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Sample records for intracellular targeting signals

  1. Sensitivity analysis of intracellular signaling pathway kinetics predicts targets for stem cell fate control.

    Directory of Open Access Journals (Sweden)

    Alborz Mahdavi

    2007-07-01

    Full Text Available Directing stem cell fate requires knowledge of how signaling networks integrate temporally and spatially segregated stimuli. We developed and validated a computational model of signal transducer and activator of transcription-3 (Stat3 pathway kinetics, a signaling network involved in embryonic stem cell (ESC self-renewal. Our analysis identified novel pathway responses; for example, overexpression of the receptor glycoprotein-130 results in reduced pathway activation and increased ESC differentiation. We used a systematic in silico screen to identify novel targets and protein interactions involved in Stat3 activation. Our analysis demonstrates that signaling activation and desensitization (the inability to respond to ligand restimulation is regulated by balancing the activation state of a distributed set of parameters including nuclear export of Stat3, nuclear phosphatase activity, inhibition by suppressor of cytokine signaling, and receptor trafficking. This knowledge was used to devise a temporally modulated ligand delivery strategy that maximizes signaling activation and leads to enhanced ESC self-renewal.

  2. Intracellular calcium homeostasis and signaling.

    Science.gov (United States)

    Brini, Marisa; Calì, Tito; Ottolini, Denis; Carafoli, Ernesto

    2013-01-01

    Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

  3. [Intracellular signaling mechanisms in thyroid cancer].

    Science.gov (United States)

    Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

    2016-01-01

    Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

  4. Intracellular signal modulation by nanomaterials.

    Science.gov (United States)

    Hussain, Salik; Garantziotis, Stavros; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Baeza-Squiban, Armelle; Boland, Sonja

    2014-01-01

    A thorough understanding of the interactions of nanomaterials with biological systems and the resulting activation of signal transduction pathways is essential for the development of safe and consumer friendly nanotechnology. Here we present an overview of signaling pathways induced by nanomaterial exposures and describe the possible correlation of their physicochemical characteristics with biological outcomes. In addition to the hierarchical oxidative stress model and a review of the intrinsic and cell-mediated mechanisms of reactive oxygen species (ROS) generating capacities of nanomaterials, we also discuss other oxidative stress dependent and independent cellular signaling pathways. Induction of the inflammasome, calcium signaling, and endoplasmic reticulum stress are reviewed. Furthermore, the uptake mechanisms can be of crucial importance for the cytotoxicity of nanomaterials and membrane-dependent signaling pathways have also been shown to be responsible for cellular effects of nanomaterials. Epigenetic regulation by nanomaterials, effects of nanoparticle-protein interactions on cell signaling pathways, and the induction of various cell death modalities by nanomaterials are described. We describe the common trigger mechanisms shared by various nanomaterials to induce cell death pathways and describe the interplay of different modalities in orchestrating the final outcome after nanomaterial exposures. A better understanding of signal modulations induced by nanomaterials is not only essential for the synthesis and design of safer nanomaterials but will also help to discover potential nanomedical applications of these materials. Several biomedical applications based on the different signaling pathways induced by nanomaterials are already proposed and will certainly gain a great deal of attraction in the near future.

  5. Radioresistant head and neck squamous cell carcinoma cells: Intracellular signaling, putative biomarkers for tumor recurrences and possible therapeutic targets

    International Nuclear Information System (INIS)

    Skvortsov, Sergej; Jimenez, Connie R.; Knol, Jaco C.; Eichberger, Paul; Schiestl, Bernhard; Debbage, Paul; Skvortsova, Ira; Lukas, Peter

