elasticity from extensibility, and those which treat the cell wall as a passive and non-stressed material. Within this review we describe experiments and models which have changed the ways in which we view the mechanical cell wall, leading to new hypotheses and research avenues. It has become increasingly apparent that while we often wish to simplify our systems, we now require more complex multi-scale experiments and models in order to gain further insight into growth mechanics. We are currently experiencing an exciting and challenging shift in the foundations of our understanding of cell wall mechanics in growth and development. PMID:26799133

  15. 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. PMID:26375864

  16. 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

  17. DNA From Dead Cancer Cells Induces TLR9-Mediated Invasion and Inflammation In Living Cancer Cells

    PubMed Central

    Tuomela, Johanna; Sandholm, Jouko; Kaakinen, Mika; Patel, Ankita; Kauppila, Joonas H.; Ilvesaro, Joanna; Chen, Dongquan; Harris, Kevin W.; Graves, David; Selander, Katri S.

    2014-01-01

    TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple negative, human MDA-MB-231 breast cancer cells stably expressing control or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy. PMID:24212717

  18. 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

  19. Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.

    PubMed Central

    Burns, A J; Lomax, A E; Torihashi, S; Sanders, K M; Ward, S M

    1996-01-01

    The structural relationships between interstitial cells of Cajal (ICC), varicose nerve fibers, and smooth muscle cells in the gastrointestinal tract have led to the suggestion that ICC may be involved in or mediate enteric neurotransmission. We characterized the distribution of ICC in the murine stomach and found two distinct classes on the basis of morphology and immunoreactivity to antibodies against c-Kit receptors. ICC with multiple processes formed a network in the myenteric plexus region from corpus to pylorus. Spindle-shaped ICC were found within the circular and longitudinal muscle layers (IC-IM) throughout the stomach. The density of these cells was greatest in the proximal stomach. IC-IM ran along nerve fibers and were closely associated with nerve terminals and adjacent smooth muscle cells. IC-IM failed to develop in mice with mutations in c-kit. Therefore, we used W/W(V) mutants to test whether IC-IM mediate neural inputs in muscles of the gastric fundus. The distribution of inhibitory nerves in the stomachs of c-kit mutants was normal, but NO-dependent inhibitory neuro-regulation was greatly reduced. Smooth muscle tissues of W/W(V) mutants relaxed in response to exogenous sodium nitroprusside, but the membrane potential effects of sodium nitroprusside were attenuated. These data suggest that IC-IM play a critical serial role in NO-dependent neurotransmission: the cellular mechanism(s) responsible for transducing NO into electrical responses may be expressed in IC-IM. Loss of these cells causes loss of electrical responsiveness and greatly reduces responses to nitrergic nerve stimulation. Images Fig. 1 Fig. 2 Fig. 4 PMID:8876253

  20. Cell-mediated immune deficiency in Hodgkin's disease.

    PubMed

    Kumar, R K; Penny, R

    1982-10-01

    Disturbances of the immune system frequently accompany the development of lymphomas in man. In the early stages of non-Hodgkin's lymphomas, abnormalities of immunological function are usually minimal, but impairment of both antibody- and cell-mediated immunity is often noted in advanced disease. In contrast, while antibody-mediated immune responses in patients with Hodgkin's disease usually remain intact until late in the course of the illness, cell-mediated immune dysfunction is an early and consistent feature. Here Rakesh Kumar and Ronald Penny discuss the abnormalities of cell-mediated immunity in Hodgkin's disease. PMID:25290229

  1. 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

  2. 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

  3. Mast Cells Condition Dendritic Cells to Mediate Allograft Tolerance

    PubMed Central

    de Vries, Victor C.; Pino-Lagos, Karina; Nowak, Elizabeth C.; Bennett, Kathy A.; Oliva, Carla; Noelle, Randolph J.

    2013-01-01

    SUMMARY Peripheral tolerance orchestrated by regulatory T cells, dendritic cells (DCs), and mast cells (MCs) has been studied in several models including skin allograft tolerance. We now define a role for MCs in controlling DC behavior (“conditioning”) to facilitate tolerance. Under tolerant conditions, we show that MCs mediated a marked increase in tumor necrosis factor (TNFα)-dependent accumulation of graft-derived DCs in the dLN compared to nontolerant conditions. This increase of DCs in the dLN is due to the local production of granulocyte macrophage colony-stimulating factor (GM-CSF) by MCs that induces a survival advantage of graft-derived DCs. DCs that migrated to the dLN from the tolerant allograft were tolerogenic; i.e., they dominantly suppress T cell responses and control regional immunity. This study underscores the importance of MCs in conditioning DCs to mediate peripheral tolerance and shows a functional impact of peripherally produced TNFα and GM-CSF on the migration and function of tolerogenic DCs. PMID:22035846

  4. Mechanisms of T cell organotropism.

    PubMed

    Fu, Hongmei; Ward, Eleanor Jayne; Marelli-Berg, Federica M

    2016-08-01

    Protective immunity relies upon T cell differentiation and subsequent migration to target tissues. Similarly, immune homeostasis requires the localization of regulatory T cells (Tregs) to the sites where immunity takes place. While naïve T lymphocytes recirculate predominantly in secondary lymphoid tissue, primed T cells and activated Tregs must traffic to the antigen rich non-lymphoid tissue to exert effector and regulatory responses, respectively. Following priming in draining lymph nodes, T cells acquire the 'homing receptors' to facilitate their access to specific tissues and organs. An additional level of topographic specificity is provided by T cells receptor recognition of antigen displayed by the endothelium. Furthermore, co-stimulatory signals (such as those induced by CD28) have been shown not only to regulate T cell activation and differentiation, but also to orchestrate the anatomy of the ensuing T cell response. We here review the molecular mechanisms supporting trafficking of both effector and regulatory T cells to specific antigen-rich tissues. PMID:27038487

  5. FTY720 increases CD74 expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death

    PubMed Central

    Alinari, Lapo; Mahoney, Emilia; Patton, John; Zhang, Xiaoli; Huynh, Lenguyen; Earl, Christian T.; Mani, Rajeswaran; Mao, Yicheng; Yu, Bo; Quinion, Carl; Towns, William H.; Chen, Ching-Shih; Goldenberg, David M.; Blum, Kristie A.; Byrd, John C.; Muthusamy, Natarajan

    2011-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL. PMID:22042694

  6. 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

  7. 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. PMID:26779834

  8. Differences in kinase-mediated regulation of cell cycle progression in normal and transformed cells

    SciTech Connect

    Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Stevenson, A.P.; Kraemer, P.M.; Bustos, L.D.; Dickson, J.A.; Bradbury, E.M. )

    1993-01-01

    Staurosporine (Stsp), a general protein kinase inhibitor, was used to investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation. Low levels of Stsp (1-2nM) prevented nontransformed cells from entering S phase, indicating that protein phosphorylation processes are essential for commitment of DNA replication in normal cells. Cells resumed cycling when Stsp was removed. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 h later than the G0/G1 boundary and extended through the G1/S boundary. The initial block point at 3 h corresponds neither to the serum nor the amino acid restriction point. In contrast, neither low nor high concentrations (100nm) of Stsp affected G1 progression of transformed cells. High drug concentrations blocked normal cells in G1 and G2 but affected only G2-progression in transformed cells. These results indicate that kinase-mediated regulation of DNA replication is lost as a result of neoplastic transformation, but the G2-arrest mechanism remains intact.

  9. Methylanthraquinone from Hedyotis diffusa WILLD induces Ca(2+)-mediated apoptosis in human breast cancer cells.

    PubMed

    Liu, Zheng; Liu, Ming; Liu, Miao; Li, Jianchun

    2010-02-01

    Methylanthraquinone from Hedyotis diffusa WILLD exhibited potent anticancer activity in many kinds of cancer cells. However, the exact mechanism and signaling pathway involved in methylanthraquinone-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of methylanthraquinone-mediated apoptosis in MCF-7 human breast cancer cells. When MCF-7 cells were co-incubated with methylanthraquinone, the percentage of apoptotic cell and S phase of cell cycle was markedly increased. In addition, a rise in intracellular calcium levels, phosphorylation of JNK and activation of calpain were found in MCF-7 cells after exposure to methylanthraquinone. With the methylanthraquinone-mediated reduction of mitochondrial membrane potential, cytochrome c was released from mitochondria to cytosol. Moreover, methylanthraquinone strongly induced cleavage of caspase-4, caspase-9 and caspase-7 in MCF-7 cells. These results suggested that methylanthraquinone from Hedyotis diffusa WILLD induced MCF-7 cells apoptosis via Ca(2+)/calpain/caspase-4 pathway. PMID:19686834

  10. Estrogen-mediated down-regulation of E-cadherin in breast cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E-cadherin is an important mediator of cell-cell interactions, and has been shown to play a crucial role in breast tumor suppression. Its inactivation occurs through instability at its chromosomal locus and mutations, but also through epigenetic mechanisms such as promoter hypermethylation and trans...

  11. Antibody-mediated red blood cell agglutination resulting in spontaneous echocardiographic contrast.

    PubMed

    Miller, M R; Thompson, W R; Casella, J F; Spevak, P J

    1999-01-01

    Spontaneous echocardiographic contrast is well reported in states of low flow and low shear stress, and the primary blood component involved has been reported as red blood cells via rouleaux formation. This report describes the occurrence of spontaneous echocardiographic contrast from a unique mechanism of IgM-mediated red blood cell agglutination and describes the clinical sequelae. PMID:10368455

  12. Wdr1-mediated cell shape dynamics and cortical tension are essential for epidermal planar cell polarity

    PubMed Central

    Pasolli, H. Amalia; Chai, Sophia; Nikolova, Maria; Stokes, Nicole; Fuchs, Elaine

    2015-01-01

    During mouse development, core planar cell polarity (PCP) proteins become polarized in the epidermal plane to guide angling/morphogenesis of hair follicles. How PCP is established is poorly understood. Here, we identify a key role for Wdr1 (also known as Aip1), an F-actin-binding protein that enhances cofilin/destrin-mediated F-actin disassembly. We show that cofilin and destrin function redundantly in developing epidermis, but their combined depletion perturbs cell adhesion, cytokinesis, apicobasal polarity and PCP. Although Wdr1 depletion accentuates single-loss-of-cofilin/destrin phenotypes, alone it resembles core PCP mutations. Seeking a mechanism, we find that Wdr1 and cofilin/destrin-mediated actomyosin remodelling are essential for generating or maintaining cortical tension within the developing epidermal sheet and driving the cell shape and planar orientation changes that accompany establishment of PCP in mammalian epidermis. Our findings suggest intriguing evolutionary parallels but mechanistic modifications to the distal wing hinge-mediated mechanical forces that drive cell shape change and orient PCP in the Drosophila wing disc. PMID:25915128

  13. Mechanically Mediated Microwave Frequency Conversion in the Quantum Regime

    NASA Astrophysics Data System (ADS)

    Lecocq, F.; Clark, J. B.; Simmonds, R. W.; Aumentado, J.; Teufel, J. D.

    2016-01-01

    We report the observation of efficient and low-noise frequency conversion between two microwave modes, mediated by the motion of a mechanical resonator subjected to radiation pressure. We achieve coherent conversion of more than 1012 photons/s with a 95% efficiency and a 14 kHz bandwidth. With less than 10-1 photons.s-1.Hz-1 of added noise, this optomechanical frequency converter is suitable for quantum state transduction. We show the ability to operate this converter as a tunable beam splitter, with direct applications for photon routing and communication through complex quantum networks.

  14. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    PubMed Central

    2009-01-01

    Background Phenylpropanoid-derived phenolic glycosides (PGs) and condensed tannins (CTs) comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM), and a negative effect on cell growth (at 10 mM). The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we identified candidate genes

  15. Dispositional mindfulness in trauma recovery: Prospective relations and mediating mechanisms.

    PubMed

    Nitzan-Assayag, Yaara; Aderka, Idan M; Bernstein, Amit

    2015-12-01

    This study examined the protective properties and candidate mediating processes (cognitive fusion and cognitive suppression) linking dispositional mindfulness to distal risk factors (negative affect, anxiety sensitivity, rumination) and psychopathology symptom outcomes (depression and posttraumatic stress symptoms) following trauma exposure. To do so, a community-based sample of adults was longitudinally studied in the six-months following exposure--within 30-days (T1), 3-months (T2), and 6-months (T3)--to a shared disaster-related potentially traumatic event (PTE). Specifically, we found that cognitive fusion predicted, and mediated, the effect of mindfulness on outcomes related to distress post-trauma including negative affect, depression and posttraumatic stress symptoms. Complementary to these effects, we found that cognitive suppression predicted, and mediated, the effect of mindfulness on distal risk factors linked to negative self-referential processes including rumination and anxiety sensitivity. Findings are discussed with respect to their theoretical and clinical implications for the potential role and mechanisms of mindfulness in recovery following trauma. PMID:26401969

  16. 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...

  17. Cell-mediated immunity in experimental Nocardia asteroides infection.

    PubMed Central

    Sundararaj, T; Agarwal, S C

    1977-01-01

    Experimental mycetoma-like lesions developed in guinea pigs after subcutaneous injection of Nocardia asteroides. Although delayed hypersensitivity appeared earlier, increased macrophage migration inhibition and microbicidal activity appeared after 7 weeks. When the lesions healed, high cell-mediated immunity was present. Cell-mediated immunity was transferred to normal recipient guinea pigs from healed donor guinea pigs by spleen cell transfer. Recipient guinea pigs showed marked protection against challenge with N. asteroides. PMID:321348

  18. 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.

  19. 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.

  20. 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

  1. Network mechanisms of grid cells.

    PubMed

    Moser, Edvard I; Moser, May-Britt; Roudi, Yasser

    2014-02-01

    One of the major breakthroughs in neuroscience is the emerging understanding of how signals from the external environment are extracted and represented in the primary sensory cortices of the mammalian brain. The operational principles of the rest of the cortex, however, have essentially remained in the dark. The discovery of grid cells, and their functional organization, opens the door to some of the first insights into the workings of the association cortices, at a stage of neural processing where firing properties are shaped not primarily by the nature of incoming sensory signals but rather by internal self-organizing principles. Grid cells are place-modulated neurons whose firing locations define a periodic triangular array overlaid on the entire space available to a moving animal. The unclouded firing pattern of these cells is rare within the association cortices. In this paper, we shall review recent advances in our understanding of the mechanisms of grid-cell formation which suggest that the pattern originates by competitive network interactions, and we shall relate these ideas to new insights regarding the organization of grid cells into functionally segregated modules. PMID:24366126

  2. Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

    PubMed

    Webb, Tonya J; Carey, Gregory B; East, James E; Sun, Wenji; Bollino, Dominique R; Kimball, Amy S; Brutkiewicz, Randy R

    2016-08-01

    Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses. PMID:27297969

  3. 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

  4. Mesenchymal stem cells as mediators of neural differentiation.

    PubMed

    Hardy, Steven A; Maltman, Daniel J; Przyborski, Stefan A

    2008-01-01

    Mesenchymal stem cells (MSCs) represent a promising source of material for autologous cell transplantation therapies, in particular, their potential use for the treatment of damaged nervous tissue. Much of the work in this area has focused on the transplantation of MSCs into animal models of neurological disorders, including stroke and spinal cord injury. Although numerous studies have reported significant functional improvements in these systems, the exact mechanism(s) by which MSCs elicit recovery remains largely undefined. While it has been proposed that 'trans'-differentiation and/or cell fusion events underly MSC-mediated neural repair, there is considerable doubt that the low frequency of these phenomena is sufficient to account for the observed levels of recovery. Furthermore, in vitro studies call into question the ability of MSCs to produce authentic neural derivatives. In this review we focus on recent evidence indicating that transplanted MSCs promote endogenous repair of neurologically damaged areas via the release of soluble trophic factors and cytokines. Through the modern analysis of MSC-conditioned media it is becoming possible to gain new insight into the release and interplay of these soluble factors and their neurogenic effects. Ultimately this understanding may lead to the rational design of new therapies for the treatment of neurological and neurodegenerative disorders. PMID:18220922

  5. 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

  6. 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.

  7. 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

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

    PubMed Central

    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/DIA-BLO 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

  9. Ebola virus mediated infectivity is restricted in canine and feline cells.

    PubMed

    Han, Ziying; Bart, Stephen M; Ruthel, Gordon; Vande Burgt, Nathan H; Haines, Kathleen M; Volk, Susan W; Vite, Charles H; Freedman, Bruce D; Bates, Paul; Harty, Ronald N

    2016-01-15

    Ebolaviruses and marburgviruses belong to the Filoviridae family and often cause severe, fatal hemorrhagic fever in humans and non-human primates. The magnitude of the 2014 outbreak in West Africa and the unprecedented emergence of Ebola virus disease (EVD) in the United States underscore the urgency to better understand the dynamics of Ebola virus infection, transmission and spread. To date, the susceptibility and possible role of domestic animals and pets in the transmission cycle and spread of EVD remains unclear. We utilized infectious VSV recombinants and lentivirus pseudotypes expressing the EBOV surface glycoprotein (GP) to assess the permissiveness of canine and feline cells to EBOV GP-mediated entry. We observed a general restriction in EBOV-mediated infection of primary canine and feline cells. To address the entry mechanism, we used cells deficient in NPC1, a host protein implicated in EBOV entry, and a pharmacological blockade of cholesterol transport, to show that an NPC1-dependent mechanism of EBOV entry is conserved in canine and feline cells. These data demonstrate that cells of canine and feline origin are susceptible to EBOV GP mediated infection; however, infectivity of these cells is reduced significantly compared to controls. Moreover, these data provide new insights into the mechanism of EBOV GP mediated entry into cells of canine and feline origin. PMID:26711035

  10. CRISPR mediated somatic cell genome engineering in the chicken.

    PubMed

    Véron, Nadège; Qu, Zhengdong; Kipen, Phoebe A S; Hirst, Claire E; Marcelle, Christophe

    2015-11-01

    Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model. PMID:26277216

  11. 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

  12. Mucin 1-mediated chemo-resistance in lung cancer cells

    PubMed Central

    Ham, S Y; Kwon, T; Bak, Y; Yu, J-H; Hong, J; Lee, S K; Yu, D-Y; Yoon, D-Y

    2016-01-01

    Paclitaxel (PTX) is a commonly used drug to treat diverse cancer types. However, its treatment can generate resistance and the mechanisms of PTX-resistance in lung cancers are still unclear. We demonstrated that non-small cell lung cancers (NSCLCs) survive PTX treatment. Compared with the progenitor NSCLC A549 cells, the PTX-resistant A549 cells (A549/PTX) displayed enhanced sphere-formation ability. The proportion of the cancer stem cell marker, aldehyde dehydrogenase-positive cells, and epithelial–mesenchymal transition signaling protein levels were also elevated in A549/PTX. Importantly, the levels of oncoproteins phosphoinositide-3 kinase/Akt, mucin 1 cytoplasmic domain (MUC1-C) and β-catenin were also significantly elevated in A549/PTX. Furthermore, nuclear translocation of MUC1-C and β-catenin increased in A549/PTX. The c-SRC protein, an activator of MUC1-C, was also overexpressed in A549/PTX. These observations led to the hypothesis that enhanced expression of MUC1-C is associated with stemness and PTX resistance in NSCLCs. To test this, we knocked down or overexpressed MUC1-C in A549/PTX and found that inhibition of MUC1-C expression coupled with PTX treatment was sufficient to reduce the sphere-forming ability and survival of A549/PTX. In summary, our in vitro and in vivo studies have revealed a potential mechanism of MUC1-C-mediated PTX resistance and provided insights into a novel therapeutic measure for lung cancers. PMID:26779808

  13. 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

  14. 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

  15. 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

  16. HDAC6-mediated EGFR stabilization and activation restrict cell response to sorafenib in non-small cell lung cancer cells.

    PubMed

    Wang, Zhihao; Hu, Pengchao; Tang, Fang; Xie, Conghua

    2016-05-01

    Sorafenib is a multi-targeted kinase inhibitor and has been the subject of extensive clinical research in advanced non-small cell lung cancer (NSCLC). However, sorafenib fails to improve overall survival of patients with advanced NSCLC. The molecular mechanisms that account for this phenomenon are unclear. Here we show that sorafenib treatment stabilizes epidermal growth factor receptor (EGFR) and activates EGFR pathway. Moreover, this is partly mediated by stabilization of histone deacetylase 6 (HDAC6), which has been shown to regulate EGFR endocytic trafficking and degradation. Overexpression of HDAC6 confers resistance to sorafenib in NSCLC cells. Inhibition of HDAC6 with selective inhibitors synergizes with sorafenib to kill NSCLC cells via inhibition of sorafenib-mediated EGFR pathway activation. Taken together, our findings might partly explain the failure of Phase III trial of sorafenib in improving overall survival of advanced NSCLC patients and bear possible implications for the improvement on the efficacy of sorafenib in treatment of NSCLC. PMID:27090797

  17. Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways.