    2011-01-01

    Purpose: Treatment of local and distant head and neck cancer recurrences after radiotherapy remains an unsolved problem. In order to identify potential targets for use in effective therapy of recurrent tumors, we have investigated protein patterns in radioresistant (FaDu-IRR and SCC25-IRR, “IRR cells”) as compared to parental (FaDu and SCC25) head and neck carcinoma cells. Methods and materials: Radiation resistant IRR cells were derived from parental cells after repeated exposure to ionizing radiation 10 times every two weeks at a single dose of 10 Gy, resulting in a total dose of 100 Gy. Protein profiling in parental and IRR cells was carried out using two-dimensional differential gel electrophoresis (2D-DIGE) followed by MALDI-TOF/TOF mass spectrometry. Cell viability, cell migration assays and Western blot analysis were used to confirm results obtained using the proteome approach. Results: Forty-five proteins that were similarly modulated in FaDu-IRR and SCC25-IRR cells compared to parental cells were selected to analyze their common targets. It was found that these either up- or down-regulated proteins are closely related to the enhancement of cell migration which is regulated by Rac1 protein. Further investigations confirmed that Rac1 is up-regulated in IRR cells, and inhibiting its action reduces the migratory abilities of these cells. Additionally, the Rac1 inhibitor exerts cytostatic effects in HNSCC cells, mostly in migratory cells. Conclusions: Based on these results, we conclude that radioresistant HNSCC cells possess enhanced metastatic abilities that are regulated by a network of migration-related proteins. Rac1 protein may be considered as a putative biomarker of HNSCC radiation resistance, and as a potential therapeutic target for treating local and distant HNSCC recurrences.

  6. Involvement of PI3K/Akt Signaling Pathway and Its Downstream Intracellular Targets in the Antidepressant-Like Effect of Creatine.

    Science.gov (United States)

    Cunha, Mauricio P; Budni, Josiane; Ludka, Fabiana K; Pazini, Francis L; Rosa, Julia Macedo; Oliveira, Ágatha; Lopes, Mark W; Tasca, Carla I; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

    2016-07-01

    Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition.

  7. Nanoparticles for intracellular-targeted drug delivery

    International Nuclear Information System (INIS)

    Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

    2011-01-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  8. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  9. Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

    Science.gov (United States)

    Hafner, Anne E.; Rieger, Heiko

    2018-03-01

    The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

  10. Intracellular Signalling by C-Peptide

    Directory of Open Access Journals (Sweden)

    Claire E. Hills

    2008-01-01

    Full Text Available C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na+/K+ ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes.

  11. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... targets for the treatment of various T-cells, immune-related diseases. We hope ... signifies the alternative routes of signal propagation. The molecules kept in ...... growth factor, mitogens for vascular cells and fibroblasts: dif- ferential ..... tumor necrosis factor contributes to CD8(+) T cell survival in the transition ...

  12. Subcellular neuropharmacology: the importance of intracellular targeting.

    Science.gov (United States)

    Miyashiro, Kevin Y; Bell, Thomas J; Sul, Jai-Yoon; Eberwine, James

    2009-04-01

    Few cell types are more adapted for cell-cell signaling than neurons. Their responsiveness lies in the formation of highly specialized compartments composed of unique repertoires of selectively distributed protein complexes generated, in part, by the local translation of mRNAs and regulated by their RNA-binding proteins. Utilizing the selective distribution of these neuronal proteins and the underlying mechanisms that generate the differential patterns of expression as central facets of drug design promises to enhance the therapeutic ratio of a drug. It is in this context that we discuss the unique arrangement of mRNAs, RNA-binding proteins and the protein macromolecular complexes at the dendrite, which is the postsynaptic site of synaptic transmission. Recent advances in identifying the function of dendritic components of the mechanisms of protein and RNA transport, non-nuclear RNA splicing and localized translation underscore their importance as targets of neuropharmacology.

  13. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

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    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

    DEFF Research Database (Denmark)

    Prats Gavalda, Clara; Gomez-Cabello, Alba; Vigelsø Hansen, Andreas

    2011-01-01

    The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism...... and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular...... compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss...

  15. ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Intracellular signaling pathways: tyrosine kinase and mTOR inhibitors).

    Science.gov (United States)

    Reinwald, M; Silva, J T; Mueller, N J; Fortún, J; Garzoni, C; de Fijter, J W; Fernández-Ruiz, M; Grossi, P; Aguado, J M