    PubMed

    Cui, Yanji; Park, Jee-Yun; Wu, Jinji; Lee, Ji Hyung; Yang, Yoon-Sil; Kang, Moon-Seok; Jung, Sung-Cherl; Park, Joo Min; Yoo, Eun-Sook; Kim, Seong-Ho; Ahn Jo, Sangmee; Suk, Kyoungho; Eun, Su-Yong

    2015-05-01

    Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91 (phox) , which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways. PMID:25954126

  18. Dieckol Attenuates Microglia-mediated Neuronal Cell Death via ERK, Akt and NADPH Oxidase-mediated Pathways

    PubMed Central

    Cui, Yanji; Park, Jee-Yun; Wu, Jinji; Lee, Ji Hyung; Yang, Yoon-Sil; Kang, Moon-Seok; Jung, Sung-Cherl; Park, Joo Min; Yoo, Eun-Sook; Kim, Seong-Ho; Ahn Jo, Sangmee; Suk, Kyoungho

    2015-01-01

    Excessive microglial activation and subsequent neuroinflammation lead to synaptic loss and dysfunction as well as neuronal cell death, which are involved in the pathogenesis and progression of several neurodegenerative diseases. Thus, the regulation of microglial activation has been evaluated as effective therapeutic strategies. Although dieckol (DEK), one of the phlorotannins isolated from marine brown alga Ecklonia cava, has been previously reported to inhibit microglial activation, the molecular mechanism is still unclear. Therefore, we investigated here molecular mechanism of DEK via extracellular signal-regulated kinase (ERK), Akt and nicotinamide adenine dinuclelotide phosphate (NADPH) oxidase-mediated pathways. In addition, the neuroprotective mechanism of DEK was investigated in microglia-mediated neurotoxicity models such as neuron-microglia co-culture and microglial conditioned media system. Our results demonstrated that treatment of anti-oxidant DEK potently suppressed phosphorylation of ERK in lipopolysaccharide (LPS, 1 µg/ml)-stimulated BV-2 microglia. In addition, DEK markedly attenuated Akt phosphorylation and increased expression of gp91phox, which is the catalytic component of NADPH oxidase complex responsible for microglial reactive oxygen species (ROS) generation. Finally, DEK significantly attenuated neuronal cell death that is induced by treatment of microglial conditioned media containing neurotoxic secretary molecules. These neuroprotective effects of DEK were also confirmed in a neuron-microglia co-culture system using enhanced green fluorescent protein (EGFP)-transfected B35 neuroblastoma cell line. Taken together, these results suggest that DEK suppresses excessive microglial activation and microglia-mediated neuronal cell death via downregulation of ERK, Akt and NADPH oxidase-mediated pathways. PMID:25954126

  19. 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

  20. 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

  1. 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.

  2. Role of scleraxis in mechanical stretch-mediated regulation of cardiac myofibroblast phenotype.

    PubMed

    Roche, Patricia L; Nagalingam, Raghu S; Bagchi, Rushita A; Aroutiounova, Nina; Belisle, Breanna M J; Wigle, Jeffrey T; Czubryt, Michael P

    2016-08-01

    The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation. PMID:27357547

  3. 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

  4. 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. PMID:26372777

  5. pH-evoked dural afferent signaling is mediated by ASIC3 and is sensitized by mast cell mediators

    PubMed Central

    Yan, Jin; Wei, Xiaomei; Bischoff, Christina; Edelmayer, Rebecca M.; Dussor, Gregory

    2013-01-01

    Background Prior studies have shown that decreased meningeal pH activates dural afferents via opening of acid-sensing ion channels (ASICs) suggesting one pathophysiological mechanism for the generation of headaches. The studies described here further examined the ASIC subtype mediating pH-induced dural-afferent activation and examined whether sensitization influences pH responses. Objective Given the potential importance of meningeal mast cells to headache, the goal of this study was to evaluate dural afferent responses to pH following sensitization with mast cell mediators. Methods Cutaneous allodynia was measured in rats following stimulation of the dura with decreased pH alone or in combination with mast cell mediators. Trigeminal ganglion neurons retrogradely-labeled from the dura were stained with an ASIC3 antibody using immunohistochemistry. Currents and action potentials evoked by changes in pH alone or in combination with mast cell mediators were measured in retrogradely-labeled dural afferents using patch-clamp electrophysiology. Results pH-sensitive dural afferents generated currents in response to the ASIC3 activator 2-guanidine-4-methylquinazoline (GMQ), approximately 80% of these neurons express ASIC3 protein, and pH-evoked behavioral responses were inhibited by the ASIC3 blocker APETx2. Following exposure to mast cell mediators, dural afferents exhibited increased pH-evoked excitability and cutaneous allodynia was observed at higher pH than with pH stimuli alone. Conclusion These data indicate that the predominant ASIC subtype responding to decreased meningeal pH is ASIC3. Additionally, they demonstrate that in the presence of inflammation, dural afferents respond to even smaller decreases in pH providing further support for the ability of small pH changes within the meninges to initiate afferent input leading to headache. PMID:23808707

  6. BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.

    PubMed

    Hatzi, Katerina; Nance, J Philip; Kroenke, Mark A; Bothwell, Marcella; Haddad, Elias K; Melnick, Ari; Crotty, Shane

    2015-04-01

    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

  7. A common clathrin-mediated machinery coordinates cell-cell adhesion and bacterial internalization

    PubMed Central

    Bonazzi, Matteo; Kühbacher, Andreas; Toledo-Arana, Alejandro; Mallet, Adeline; Vasudevan, Lavanya; Pizarro-Cerdá, Javier; Brodsky, Frances M.; Cossart, Pascale

    2013-01-01

    Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction machinery, and invade non-phagocytic cells by a clathrin-dependent mechanism. Here we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of adherens junction formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of adherens junctions. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form adherens junctions with cells expressing E-cadherin. To model bacterial invasion, we demonstrate that non-adherent cells expressing the InlA chimera can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes. PMID:22984946

  8. 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

  9. Ca-mediated electroformation of cell-sized lipid vesicles.

    PubMed

    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 (Ca(2+)) and calcium carbonate (CaCO3) mineral adsorption layers on a lipid film surface respectively during the electroformation of GUVs. It is found that Ca(2+) 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

  10. 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

  11. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

    PubMed

    Lindemans, Caroline A; Calafiore, Marco; Mertelsmann, Anna M; O'Connor, Margaret H; 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 E; Nieuwenhuis, Edward E; Shroyer, Noah F; Liu, Chen; Kolesnick, Richard; van den Brink, Marcel R M; Hanash, Alan M

    2015-12-24

    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 maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  12. 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

  13. 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.

  14. Adherence of non-O157 Shiga-toxin Escherichia coli to bovine recto-anal junction squamous epithelial cells appears to be mediated by mechanisms distinct from those used by O157

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study presents evidence that the pattern of adherence of clinically relevant non-O157 Shiga-toxin producing Escherichia coli (STEC) to bovine recto-anal junction squamous epithelial cells (RSE) is similar to that of O157, although the mechanisms of adherence appear to be distinct. Our results f...

  15. 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. PMID:27060907

  16. 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

  17. Temporal Visual Mechanisms May Mediate Compensation for Macular Pigment.

    PubMed

    Stringham, Nicole T; Stringham, James M

    2015-12-01

    Macular pigment (MP) is a pre-receptoral filter that is diet derived and deposited in relatively high optical density in the foveal region of the retina. Due to its yellow coloration, MP absorbs light of relatively short wavelengths, ranging from 400 nm to 520 nm. Despite the spectral and spatial nonuniformity imposed upon the sensory retina by MP, perception appears to be relatively uniform across the central visual field. MP therefore offers an opportunity to determine experimentally potential mechanisms responsible for mediating this uniformity. After assessing, in 14 subjects, MP's effects on the temporal sensitivity of both the short-wavelength- and middle-/long-wavelength-sensitive visual pathways, it appears that the visual system compensates for absorption of short-wavelength light by MP by slowing the sampling rate of short-wavelength cones and by increasing the processing speed of middle-/long-wavelength-sensitive cones. This mechanism could work via temporal summation or a temporal neural code, whereby slower response dynamics lead to amplification of relatively weak signals. PMID:26562864

  18. 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

  19. Immunomodulatory functions of mesenchymal stem cells and possible mechanisms.

    PubMed

    Wang, Qing; Ding, Gang; Xu, Xin

    2016-09-01

    In addition to their well-studied self-renewal capabilities and multipotent differentiation properties, mesenchymal stem cells (MSCs) have been reported to possess profound immunomodulatory functions both in vitro and in vivo. More and more studies have shown that MSCs are capable of interacting closely with almost all subsets of immune cells, such as T cells, B cells, dendritic cells, natural killer cells, macrophages, and neutrophils etc. The immunomodulatory property of MSCs may shed light on the treatment of a variety of autoimmune and inflammation-related diseases. In this article, we will review the studies on the immunomodulatory and anti-inflammatory functions of MSCs and the mechanisms responsible for the interaction between immune cells and MSCs, which could improve the development of promising approaches for cell-mediated immune therapies. PMID:26932157

  20. [NKT cells: their development, mechanisms and effects of action].

    PubMed

    Bojarska-Junak, Agnieszka; Tabarkiewicz, Jacek; Roliński, Jacek

    2013-01-01

    NKT cells are a heterogeneous subset of T lymphocytes that share surface markers and functional characteristics with both conventional T lymphocytes and natural killer cells. Most NKT cells express a semi-invariant T cell receptor that reacts with glycolipid antigens presented by the CD1d molecule on the surface of antigen-presenting cells. NKT cells can modulate the immune response against infectious agents, autoantigens, tumors, tissue grafts and allergens. NKT cells mediate the activities through their ability to express pro- and anti-inflammatory cytokines that influence the type and magnitude of the immune response. The manuscript summarizes current views on development of NKT cells as well as mechanisms and effects of their action.  PMID:23475484

  1. Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

    NASA Astrophysics Data System (ADS)

    Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

    2015-12-01

    To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, 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 fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

  2. 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

  3. Nicotine-Mediated Ca(2+)-Influx Induces IL-8 Secretion in Oral Squamous Cell Carcinoma Cell.

    PubMed

    Tsunoda, Kou; Tsujino, Ichiro; Koshi, Ryosuke; Sugano, Naoyuki; Sato, Shuichi; Asano, Masatake

    2016-04-01

    Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin-8 (IL-8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine-mediated IL-8 induction in OSCC was investigated. Augmented IL-8 secretion by Ca9-22 cells was blocked by the NF-κB inhibitor L-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)-specific inhibitor α-bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre-incubating the cells with inhibitors against mitogen-activated protein kinase (MEK), protein kinase C (PKC), and Ca(2+)/calmodulin-dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine-mediated IL-8 secretion. Pre-treatment of the Ca9-22 cells with the Ca(2+) chelator BAPTA-AM drastically inhibited IL-8 secretion. Although nicotine stimulation induced the phosphorylation of the NF-κB p65 subunit, pre-treatment with BAPTA-AM was found to inhibit this activity significantly. CaMK II-dependent p65 phosphorylation was confirmed by pre-incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca(2+) influx, which results in the activation and phosphorylation of CaMK II and NF-κB p65, respectively. Nicotine-mediated IL-8 induction should be a trigger for the initiation of various diseases. PMID:26418512

  4. IL-4 attenuates pulmonary epithelial cell-mediated suppression of T cell priming.

    PubMed

    Albrecht, Melanie; Arnhold, Markus; Lingner, Sandra; Mahapatra, Subhashree; Bruder, Dunja; Hansen, Gesine; Dittrich, Anna-Maria

    2012-01-01

    We have previously shown that Th2-polarized airway inflammation facilitates sensitization towards new, protein antigens. In this context, we could demonstrate that IL-4 needs to act on cells of the hematopoetic and the structural compartment in order to facilitate sensitization towards new antigens. We thus aimed to elucidate possible mechanisms of action of IL-4 on structural cells choosing to analyze pulmonary epithelial cells as an important part of the lung's structural system. We used a co-culture system of DC- or APC-dependent in vitro priming of T cells, co-cultivated on a layer of cells of a murine pulmonary epithelial cell line (LA-4) pretreated with or without IL-4. Effects on T cell priming were analyzed via CFSE-dilution and flow cytometric assessment of activation status. Pulmonary epithelial cells suppressed T cell proliferation in vitro but this effect was attenuated by pre-treatment of the epithelial cells with IL-4. Transwell experiments suggest that epithelial-mediated suppression of T cell activation is mostly cell-contact dependent and leads to attenuation in an early naive T cell phenotype. Secretion of soluble factors like TARC, TSLP, GM-CSF and CCL20 by epithelial cells did not change after IL-4 treatment. However, analysis of co-stimulatory expression on pulmonary epithelial cells revealed that pre-treatment of epithelial cells with IL-4 changed expression GITR-L, suggesting a possible mechanism for the effects observed. Our studies provide new insight into the role of IL-4 during the early phases of pulmonary sensitization: The inhibitory activity of pulmonary epithelial cells in homeostasis is reversed in the presence of IL-4, which is secreted in the context of Th2-dominated allergic airway inflammation. This mechanism might serve to explain facilitated sensitization in the clinical context of polysensitization where due to a pre-existing sensitization increased levels of IL-4 in the airways might facilitate T cell priming towards new

  5. 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.

  6. 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. PMID:26276275

  7. 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

  8. Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms.

    PubMed

    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

  9. Mechanism of Plasmid-Mediated Resistance to Cadmium in Staphylococcus aureus

    PubMed Central

    Chopra, I.

    1975-01-01

    The mechanism of plasmid-mediated resistance to cadmium in Staphylococcus aureus was investigated. Protein synthesis in cell-free extracts from resistant or susceptible bacteria was equally susceptible to inhibition by Cd2+, but spheroplasts from resistant bacteria retained their resistance. Resistant bacteria did not have a decreased affinity for cations in general, nor was active metabolism required for exclusion of Cd2+. The kinetics of Cd2+ uptake into susceptible and resistant bacteria suggested that the conformation of membrane proteins in resistant bacteria may be important in the exclusion of Cd2+. PMID:1137361

  10. 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

  11. 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

  12. Freeze-dried grape powder attenuates mitochondria- and oxidative stress-mediated apoptosis in liver cells.

    PubMed

    Xu, Yumin; Khaoustov, Vladimir I; Wang, Hui; Yu, Jing; Tabassam, Fazal; Yoffe, Boris

    2009-10-14

    The beneficial effects of grape consumption have been attributed to the antioxidant activity of its polyphenols. This study was conducted to investigate the cytoprotective effects of a freeze-dried grape powder (FDGP) on liver cells. FDGP treatment of primary hepatocytes and hepatoma cells revealed increased metabolic activity of cells and phosphorylation of Akt and IkappaBalpha, as well as up-regulation of proliferating cell nuclear antigen (PCNA) level. To study the molecular mechanisms of FDGP effects, cells were treated with TNF-related apoptosis-inducing ligand (TRAIL); taurodeoxycholic acid (TDCA); thapsigargin (TG), to induce cell apoptosis through death receptor-, mitochondria-, or ER-mediated pathway; and H(2)O(2), to induce oxidative stress, respectively. TDCA-induced activation of caspase-3, caspase-7, caspase-9, and Bax was dramatically decreased with cotreatment of FDGP. Furthermore, FDGP reduced levels of annexin V positive cells by 4-fold. Also, FDGP pretreatment restored cellular glutathione content by 71% in cells treated with H(2)O(2). However, FDGP did not inhibit ER-mediated apoptosis. In conclusion, FDGP increased the viability and metabolic activity of liver cells and attenuated oxidative stress- and mitochondria-mediated apoptosis. These data may contribute to the understanding of the mechanisms involved in protective effects of grape in a variety of liver conditions associated with cellular stress. PMID:19754144

  13. Sphingosine-1-phosphate and other lipid mediators generated by mast cells as critical players in allergy and mast cell function.

    PubMed

    Kulinski, Joseph M; Muñoz-Cano, Rosa; Olivera, Ana

    2016-05-01

    Sphingosine-1-phosphate (S1P), platelet activating factor (PAF) and eicosanoids are bioactive lipid mediators abundantly produced by antigen-stimulated mast cells that exert their function mostly through specific cell surface receptors. Although it has long been recognized that some of these bioactive lipids are potent regulators of allergic diseases, their exact contributions to disease pathology have been obscured by the complexity of their mode of action and the regulation of their metabolism. Indeed, the effects of such lipids are usually mediated by multiple receptor subtypes that may differ in their signaling mechanisms and functions. In addition, their actions may be elicited by cell surface receptor-independent mechanisms. Furthermore, these lipids may be converted into metabolites that exhibit different functionalities, adding another layer of complexity to their overall biological responses. In some instances, a second wave of lipid mediator synthesis by both mast cell and non-mast cell sources may occur late during inflammation, bringing about additional roles in the altered environment. New evidence also suggests that bioactive lipids in the local environment can fine-tune mast cell maturation and phenotype, and thus their responsiveness. A better understanding of the subtleties of the spatiotemporal regulation of these lipid mediators, their receptors and functions may aid in the pursuit of pharmacological applications for allergy treatments. PMID:25941085

  14. 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

  15. PIKE mediates EGFR proliferative signaling in squamous cell carcinoma cells.

    PubMed

    Xie, Z; Jiang, Y; Liao, E-Y; Chen, Y; Pennypacker, S D; Peng, J; Chang, S M

    2012-12-01

    One of the key drivers for squamous cell carcinoma (SCC) proliferation is activation of the epidermal growth factor receptor (EGFR), a known proto-oncogene. However, the mechanism of EGFR-dependent SCC proliferation remains unclear. Our previous studies indicate that epidermal growth factor (EGF)-induced SCC cell proliferation requires the SH3 domain of phospholipase C-γ1 (PLC-γ1), but not its catalytic activity. The SH3 domain of PLC-γ1 is known to activate the short form of nuclear phosphatidylinositol 3-kinase enhancer (PIKE) that enhances the activity of nuclear class Ia phosphatidylinositol 3-kinase (PI3K) required for proliferation. However, PIKE has been described for more than a decade to be present exclusively in neuronal cells. In the present study, we found that PIKE was highly expressed in malignant human keratinocytes (SCC4 and SCC12B2) but had low expression in normal human keratinocytes. Immunohistochemical analysis showed strong nuclear staining of PIKE in human epidermal and tongue SCC specimens but little staining in the adjacent non-cancerous epithelium. Treatment of SCC4 cells with EGF-induced translocation of PLC-γ1 to the nucleus and binding of PLC-γ1 to the nuclear PIKE. Knockdown of PLC-γ1 or PIKE blocked EGF-induced activation of class Ia PI3K and protein kinase C-ζ and phosphorylation of nucleolin in the nucleus as well as EGF-induced SCC cell proliferation. However, inhibition of the catalytic activity of PLC-γ1 had little effect. These data suggest that PIKE has a critical role in EGF-induced SCC cell proliferation and may function as a proto-oncogene in SCC. PMID:22349826

  16. 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

  17. 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.

  18. 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

  19. Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

    SciTech Connect

    Yoon, Hana; Oh, Young Taek; Lee, Jung Yeon; Choi, Ji Hyun; Lee, Ju Hie; Baik, Hyung Hwan; Kim, Sung Soo; Choe, Wonchae; Yoon, Kyung-Sik; Ha, Joohun; Kang, Insug

    2008-07-04

    AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca{sup 2+}/calmodulin-dependent protein kinase kinase (CaMKK) {beta}. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPK{alpha}1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-{kappa}B) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-{kappa}B activation.

  20. 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. PMID:25934926

  1. Characterization of a serine protease-mediated cell death program activated in human leukemia cells

    SciTech Connect

    O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C. . E-mail: catherine.stenson@nuigalway.ie

    2006-01-01

    Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis.

  2. 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.

  3. 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

  4. Suppression of Th1-Mediated Autoimmunity by Embryonic Stem Cell-Derived Dendritic Cells

    PubMed Central

    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

  5. 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

  6. Optically absorbing nanoparticle mediated cell membrane permeabilization.

    PubMed

    Bhattacharyya, Kiran; Mehta, Smit; Viator, John

    2012-11-01

    Membrane permeabilization is imperative for gene and drug delivery systems, along with other cell manipulation methods, since the average eukaryotic cell membrane is not permeable to polar and large nonpolar molecules. Antibody conjugated optically absorbing gold nanospheres are targeted to the cell membrane of T47D breast cancer cell line and irradiated with 5 ns pulse, 20 Hz, 532 nm light to increase membrane permeability. Up to 90% permeabilization with less than 6% death is reported at radiant exposures up to 10 times lower than those of other comparable studies. PMID:23114334

  7. 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. PMID:27111515

  8. 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.

  9. Monolithic and mechanical multijunction space solar cells

    SciTech Connect

    Jain, R.K.; Flood, D.J. )

    1993-05-01

    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.

  10. Monolithic and mechanical multijunction space solar cells

    SciTech Connect

    Jain, R.K.; Flood, D.J.

    1992-08-01

    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.