    2018-06-01

    The present review is part of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biologic therapies. To review, from an infectious diseases perspective, the safety profile of therapies targeting different intracellular signaling pathways and to suggest preventive recommendations. Computer-based Medline searches with MeSH terms pertaining to each agent or therapeutic family. Although BCR-ABL tyrosine kinase inhibitors modestly increase the overall risk of infection, dasatinib has been associated with cytomegalovirus and hepatitis B virus reactivation. BRAF/MEK kinase inhibitors do not significantly affect infection susceptibility. The effect of Bruton tyrosine kinase inhibitors (ibrutinib) among patients with B-cell malignancies is difficult to distinguish from that of previous immunosuppression. However, cases of Pneumocystis jirovecii pneumonia (PCP), invasive fungal infection and progressive multifocal leukoencephalopathy have been occasionally reported. Because phosphatidylinositol-3-kinase inhibitors (idelalisib) may predispose to opportunistic infections, anti-Pneumocystis prophylaxis and prevention strategies for cytomegalovirus are recommended. No increased rates of infection have been observed with venetoclax (antiapoptotic protein Bcl-2 inhibitor). Therapy with Janus kinase inhibitors markedly increases the incidence of infection. Pretreatment screening for chronic hepatitis B virus and latent tuberculosis infection must be performed, and anti-Pneumocystis prophylaxis should be considered for patients with additional risk factors. Cancer patients receiving mTOR inhibitors face an increased incidence of overall infection, especially those with additional risk factors (prior therapies or delayed wound healing). Specific preventive approaches are warranted in view of the increased risk of infection associated with some of the

  16. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits......Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  17. Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells - high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation.

    Science.gov (United States)

    Bruserud, Øystein; Reikvam, Håkon; Fredly, Hanne; Skavland, Jørn; Hagen, Karen-Marie; van Hoang, Tuyen Thy; Brenner, Annette K; Kadi, Amir; Astori, Audrey; Gjertsen, Bjørn Tore; Pendino, Frederic

    2015-02-20

    The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication.

  18. Fatty Acid Signaling: The New Function of Intracellular Lipases

    Directory of Open Access Journals (Sweden)

    Zuzana Papackova

    2015-02-01

    Full Text Available Until recently, intracellular triacylglycerols (TAG stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

  19. Radiation-induced adaptive response and intracellular signal transduction pathways

    International Nuclear Information System (INIS)

    Tachibana, Akira

    2009-01-01

    As an essential biological function, cells can sense the radiation even at low dose and respond to it, and which is one of bases of the radiation-induced adaptive response (AR) where effects caused by high dose radiation are reduced by prior exposure to low dose radiation (LDR). Here described are studies of AR in well established m5S cells on the intracellular signal transduction that involves sensing of LDR and transmitting of its signal within the cell network. The first signal for AR yielded by LDR on the cell membrane is exactly unknown though hydrogen peroxide and phorbol ester (PMA) can reportedly cause AR. As PMA activates protein kinase C (PKC) and its inhibitors suppress AR, participation of PKC in AR has been suggested and supported by studies showing PKCα activation by LDR. In addition, p38 mitogen-activated protein kinase (MAPK) is shown to participate in AR by those facts that the enzyme is activated by LDR, a p38 MAPK inhibitor suppresses AR, and PKC inhibitors suppress the enzyme activation, which also suggesting that the signaling from PKC to p38 MAPK can become operative by LDR. However, the possible reverse signaling is also suggested, and thus the activation of positive feedback mechanism is postulated in PKC/p38 MAPK/phospholipase δ1/ PKC pathway. Cells introduced with siRNA against Prkca gene (coding PKCs) produce reduced amount of the enzyme, particularly, of PKCα. In those cells, AR by 5 Gy X-ray is not observed and thereby PKCα is involved in AR. The signaling in AR is only partly elucidated at present as above, and more detailed studies including identification of more PKC subtypes and signaling to DNA repair system are considered necessary. (K.T.)

  20. Intracellular Signaling Mediators in the Circulatory and Ventilatory Systems

    CERN Document Server

    Thiriet, Marc

    2013-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to phenomenological models of nano- and microscopic events in a corrector scheme of regulated mechanisms when the vessel lumen caliber varies markedly. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning. Volume 4 is devoted to major sets of intracellular mediators that transmit signals upon stimulation of cell-surface receptors.  Activation of...

  1. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis.

    Science.gov (United States)

    Micheva-Viteva, Sofiya N; Shou, Yulin; Ganguly, Kumkum; Wu, Terry H; Hong-Geller, Elizabeth

    2017-01-01

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis , we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted

  2. Model-based control of the temporal patterns of intracellular signaling in silico

    Science.gov (United States)