  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. 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

  13. 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

  14. Mechanical stress triggers selective release of fibrotic mediators from bronchial epithelium.

    PubMed

    Tschumperlin, Daniel J; Shively, Jonathan D; Kikuchi, Tadashi; Drazen, Jeffrey M

    2003-02-01

    Transforming growth factor-beta (TGF-beta) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-beta2 from a preformed cell-associated pool. TGF-beta2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-beta2. These data are consistent with a primary pathogenic role for mechanical stress-induced release of both TGF-beta2 and ET in the subepithelial fibrosis that characterizes chronic asthma. PMID:12540481

  15. Molecular mechanisms mediating vascular calcification: role of matrix Gla protein.

    PubMed

    Proudfoot, Diane; Shanahan, Catherine M

    2006-10-01

    Patients with chronic kidney disease (CKD) have a higher incidence of vascular calcification and a greatly increased risk of cardiovascular death. The mechanisms involved in the accelerated vascular calcification observed in CKD have recently become clearer, leading to the hypothesis that a lack of natural inhibitors of calcification may trigger calcium deposition. One of these inhibitory factors, matrix Gla protein (MGP), is the focus of the present review. MGP, originally isolated from bone, is a vitamin K-dependent protein that is also highly expressed by vascular smooth muscle cells. MGP has been confirmed as a calcification-inhibitor in numerous studies; however, its mechanism of action is not completely understood. It potentially acts in several ways to regulate calcium deposition including: (i) binding calcium ions and crystals; (ii) antagonizing bone morphogenetic protein and altering cell differentiation; (iii) binding to extracellular matrix components; and (iv) regulating apoptosis. Its expression is regulated by several factors including retinoic acid, vitamin D and extracellular calcium ions, and a reduced form of vitamin K (KH2) is important in maintaining MGP in an active form. Therefore, strategies aimed at increasing its expression and activity may be beneficial in tipping the balance in favour of inhibition of calcification in CKD. PMID:17014561

  16. Myofibroblast-mediated mechanisms of pathological remodelling of the heart.

    PubMed

    Weber, Karl T; Sun, Yao; Bhattacharya, Syamal K; Ahokas, Robert A; Gerling, Ivan C

    2013-01-01

    The syncytium of cardiomyocytes in the heart is tethered within a matrix composed principally of type I fibrillar collagen. The matrix has diverse mechanical functions that ensure the optimal contractile efficiency of this muscular pump. In the diseased heart, cardiomyocytes are lost to necrotic cell death, and phenotypically transformed fibroblast-like cells-termed 'myofibroblasts'-are activated to initiate a 'reparative' fibrosis. The structural integrity of the myocardium is preserved by this scar tissue, although at the expense of its remodelled architecture, which has increased tissue stiffness and propensity to arrhythmias. A persisting population of activated myofibroblasts turns this fibrous tissue into a living 'secretome' that generates angiotensin II and its type 1 receptor, and fibrogenic growth factors (such as transforming growth factor-β), all of which collectively act as a signal-transducer-effector signalling pathway to type I collagen synthesis and, therefore, fibrosis. Persistent myofibroblasts, and the resultant fibrous tissue they produce, cause progressive adverse myocardial remodelling, a pathological hallmark of the failing heart irrespective of its etiologic origin. Herein, we review relevant cellular, subcellular, and molecular mechanisms integral to cardiac fibrosis and consequent remodelling of atria and ventricles with a heterogeneity in cardiomyocyte size. Signalling pathways that antagonize collagen fibrillogenesis provide novel strategies for cardioprotection. PMID:23207731

  17. Morphogenesis of protrusions from confined lipid bilayers mediated by mechanics

    NASA Astrophysics Data System (ADS)

    Arroyo, Marino; Staykova, Margarita; Rahimi, Mohammad; Stone, Howard A.

    2012-02-01

    Biological membranes adopt a wide range of shapes that structure and give functionality to cells, compartmentalizing the cytosol, forming organelles, or regulating their area. The formation, stabilization, and remodeling of these structures is generally attributed to localized forces or to biochemical processes (insertion of proteins, active compositional regulation). Noting that in the crowded intra and extra-cellular environments membranes are highly constrained, we explore to what extent can mechanics explain the shape of protrusions out of confined membranes. For this purpose, we developed an in-vitro system coupling a lipid bilayer to the strain-controlled deformation of an elastic sheet (Staykova et al, PNAS 108, 2011). We show that upon contracting the elastic support, tubular or spherical protrusions grow out of the adhered membrane, which can be reversibly controlled with strain and osmolarity without resorting to localized forces or chemical alterations of the bilayer. The morphologies produced by our minimal system are ubiquitous in cells, suggesting mechanics may be a simple and generic organizing principle. We can understand most of our observations in terms of a phase diagram accounting for elasticity, adhesion, and the limited amount of area and volume available.

  18. 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

  19. Fc Receptor-Mediated Phagocytosis in Tissues as a Potent Mechanism for Preventive and Therapeutic HIV Vaccine Strategies

    PubMed Central

    Sips, Magdalena; Krykbaeva, Marina; Diefenbach, Thomas J.; Ghebremichael, Musie; Bowman, Brittany A.; Dugast, Anne-Sophie; Boesch, Austin W.; Streeck, Hendrik; Kwon, Douglas S.; Ackerman, Margaret E.; Suscovich, Todd J.; Brouckaert, Peter; Schacker, Timothy W.; Alter, Galit

    2016-01-01

    While the development of a protective HIV vaccine is the ultimate goal of HIV research, to date only one HIV vaccine trial, the RV144, has successfully induced a protective response. The 31% protection from infection achieved in the RV144 trial was linked to the induction of non-neutralizing antibodies, able to mediate ADCC, suggestive of an important role of Fc-mediated functions in protection. Likewise, Fc-mediated antiviral activity was recently shown to play a critical role in actively suppressing the viral reservoir, however, the Fc-effector mechanisms within tissues that provide protection from or after infection are largely unknown. Here we aimed to define the landscape of effector cells and Fc-receptors present within vulnerable tissues. We found negligible Fc-receptor expressing NK cells in the female reproductive and gastrointestinal mucosa. Conversely, Fc-receptor expressing macrophages were highly enriched in most tissues, but neutrophils mediated superior antibody-mediated phagocytosis. Modifications in Fc domain of VRC01 antibody increased phagocytic responses in both phagocytes. These data suggest that non-ADCC mediated mechanisms, such as phagocytosis and neutrophil activation, are more likely to play a role in preventative vaccine or reservoir-eliminating therapeutic approaches. PMID:26883728

  20. 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

  1. 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.

  2. Cytoskeleton mediated spreading dynamics of immune cells

    NASA Astrophysics Data System (ADS)

    Hui, King-Lam; Wayt, Jessica; Grooman, Brian; Upadhyaya, Arpita

    2009-03-01

    We have studied the spreading of Jurkat T-cells on anti-CD3 antibody-coated substrates as a model of immune synapse formation. Cell adhesion area versus time was measured via interference reflection contrast microscopy. We found that the spread area exhibited a sigmoidal growth as a function of time in contrast to the previously proposed universal power-law growth for spreading cells. We used high-resolution total internal reflection fluorescence microscopy of these cells transfected with GFP-actin to study cytoskeletal dynamics during the spreading process. Actin filaments spontaneously organized into a variety of structures including traveling waves, target patterns, and mobile clusters emanating from an organizing center. We quantify these dynamic structures and relate them to the spreading rates. We propose that the spreading kinetics are determined by active rearrangements of the cytoskeleton initiated by signaling events upon antibody binding by T-cell receptors. Membrane deformations induced by such wavelike organization of the cytoskeleton may be a general phenomenon that underlies cell movement and cell-substrate interactions.

  3. 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.

  4. Biphosphinic palladacycle complex mediates lysosomal-membrane permeabilization and cell death in K562 leukaemia cells.

    PubMed

    Barbosa, Christiano M V; Oliveira, Carlos R; Nascimento, Fábio D; Smith, Mickaela C M; Fausto, Daniela M; Soufen, Marco Antonio; Sena, Eliana; Araújo, Ronaldo C; Tersariol, Ivarne L S; Bincoletto, Claudia; Caires, Antonio C F

    2006-08-01

    The cell death mechanism of cytotoxicity induced by the Biphosphinic Palladacycle Complex (BPC) was studied using a K562 leukaemia cell line. The IC50 values obtained for K562 cells post-72 h of BPC were less than 5.0 microM by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue assays. Using the Acridine Orange vital staining combining fluorescence microscopy it was observed that the complex triggers apoptosis in K562 cells, inducing DNA fragmentation, as analysed through electrophoresis. Lysosomal-membrane permeabilization was also observed in K562 cells post-5 h of BPC, which suggests intralysosomal accumulation by proton-trapping, since its pKa value ranged from 5.1 to 6.5. Caspase-3, and -6 activity induced by BPC in K562 cells was prevented by the cathepsin-B inhibitor [N-(L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-proline] (CA074). These events occurred in the presence of endogenous bcl-2 and bax expression. Acute toxicological studies demonstrated that BPC produces no lesions for liver and kidney fourteen-days after drug administration (100 mg/kg--i.p.). White and red blood cells of BPC-treated mice presented normal morphological characteristics. Taken together, these data suggest a novel lysosomal pathway for BPC-induced apoptosis, in which lysosomes are the primary target and cathepsin B acts as death mediator. PMID:16831419

  5. 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

  6. Stem cell mechanics: Auxetic nuclei

    NASA Astrophysics Data System (ADS)

    Wang, Ning

    2014-06-01

    The nuclei of naive mouse embryonic stem cells that are transitioning towards differentiation expand when the cells are stretched and contract when they are compressed. What drives this auxetic phenotype is, however, unclear.

  7. Hierarchical mechanisms for transcription factor-mediated reprogramming of fibroblasts to neurons

    PubMed Central

    Wapinski, Orly L.; Vierbuchen, Thomas; Qu, Kun; Lee, Qian Yi; Chanda, Soham; Fuentes, Daniel R.; Giresi, Paul G.; Ng, Yi Han; Marro, Samuele; Neff, Norma F.; Drechsel, Daniela; Martynoga, Ben; Castro, Diogo S.; Webb, Ashley E.; Brunet, Anne; Guillemot, Francois; Chang, Howard Y.; Wernig, Marius

    2013-01-01

    SUMMARY Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine with poorly understood mechanisms. Here we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an “on target” pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead Ascl1 recruits Brn2 to Ascl1 sites genome-wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, precise match between pioneer factor and the chromatin context at key target genes is determinative for trans-differentiation to neurons and likely other cell types. PMID:24243019

  8. 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

  9. 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

  10. Sphingolipid mediators in cardiovascular cell biology and pathology.

    PubMed

    Levade, T; Augé, N; Veldman, R J; Cuvillier, O; Nègre-Salvayre, A; Salvayre, R

    2001-11-23

    Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications. PMID:11717151

  11. 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

  12. 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. PMID:26079887

  13. The Battle Against Immunopathology: Infectious Tolerance Mediated by Regulatory T Cells

    PubMed Central

    Gravano, David M.; Vignali, Dario A.A.

    2012-01-01

    Infectious tolerance is a process whereby one regulatory lymphoid population confers suppressive capacity on another. Diverse immune responses are induced following infection or inflammatory insult that can protect the host, or potentially cause damage if not properly controlled. Thus, the process of infectious tolerance may be critical in vivo for exerting effective immune control and maintaining immune homeostasis by generating specialized regulatory subpopulations with distinct mechanistic capabilities. Foxp3+ regulatory T cells (Tregs) are a central mediator of infectious tolerance through their ability to convert conventional T cells into induced regulatory T cells (iTregs) directly by secretion of the suppressive cytokines TGF-β, IL-10 or IL-35, or indirectly via dendritic cells. In this review we will discuss the mechanisms and cell populations that mediate and contribute to infectious tolerance, with a focus on the intestinal environment, where tolerance induction to foreign material is critical. PMID:22205213

  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. Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins.

    PubMed

    Litvinov, S V; Balzar, M; Winter, M J; Bakker, H A; Briaire-de Bruijn, I H; Prins, F; Fleuren, G J; Warnaar, S O

    1997-12-01

    The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association

  16. 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

  17. 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

  18. Cell mechanics: principles, practices, and prospects.

    PubMed

    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. Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation

    PubMed Central

    Kumar, Suresh M.; Liu, Shujing; Lu, Hezhe; Zhang, Hongtao; Zhang, Paul J.; Gimotty, Phyllis A.; Guerra, Matthew; Guo, Wei; Xu, Xiaowei

    2012-01-01

    There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression. PMID:22286766

  20. Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vitro.

    PubMed Central

    Kaufmann, Y; Berke, G; Eshhar, Z

    1981-01-01

    Cytotoxic hybridomas were generated by polyethylene glycol-induced fusion of cytotoxic T lymphocytes (CTL) and BW5147 lymphoma cells. The CTL populations used for fusion were obtained from BALB/c (H-2d) mice primed with leukemia EL4 of C57BL/6 (H-2b) and restimulated either in vivo or in vitro. To circumvent possible CTL-mediated nonspecific lysis of BW5147 cells during fusion, the CTL were transiently inactivated by trypsin prior to fusion. Four cytolytically active hybridomas were obtained, cloned, and subcloned. Hybrid clones lysed all H-2b leukemic target cells tested but not lipopolysaccharide- or concavanalin A-stimulated C57BL/6 lymphoblasts or non-H2b target tumor cells. The mechanism of hybridoma-mediated killing of target cells in vitro appears to be similar to that of parental CTL, although some differences have been observed. The hybridomas appear to possess neither natural killing nor antibody-dependent cytolytic activity. Clones of hybrids propagated in culture for over 6 months without the addition of known external stimulus (i.e., independent of cell growth factor and antigen) exhibit specific lytic activity against H-2b tumor cells. Such autonomous hybridomas will provide a tool for studying the mechanism of CTL-mediated lysis and the nature of the CTL receptors. PMID:6972538

  1. Melatonin protects mast cells against cytotoxicity mediated by chemical stimuli PMACI: possible clinical use.

    PubMed

    Maldonado, M D; Garcia-Moreno, H; Calvo, J R

    2013-09-15

    Melatonin has documented cytoprotective effects on a wide variety of immune cells. The mechanism of action on mast cells (RBL-2H3) still remains in the dark. We found that melatonin significantly attenuated phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced cytotoxicity in a concentration and time-dependent manner. It appears that the effect of melatonin on mast cells is two-fold: dependent (MT1 and MT2) and independent membrane receptors. In conclusion, melatonin treatment reduced the cytotoxicity, mediated by PMACI, and could provide a useful therapeutic option in processes where an excessive activation of mast cells occurs. PMID:23870536

  2. Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia

    PubMed Central

    Baginska, Joanna; Viry, Elodie; Berchem, Guy; Poli, Aurélie; Noman, Muhammad Zaeem; van Moer, Kris; Medves, Sandrine; Zimmer, Jacques; Oudin, Anaïs; Niclou, Simone P.; Bleackley, R. Chris; Goping, Ing Swie; Chouaib, Salem; Janji, Bassam

    2013-01-01

    Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies. PMID:24101526

  3. Acetylation regulates DNA repair mechanisms in human cells.

    PubMed

    Piekna-Przybylska, Dorota; Bambara, Robert A; Balakrishnan, Lata

    2016-06-01

    The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation. PMID:27104361

  4. In Vivo Mechanical Loading Rapidly Activates β–catenin Signaling in Osteocytes through a Prostaglandin Mediated Mechanism

    PubMed Central

    Lara-Castillo, N; Kim-Weroha, NA; Kamel, MA; Javaheri, B; Ellies, DL; Krumlauf, RE; Thiagarajan, G; Johnson, ML

    2015-01-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in the in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1 hour post loading, spreading to additional osteocytes and finally to cells on the bone surface by 24 hrs. This early activation at 1 hour appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly down regulated in osteocytes in vivo at 24 hours post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  5. In vivo mechanical loading rapidly activates β-catenin signaling in osteocytes through a prostaglandin mediated mechanism.

    PubMed

    Lara-Castillo, N; Kim-Weroha, N A; Kamel, M A; Javaheri, B; Ellies, D L; Krumlauf, R E; Thiagarajan, G; Johnson, M L

    2015-07-01

    The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo. PMID:25836764

  6. 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

  7. 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

  8. 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

  9. Immune-Mediated Complications after Hematopoietic Stem Cell Transplantation.

    PubMed

    Li, Zhuoyan; Rubinstein, Samuel M; Thota, Ramya; Savani, Malvi; Brissot, Eolia; Shaw, Bronwen E; Majhail, Navneet S; Mohty, Mohamad; Savani, Bipin N

    2016-08-01

    Hematopoietic stem cell transplantation (HSCT) has an integral role in the treatment of malignant and nonmalignant diseases. Long-term complications after HSCT have been well established and include graft-versus-host disease (GVHD), conditioning regimen-related toxicities, disease relapse, and infections. Immune-mediated phenomena are increasingly described after HSCT with clinically significant sequelae. Diagnosis is challenging because of features that overlap with other commonly reported post-transplantation complications. Patients who experience immune-mediated disease after HSCT tend to have poor outcomes. Early recognition of immune-mediated complications is imperative to reduce preventable morbidity and mortality. This review looks at the currently available literature on pathogenesis, incidence, risk factors, treatment, and outcomes of immune-mediated disease (other than GVHD) after HSCT. PMID:27095688

  10. Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation

    PubMed Central

    Klumpers, Darinka D.; Zhao, Xuanhe; Mooney, David J.; Smit, Theo H.

    2013-01-01

    During embryonic development, morphogenetic processes give rise to a variety of shapes and patterns that lead to functional tissues and organs. While the impact of chemical signals in these processes is widely studied, the role of physical cues is less understood. The aim of this study was to test the hypothesis that the interplay of cell mediated contraction and mechanical boundary conditions alone can result in spatially regulated differentiation in simple 3D constructs. An experimental model consisting of a 3D cell-gel construct and a finite element (FE) model were used to study the effect of cellular traction exerted by mesenchymal stem cells (MSCs) on an initially homogeneous matrix under inhomogeneous boundary conditions. A robust shape change is observed due to contraction under time-varying mechanical boundary conditions, which is explained by the finite element model. Furthermore, distinct local differences of osteogenic differentiation are observed, with a spatial pattern independent of osteogenic factors in the culture medium. Regions that are predicted to have experienced relatively high shear stress at any time during contraction, correlate with the regions of distinct osteogenesis. Taken together, these results support the underlying hypothesis that cellular contractility and mechanical boundary conditions alone can result in spatially regulated differentiation. These results will have important implications for tissue engineering and regeneration. PMID:23925497

  11. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles

    PubMed Central

    Hu, Xiaoming; Liou, Anthony K.F.; Leak, Rehana K.; Xu, Mingyue; An, Chengrui; Suenaga, Jun; Shi, Yejie; Gao, Yanqin; Zheng, Ping; Chen, Jun

    2014-01-01

    Microglia are the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery and are known for their ability to assume diverse phenotypes. A broad range of surface receptors are expressed on microglia and mediate microglial ‘On’ or ‘Off’ responses to signals from other host cells as well as invading microorganisms. The integrated actions of these receptors result in tightly regulated biological functions, including cell mobility, phagocytosis, the induction of acquired immunity, and trophic factor/inflammatory mediator release. Over the last few years, significant advances have been made towards deciphering the signaling mechanisms related to these receptors and their specific cellular functions. In this review, we describe the current state of knowledge of the surface receptors involved in microglial activation, with an emphasis on their engagement of distinct functional programs and their roles in CNS injuries. It will become evident from this review that microglial homeostasis is carefully maintained by multiple counterbalanced strategies, including, but not limited to, ‘On’ and ‘Off’ receptor signaling. Specific regulation of theses microglial receptors may be a promising therapeutic strategy against CNS injuries. PMID:24923657

  12. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling.

    PubMed

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I; Nienhaus, G Ulrich; Gierschik, Peter

    2015-07-10

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling. PMID:25903139

  13. Fas-mediated elimination of antigen-presenting cells and autoreactive T cells contribute to prevention of autoimmunity

    PubMed Central

    Stranges, Peter. B.; Watson, Jessica; Cooper, Cristie J.; Choisy-Rossi, Caroline-Morgane; Stonebraker, Austin C.; Beighton, Ryan A.; Hartig, Heather; Sundberg, John P.; Servick, Stein; Kaufmann, Gunnar; Fink, Pamela J.; Chervonsky, Alexander V.

    2008-01-01

    Summary Fas (Apo-1, CD95) receptor has been suggested to control T cell expansion by triggering T cell-autonomous apoptosis. This paradigm is based on the extensive lymphoproliferation and systemic autoimmunity in mice and humans lacking Fas or its ligand. However, with systemic loss of Fas, it is unclear whether T cell-extrinsic mechanisms contribute to autoimmunity. We found that tissue-specific deletion of Fas in mouse antigen presenting cells (APC) was sufficient to cause systemic autoimmunity, implying that normally APC are destroyed during immune responses via a Fas-mediated mechanism. Fas expression by APC was increased by exposure to microbial stimuli. Analysis of mice with Fas loss restricted to T cells revealed that Fas indeed controls autoimmune T cells, but not T cells responding to strong antigenic stimulation. Thus, Fas-dependent elimination of APC is a major regulatory mechanism curbing autoimmune responses and acts in concert with Fas-mediated regulation of chronically activated autoimmune T cells. PMID:17509906

  14. Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation.