    Murakami, Yohei; Koyama, Masanori; Oba, Shigeyuki; Kuroda, Shinya; Ishii, Shin

    2017-01-01

    The functions of intracellular signal transduction systems are determined by the temporal behavior of intracellular molecules and their interactions. Of the many dynamical properties of the system, the relationship between the dynamics of upstream molecules and downstream molecules is particularly important. A useful tool in understanding this relationship is a methodology to control the dynamics of intracellular molecules with an extracellular stimulus. However, this is a difficult task because the relationship between the levels of upstream molecules and those of downstream molecules is often not only stochastic, but also time-inhomogeneous, nonlinear, and not one-to-one. In this paper, we present an easy-to-implement model-based control method that makes the target downstream molecule to trace a desired time course by changing the concentration of a controllable upstream molecule. Our method uses predictions from Monte Carlo simulations of the model to decide the strength of the stimulus, while using a particle-based approach to make inferences regarding unobservable states. We applied our method to in silico control problems of insulin-dependent AKT pathway model and EGF-dependent Akt pathway model with system noise. We show that our method can robustly control the dynamics of the intracellular molecules against unknown system noise of various strengths, even in the absence of complete knowledge of the true model of the target system. PMID:28275530

  3. Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

    Science.gov (United States)

    Shah, Dhiral Ashwin

    Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

  4. Sigma-1 receptor: The novel intracellular target of neuropsychotherapeutic drugs

    Directory of Open Access Journals (Sweden)

    Teruo Hayashi

    2015-01-01

    Full Text Available Sigma-1 receptor ligands have been long expected to serve as drugs for treatment of human diseases such as neurodegenerative disorders, depression, idiopathic pain, drug abuse, and cancer. Recent research exploring the molecular function of the sigma-1 receptor started unveiling underlying mechanisms of the therapeutic activity of those ligands. Via the molecular chaperone activity, the sigma-1 receptor regulates protein folding/degradation, ER/oxidative stress, and cell survival. The chaperone activity is activated or inhibited by synthetic sigma-1 receptor ligands in an agonist-antagonist manner. Sigma-1 receptors are localized at the endoplasmic reticulum (ER membranes that are physically associated with the mitochondria (MAM: mitochondria-associated ER membrane. In specific types of neurons (e.g., those at the spinal cord, sigma-1 receptors are also clustered at ER membranes that juxtapose postsynaptic plasma membranes. Recent studies indicate that sigma-1 receptors, partly in sake of its unique subcellular localization, regulate the mitochondria function that involves bioenergetics and free radical generation. The sigma-1 receptor may thus provide an intracellular drug target that enables controlling ER stress and free radical generation under pathological conditions.

  5. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    Science.gov (United States)

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  6. Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation.

    Science.gov (United States)

    Abdallah, Basem M; Jafari, Abbas; Zaher, Walid; Qiu, Weimin; Kassem, Moustapha

    2015-01-01

    Skeletal (marrow stromal) stem cells (BMSCs) are a group of multipotent cells that reside in the bone marrow stroma and can differentiate into osteoblasts, chondrocytes and adipocytes. Studying signaling pathways that regulate BMSC differentiation into osteoblastic cells is a strategy for identifying druggable targets for enhancing bone formation. This review will discuss the functions and the molecular mechanisms of action on osteoblast differentiation and bone formation; of a number of recently identified regulatory molecules: the non-canonical Notch signaling molecule Delta-like 1/preadipocyte factor 1 (Dlk1/Pref-1), the Wnt co-receptor Lrp5 and intracellular kinases. This article is part of a Special Issue entitled: Stem Cells and Bone. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Preparation and characterization of vinculin-targeted polymer-lipid nanoparticle as intracellular delivery vehicle.

    Science.gov (United States)

    Wang, Junping; Ornek-Ballanco, Ceren; Xu, Jiahua; Yang, Weiguo; Yu, Xiaojun

    2013-01-01

    Intracellular delivery vehicles have been extensively investigated as these can serve as an effective tool in studying the cellular mechanism, by delivering functional protein to specific locations of the cells. In the current study, a polymer-lipid nanoparticle (PLN) system was developed as an intracellular delivery vehicle specifically targeting vinculin, a focal adhesion protein associated with cellular adhesive structures, such as focal adhesions and adherens junctions. The PLNs possessed an average size of 106 nm and had a positively charged surface. With a lower encapsulation efficiency 32% compared with poly(lactic-co-glycolic) acid (PLGA) nanoparticles (46%), the PLNs showed the sustained release profile of model drug BSA, while PLGA nanoparticles demonstrated an initial burst-release property. Cell-uptake experiments using mouse embryonic fibroblasts cultured in fibrin-fibronectin gels observed, under confocal microscope, that the anti-vinculin conjugated PLNs could successfully ship the cargo to the cytoplasm of fibroblasts, adhered to fibronectin-fibrin. With the use of cationic lipid, the unconjugated PLNs were shown to have high gene transfection efficiency. Furthermore, the unconjugated PLNs had nuclear-targeting capability in the absence of nuclear-localization signals. Therefore, the PLNs could be manipulated easily via different type of targeting ligands and could potentially be used as a powerful tool for cellular mechanism study, by delivering drugs to specific cellular organelles.

  8. Membrane mechanisms and intracellular signalling in cell volume regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay; Dunham, Philip B.

    1995-01-01

    Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation.......Volume regulation, Signal transduction, Calcium-calmodulin, Stretch-activated channels, Eicosanoids, Macromolecular crowding, Cytoskeleton, Protein phosphorylation, dephosphorylation....

  9. Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

    Science.gov (United States)

    Sánchez-Gómez, F J; González-Morena, J M; Vida, Y; Pérez-Inestrosa, E; Blanca, M; Torres, M J; Pérez-Sala, D

    2017-03-01

    Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Lung cancer, intracellular signaling pathways, and preclinical models

    International Nuclear Information System (INIS)

    Mordant, P.

    2012-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the consequences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced anti-tumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the anti-tumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing anti-tumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative

  11. DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

  12. Quantitative imaging of intracellular signaling for personalized pancreatic cancer therapy in an in vivo avatar (Conference Presentation)

    Science.gov (United States)

    Samkoe, Kimberley S.; Schultz, Emily; Park, Yeonjae; Fischer, Dawn; Pogue, Brian W.; Smith, Kerrington; Tichauer, Kenneth M.; Gibbs, Summer L.

    2017-02-01

    Pancreatic ductal adenocarcinomas (PDAC) are notoriously difficult to treat and in general, molecular targeted therapies have failed even when the targeted protein is overexpressed in the tumor tissue. Genetic mutations in extracellular receptors and downstream signaling proteins (i.e., RAS signaling pathway) and convoluted intracellular cross-talk between cell signaling pathways are likely reasons that these promising therapies fail. Monitoring the complex relationship between intracellular protein signaling is difficult and to-date, standard techniques that are used (Western blot, flow cytometry, immunohistochemistry, etc.) are invasive, static and do not accurately represent in vivo structure-function relationships. Here, we describe the development of an in ovo avatar using patient derived tumors grown on the chicken chorioallantoic membrane (CAM) and the novel fluorescence-based Quantitative Protein Expression Tracking (QUIET) methodology to bridge the gap between oncology, genomics and patient outcomes. Previously developed paired-agent imaging, was extended to a three-compartment model system in QUIET, which utilizes three types of imaging agents: novel fluorophore conjugated cell permeable targeted and untargeted small molecule paired-agents, in addition to a tumor perfusion agent that is not cell membrane permeable. We have demonstrated the ability to quantify the intracellular binding domain of a trans-membrane protein in vitro using cell permeable fluorescent agents (erlotinib-TRITC and control isotype-BODIPY FL). In addition, we have demonstrated imaging protocols to simultaneously image up to 6 spectrally distinct organic fluorophores in in ovo avatars using the Nuance EX (Perkin Elmer) and established proof-of-principle intracellular and extracellular protein concentrations of epidermal growth factor receptor using QUIET and traditional paired-agent imaging.

  13. Intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory.

    Science.gov (United States)

    Takeda, Atsushi; Tamano, Haruna; Ogawa, Taisuke; Takada, Shunsuke; Nakamura, Masatoshi; Fujii, Hiroaki; Ando, Masaki

    2014-11-01

    The role of perforant pathway-dentate granule cell synapses in cognitive behavior was examined focusing on synaptic Zn(2+) signaling in the dentate gyrus. Object recognition memory was transiently impaired when extracellular Zn(2+) levels were decreased by injection of clioquinol and N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylendediamine. To pursue the effect of the loss and/or blockade of Zn(2+) signaling in dentate granule cells, ZnAF-2DA (100 pmol, 0.1 mM/1 µl), an intracellular Zn(2+) chelator, was locally injected into the dentate molecular layer of rats. ZnAF-2DA injection, which was estimated to chelate intracellular Zn(2+) signaling only in the dentate gyrus, affected object recognition memory 1 h after training without affecting intracellular Ca(2+) signaling in the dentate molecular layer. In vivo dentate gyrus long-term potentiation (LTP) was affected under the local perfusion of the recording region (the dentate granule cell layer) with 0.1 mM ZnAF-2DA, but not with 1-10 mM CaEDTA, an extracellular Zn(2+) chelator, suggesting that the blockade of intracellular Zn(2+) signaling in dentate granule cells affects dentate gyrus LTP. The present study demonstrates that intracellular Zn(2+) signaling in the dentate gyrus is required for object recognition memory, probably via dentate gyrus LTP expression. Copyright © 2014 Wiley Periodicals, Inc.