    PubMed

    Du, Yibin; Zhang, Shuo; Yu, Tao; Du, Gongwen; Zhang, Hui; Yin, Zongsheng

    2016-09-01

    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

  15. 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

  16. MFG-E8 Is Critical for Embryonic Stem Cell-Mediated T Cell Immunomodulation

    PubMed Central

    Tan, Yuan; AlKhamees, Bodour; Jia, Deyong; Li, Li; Couture, Jean-François; Figeys, Daniel; Jinushi, Masahisa; Wang, Lisheng

    2015-01-01

    Summary The molecules and mechanisms pertinent to the low immunogenicity of undifferentiated embryonic stem cells (ESCs) remain poorly understood. Here, we provide evidence that milk fat globule epidermal growth factor 8 (MFG-E8) is a vital mediator in this phenomenon and directly suppresses T cell immune responses. MFG-E8 is enriched in undifferentiated ESCs but diminished in differentiated ESCs. Upregulation of MFG-E8 in ESCs increases the successful engraftment of both undifferentiated and differentiated ESCs across major histocompatibility complex barriers. MFG-E8 suppresses T cell activation/proliferation and inhibits Th1, Th2, and Th17 subpopulations while increasing regulatory T cell subsets. Neutralizing MFG-E8 substantially abrogates these effects, whereas addition of recombinant MFG-E8 to differentiated ESCs restores immunosuppression. Furthermore, we provide the evidence that MFG-E8 suppresses T cell activation and regulates T cell polarization by inhibiting PKCθ phosphorylation through the α3/5βV integrin receptor. Our findings offer an approach to facilitate transplantation acceptance. PMID:26455415

  17. Cell membrane mediated (-)-epicatechin effects on upstream endothelial cell signaling: evidence for a surface receptor.

    PubMed

    Moreno-Ulloa, Aldo; Romero-Perez, Diego; Villarreal, Francisco; Ceballos, Guillermo; Ramirez-Sanchez, Israel

    2014-06-15

    The consumption of cacao-derived products, particularly in the form of dark chocolate is known to provide beneficial cardiovascular effects in normal individuals and in those with vascular dysfunction (reduced nitric oxide [NO] bioavailability and/or synthesis). Upstream mechanisms by which flavonoids exert these effects are poorly understood and may involve the participation of cell membrane receptors. We previously demonstrated that the flavanol (-)-epicatechin (EPI) stimulates NO production via Ca(+2)-independent eNOS activation/phosphorylation. We wished to investigate the plausible participation of a cell surface receptor using a novel cell-membrane impermeable EPI-Dextran conjugate (EPI-Dx). Under Ca(2+)-free conditions, human coronary artery endothelial cells (HCAEC) were treated for 10min with EPI or EPI-Dx at equimolar concentrations (100nM). Results demonstrate that both EPI and EPI-Dx induced the phosphorylation/activation of PI3K, PDK-1, AKT and eNOS. Interestingly, EPI-Dx effects were significantly higher in magnitude than those of EPI alone. The capacity of EPI-Dx to stimulate cell responses supports the existence of an EPI cell membrane receptor mediating eNOS activation. PMID:24794111

  18. Leelamine mediates cancer cell death through inhibition of intracellular cholesterol transport.

    PubMed

    Kuzu, Omer F; Gowda, Raghavendra; Sharma, Arati; Robertson, Gavin P

    2014-07-01

    Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray-based analyses of the intracellular signaling cascades showed alterations in RTK-AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers. PMID:24688051

  19. Leelamine mediates cancer cell death through inhibition of intracellular cholesterol transport

    PubMed Central

    Kuzu, Omer F.; Gowda, Raghavendra; Sharma, Arati; Robertson, Gavin P.

    2015-01-01

    Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray–based analyses of the intracellular signaling cascades showed alterations in RTK–AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers. PMID:24688051

  20. Micheliolide overcomes KLF4-mediated cisplatin resistance in breast cancer cells by downregulating glutathione

    PubMed Central

    Jia, Yongsheng; Zhang, Chunze; Zhou, Liyan; Xu, Huijun; Shi, Yehui; Tong, Zhongsheng

    2015-01-01

    Micheliolide (MCL) is a promising novel compound with broad-spectrum anticancer activity. However, little is known regarding its action and mechanism in breast cancer. To explore the potential therapeutic application of MCL as a chemosensitivity modulator, this study investigated the effects of MCL on cisplatin sensitivity in breast cancer and the underlying mechanisms. In the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay and a xenograft tumor model, MCL enhanced the cisplatin sensitivity of the breast cancer cell line MCF-7 both in vitro and in vivo. Treatment of MCF-7 cells with low-dose cisplatin (10 µM) was sufficient to enrich the proportion of ALDH+ cells and upregulate Krüppel-like factor 4 (KLF4) expression. The results obtained from knockdown and overexpression experiments demonstrate that KLF4 is both necessary and sufficient to induce a cisplatin resistance phenotype in breast cancer cells. Furthermore, the glutathione (GSH) content was elevated in MCF-7 cells after overexpression of KLF4. KLF4-mediated resistance to cisplatin was found to be abrogated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis. MCL induced GSH depletion and severe cell death in KLF4-overexpressing MCF-7 cells following exposure to cisplatin. Therefore, these results suggest that MCL-mediated direct depletion of GSH represents a major mechanism in reversing KLF4-induced cisplatin resistance in MCF-7 cells. PMID:26356142

  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. PMID:23959171

  2. Outer hair cells as potential targets of inflammatory mediators.

    PubMed

    Huang, M; Dulon, D; Schacht, J

    1990-06-01

    Inner ear sequelae with temporary or permanent sensorineural hearing loss can result from inflammatory processes in the middle ear. Loss of outer hair cells in the base of the cochlea has been noted in otitis media, but it is not known how this damage occurs. Evidence supports the permeability of the round window membrane to substances mediating inflammation in the middle ear, and the presence of white blood cells has been reported in the perilymph. In the present study, the potential cytotoxic effects of two representative inflammatory mediators, endotoxin and free radicals, have been evaluated by use of short-term culture of isolated outer hair cells from the guinea pig cochlea model. Incubation with endotoxins from two gram-negative pathogens increased the rate of hair cell death fourfold to sixfold. Free radicals (generated by exposure of cells to UV light or by excitation of intracellular fluorescent dyes) produced morphologic damage to hair cells within 60 seconds. These latter effects were delayed by addition of free-radical scavengers. It is concluded that inflammatory mediators are cytotoxic to hair cells and therefore are potentially ototoxic if permeating the round window membrane. PMID:2112361

  3. Resistance to transforming growth factor β-mediated tumor suppression in melanoma: are multiple mechanisms in place?

    PubMed Central

    Lasfar, Ahmed; Cohen-Solal, Karine A.

    2010-01-01

    Resistance to transforming growth factor (TGF) β-mediated tumor suppression in melanoma appears to be a crucial step in tumor aggressiveness since it is usually coupled with the ability of TGFβ to drive the oncogenic process via autocrine and paracrine effects. In this review, we will focus mainly on the mechanisms of escape from TGFβ-induced cell cycle arrest because the mechanisms of resistance to TGFβ-mediated apoptosis are still essentially speculative. As expected, some of these mechanisms can directly affect the function of the main downstream effectors of TGFβ, Smad2 and Smad3, resulting in compromised Smad-mediated antiproliferative activity. Other mechanisms can counteract or overcome TGFβ-mediated cell cycle arrest independently of the Smads. In melanoma, some models of resistance to TGFβ have been suggested and will be described. In addition, we propose additional models of resistance taking into consideration the information available on the dysregulation of fundamental cellular effectors and signaling pathways in melanoma. PMID:20656791

  4. 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.

  5. 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

  6. Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

    SciTech Connect

    Glass, J.R.; Gerner, E.W.

    1987-01-01

    The mechanism of spermidine-induced ornithine decarboxylase (OCD, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine. Treatment of cells with 10 ..mu..M exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of (/sup 35/S)methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37/sup 0/C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22/sup 0/C for 3 hours with 10 ..mu.. M spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents had no effect of ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway.

  7. 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

  8. [Progress of study on antitumor effects of antibody dependent cell mediated cytotoxicity--review].

    PubMed

    Qu, Yu-Hua; Li, Yang

    2010-10-01

    In recent years, as increasing of monoclonal antibody application in clinic, the antitumor effect of antibody dependent cell-mediated cytotoxicity (ADCC) get increasing attention. The natural killer (NK) cells are the most important effector cells mediating specific antitumor of ADCC; the phagocytes, T-cells and granulocytes have the definite effect on antitumor of ADCC. ADCC is confirmed as the important mechanism and means for clinically treating the cancers with monoclonal antibodies. The IgG antibody firstly combines with target cells (tumor cells) through antigen-binding sites, and then FcγR on effector cells identifies its Fc fragment and mediates ADCC. Today many kinds of monoclonal antibodies have been put into clinical application such as rituximab and other new anti-CD20 monoclonal antibodies including trastuzumab, erbitux, cetuximab, edrecolomab, nimotuzumab, gemtuzumab ozogamicin and so on, which all can mediate ADCC. The antitumor effects of ADCC mediated by monoclonal antibody can be influenced by IgG Fc receptor gene polymorphism, tumor cell antigen, serum antibody levels, cytokines and drugs etc. As to peripheral blood mononuclear cells, ADCC efficacies of FcγRIIIa-158V/V and FcγRIIa-131H/H are higher than that of other genotypes, while increasing the level of tumor antigen and decreasing the level of serum antibody or adding some cytokines (IL-2, IL-21, IL-15, etc) may elevate the ADCC effect mediated by monoclonal antibodies. Avoiding use of certain drugs (dexamethasone, TNF antagonist) or appropriately using of ondansetron and clemastine also can enhance the anti-tumor effect of ADCC mediated by monoclonal antibodies. In short, ADCC is very important in clinical application for anti-tumor treatment, but its efficacy may be impacted by multiple factors.In this article, the killing mechanisms of ADCC, the clinical use of monoclonal antibodies with antitumor effect of ADCC, the factors influencing anti-tumor efficacy of ADCC, and the antitumor

  9. Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition.

    PubMed

    Di Modica, Martina; Sfondrini, Lucia; Regondi, Viola; Varchetta, Stefania; Oliviero, Barbara; Mariani, Gabriella; Bianchi, Giulia Valeria; Generali, Daniele; Balsari, Andrea; Triulzi, Tiziana; Tagliabue, Elda

    2016-01-01

    Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity. PMID:26595802

  10. Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition

    PubMed Central

    Regondi, Viola; Varchetta, Stefania; Oliviero, Barbara; Mariani, Gabriella; Bianchi, Giulia Valeria; Generali, Daniele; Balsari, Andrea

    2016-01-01

    Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15–40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity. PMID:26595802

  11. 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

  12. 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.

  13. Mast cell mediators in the blood of patients with asthma.

    PubMed

    Wasserman, S I

    1985-01-01

    Mast cell activation occurs in allergic asthma and may play a role in a variety of nonallergic asthmatic states. The defined mast cell constituent histamine has been identified in blood of antigen-sensitive challenged asthma patients, while other mediators, whose cell of origin is not fully defined, accompany this amine in blood (Table 1). Due to technical difficulty in accurate assessment, the rapid metabolism of various constituents, and the need for biologic rather than chemical assay of some mediators, it is not yet possible to assess blood constituents for the unequivocal attribution of asthma to activation of a particular cell type. Likewise, the usefulness of blood studies in the prediction of the course of asthma or as serial measurements to define the severity of asthma remains limited. However, it is only with analysis of the appropriate biologic fluids, blood and/or bronchoalveolar lavage materials, that it will be possible to define which potential mediators are, in fact, present and active in asthma. Until such analysis is completed, it is not possible to assign a function in this disease to the numerous potent inflammatory mediators known to be active in in vitro or in vivo models of asthma. PMID:3880526

  14. EFFECTS OF ENVIRONMENTAL CONTAMINANTS ON CELL MEDIATED IMMUNITY

    EPA Science Inventory

    The effect of lead and cadmium on cell-mediated immunity was studied in peritoneal macrophages, B-, and T-lymphocytes of mice. Lead and cadmium were administered in drinking water for 10 weeks in short-term experiments and up to 18 months to deal with immune responses in aged mic...

  15. 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. ...

  16. Electrodeformation for single cell mechanical characterization

    NASA Astrophysics Data System (ADS)

    Chen, Jian; Abdelgawad, Mohamed; Yu, Liming; Shakiba, Nika; Chien, Wei-Yin; Lu, Zhe; Geddie, William R.; Jewett, Michael A. S.; Sun, Yu

    2011-05-01

    This paper presents the use of electrodeformation as a method for single cell mechanical characterization in which mechanical properties of SiHa and ME180 cells (two cervical cancer cell lines) were quantified. Cells were directly placed between two microelectrodes with a rectangular ac electric field applied, and cell deformation was recorded under certain experimental conditions. Numerical simulations were performed to model cell electrodeformation based on the Maxwell stress tensor formulation. In these simulations, effects of cell electrical property variations on their electrodeformed behavior were investigated. By comparing the measured morphological changes with those obtained from numerical simulations, we were able to quantify Young's modulus of SiHa cells (601 ± 183 Pa) and ME180 cells (1463 ± 649 Pa). These values were consistent with Young's modulus values (SiHa: 400 ± 290 Pa and ME180: 1070 ± 580 Pa) obtained from conventional micropipette aspiration.

  17. 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

  18. Natural killer cell mediated cytotoxic responses in the Tasmanian devil.

    PubMed

    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

  19. Explanatory style and cell-mediated immunity in elderly men and women.

    PubMed

    Kamen-Siegel, L; Rodin, J; Seligman, M E; Dwyer, J

    1991-01-01

    Correlated pessimistic explanatory style--the belief that negative events are caused by internal, stable, and global factors--with lowered immunocompetence in a sample of 26 older adults. Two measures of cell-mediated immunity--T-helper cell/T-suppressor cell ratio and T-lymphocyte response to mitogen challenge--were lower in individuals with a pessimistic style, controlling for the influence of current health, depression, medication, recent weight change, sleep, and alcohol use. A relative increase in the percentage of T-suppressor cells seemed to underlie this immunosuppression. Although the mechanism by which explanatory style might influence immune function remains unknown, we speculate that a pessimistic style might be an important psychological risk factor--at least among older people--in the early course of certain immune-mediated diseases. PMID:1915208

  20. 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

  1. Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

    PubMed Central

    Chávez-Galán, L; Arenas-Del Angel, M C; Zenteno, E; Chávez, R; Lascurain, R

    2009-01-01

    One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+ T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lytic molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis. PMID:19254476

  2. 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.

  3. Enhancement of oxaliplatin sensitivity in human colorectal cancer by hypericin mediated photodynamic therapy via ROS-related mechanism.

    PubMed

    Lin, Shengchao; Lei, Kecheng; Du, Wenpei; Yang, Liyan; Shi, Haiyang; Gao, Yuwei; Yin, Peihao; Liang, Xin; Liu, Jianwen

    2016-02-01

    The resistance to oxaliplatin (L-OHP) is a major obstacle to ideal therapeutic outcomes in colorectal cancer. Photodynamic therapy (PDT) induces tumor damage through photosensitizer-mediated oxidative cytotoxicity. Hypericin is a well-studied photosensitizer. In this study, we explored the role of hypericin-mediated PDT (HY-PDT) in sensitizing human colorectal cancer cells towards L-OHP. Pre-treatment with HY-PDT enhanced the anti-tumor activity of L-OHP via decreasing drug efflux and increasing platinum accumulation. Further research showed that HY-PDT-mediated resensitization of resistance cells towards L-OHP was dependent on regulation of MRP-2, instead of p-gp. HY-PDT was also found to inhibit intracellular glutathione (GSH) and Glutathione S-transferase (GST), suggesting the involvement of GSH-related detoxification in the sensitization effect. Additionally, enhanced DNA double-strand breaks (DSBs) was observed following HY-PDT/L-OHP combined treatment. HY-PDT lowered the removing rate of platinum from DNA and down-regulated the expression of ERCC1 and XPF, two critical enzymes involved in nucleotide excision repair (NER) pathway. GSH monoethyl ester (GSH-EE) antagonized HY-PDT-induced ROS and repressed sensitization to platinum. Taken together, HY-PDT mediated sensitization of L-OHP in human colorectal cancer is mediated by ROS, whose mechanism involves affecting drug efflux, GSH-related detoxification and NER-mediated DNA repair. PMID:26673998

  4. 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

  5. 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

  6. CHOP Contributes to, But Is Not the Only Mediator of, IAPP Induced β-Cell Apoptosis.

    PubMed

    Gurlo, T; Rivera, J F; Butler, A E; Cory, M; Hoang, J; Costes, S; Butler, Peter C

    2016-04-01

    The islet in type 2 diabetes is characterized by β-cell loss, increased β-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). When protein misfolding protective mechanisms are overcome, human IAPP (h-IAPP) forms membrane permeant toxic oligomers that induce β-cell dysfunction and apoptosis. In humans with type 2 diabetes (T2D) and mice transgenic for h-IAPP, endoplasmic reticulum (ER) stress has been inferred from nuclear translocation of CCAAT/enhancer-binding protein homologous protein (CHOP), an established mediator of ER stress. To establish whether h-IAPP toxicity is mediated by ER stress, we evaluated diabetes onset and β-cell mass in h-IAPP transgenic (h-TG) mice with and without deletion of CHOP in comparison with wild-type controls. Diabetes was delayed in h-TG CHOP(-/-) mice, with relatively preserved β-cell mass and decreased β-cell apoptosis. Deletion of CHOP attenuates dysfunction of the autophagy/lysosomal pathway in β-cells of h-TG mice, uncovering a role for CHOP in mediating h-IAPP-induced dysfunction of autophagy. As deletion of CHOP delayed but did not prevent h-IAPP-induced β-cell loss and diabetes, we examined CHOP-independent stress pathways. JNK, a target of the IRE-1pTRAF2 complex, and the Bcl-2 family proapoptotic mediator BIM, a target of ATF4, were comparably activated by h-IAPP expression in the presence and absence of CHOP. Therefore, although these studies affirm that CHOP is a mediator of h-IAPP-induced ER stress, it is not the only one. Therefore, suppression of CHOP alone is unlikely to be a durable therapeutic strategy to protect against h-IAPP toxicity because multiple stress pathways are activated. PMID:26900721

  7. 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

  8. Histone deacetylase 10 promotes autophagy-mediated cell survival

    PubMed Central

    Oehme, Ina; Linke, Jan-Peter; Böck, Barbara C.; Milde, Till; Lodrini, Marco; Hartenstein, Bettina; Wiegand, Inga; Eckert, Christian; Roth, Wilfried; Kool, Marcel; Kaden, Sylvia; Gröne, Hermann-Josef; Schulte, Johannes H.; Lindner, Sven; Hamacher-Brady, Anne; Brady, Nathan R.; Deubzer, Hedwig E.; Witt, Olaf

    2013-01-01

    Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate that HDAC10 protects cancer cells from cytotoxic agents by mediating autophagy and identify this HDAC isozyme as a druggable regulator of advanced-stage tumor cell survival. Moreover, these results propose a promising way to considerably improve treatment response in the neuroblastoma patient subgroup with the poorest outcome. PMID:23801752

  9. Histone deacetylase 10 promotes autophagy-mediated cell survival.

    PubMed

    Oehme, Ina; Linke, Jan-Peter; Böck, Barbara C; Milde, Till; Lodrini, Marco; Hartenstein, Bettina; Wiegand, Inga; Eckert, Christian; Roth, Wilfried; Kool, Marcel; Kaden, Sylvia; Gröne, Hermann-Josef; Schulte, Johannes H; Lindner, Sven; Hamacher-Brady, Anne; Brady, Nathan R; Deubzer, Hedwig E; Witt, Olaf

    2013-07-01

    Tumor cells activate autophagy in response to chemotherapy-induced DNA damage as a survival program to cope with metabolic stress. Here, we provide in vitro and in vivo evidence that histone deacetylase (HDAC)10 promotes autophagy-mediated survival in neuroblastoma cells. We show that both knockdown and inhibition of HDAC10 effectively disrupted autophagy associated with sensitization to cytotoxic drug treatment in a panel of highly malignant V-MYC myelocytomatosis viral-related oncogene, neuroblastoma derived-amplified neuroblastoma cell lines, in contrast to nontransformed cells. HDAC10 depletion in neuroblastoma cells interrupted autophagic flux and induced accumulation of autophagosomes, lysosomes, and a prominent substrate of the autophagic degradation pathway, p62/sequestosome 1. Enforced HDAC10 expression protected neuroblastoma cells against doxorubicin treatment through interaction with heat shock protein 70 family proteins, causing their deacetylation. Conversely, heat shock protein 70/heat shock cognate 70 was acetylated in HDAC10-depleted cells. HDAC10 expression levels in high-risk neuroblastomas correlated with autophagy in gene-set analysis and predicted treatment success in patients with advanced stage 4 neuroblastomas. Our results demonstrate