  14. Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

    Science.gov (United States)

    Tomiyama, Tetsuro; Toita, Riki; Kang, Jeong-Hun; Koga, Haruka; Shiosaki, Shujiro; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

    2011-09-01

    We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

  15. Multiple-targeted graphene-based nanocarrier for intracellular imaging of mRNAs

    International Nuclear Information System (INIS)

    Wang, Ying; Li, Zhaohui; Liu, Misha; Xu, Jinjin; Hu, Dehong; Lin, Yuehe; Li, Jinghong

    2017-01-01

    Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labelled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis. - Graphical abstract: Schematic illustration of simultaneously multiple mRNAs monitoring inside single living breast cancer cell based on GO nanocarrier. In particular, the fluorescent signals could be monitored when Mn-SOD probe (red) and β-actin probe (green) hybridizes with their mRNA targets inside the living cells. Random probe (orange) was regarded as control probe for the sensing strategy. - Highlights: • A multiple-targeted GO nanocarrier was used for mRNAs imaging and expression changes after drug treatment can be monitored successfully. • Sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) m

  16. Hierarchic stochastic modelling applied to intracellular Ca(2+ signals.

    Directory of Open Access Journals (Sweden)

    Gregor Moenke

    Full Text Available Important biological processes like cell signalling and gene expression have noisy components and are very complex at the same time. Mathematical analysis of such systems has often been limited to the study of isolated subsystems, or approximations are used that are difficult to justify. Here we extend a recently published method (Thurley and Falcke, PNAS 2011 which is formulated in observable system configurations instead of molecular transitions. This reduces the number of system states by several orders of magnitude and avoids fitting of kinetic parameters. The method is applied to Ca(2+ signalling. Ca(2+ is a ubiquitous second messenger transmitting information by stochastic sequences of concentration spikes, which arise by coupling of subcellular Ca(2+ release events (puffs. We derive analytical expressions for a mechanistic Ca(2+ model, based on recent data from live cell imaging, and calculate Ca(2+ spike statistics in dependence on cellular parameters like stimulus strength or number of Ca(2+ channels. The new approach substantiates a generic Ca(2+ model, which is a very convenient way to simulate Ca(2+ spike sequences with correct spiking statistics.

  17. Dopamine signaling: target in glioblastoma

    Czech Academy of Sciences Publication Activity Database

    Bartek, Jiří; Hodný, Zdeněk

    2014-01-01

    Roč. 5, č. 5 (2014), 1116-1117 ISSN 1949-2553 Institutional support: RVO:68378050 Keywords : Dopamine signaling * glioblastoma * MAPK Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.359, year: 2014

  18. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    Directory of Open Access Journals (Sweden)

    Taslima T. Lina

    2016-07-01

    Full Text Available Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40% were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2 and p38 mitogen-activated protein kinase (MAPK pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4 expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

  19. Specific intracellular signal transduction pathways downstream of CSF-1 receptors: their relationship to breast cancer local recurrence and distant relapse in vivo. Potential targets for the development of new, specific anti-breast cancer therapies to improve local control and block metastatic spread?

    International Nuclear Information System (INIS)

    Kacinski, Barry M.; Sapi, Eva; Flick, Maryann B.; Turner, Bruce; Perrotta, Peter; Maher, M. Grey; Carter, Darryl; Haffy, Bruce

    1997-01-01

    analogues such as Matrigel. Agents which pharmacologically mimic a TYR-721 →PHE mutation by interfering with the activation of elements of intracellular signal transduction downstream of this tyrosine (PI-3 kinase. pp70-S6kinase) produced similar effects. In contrast, a TYR-809→PHE mutation was without effect on anchorage independent growth or the generation of pulmonary metastases but did completely abolish protease production and rendered the transfected cells unable to invade basement membrane analogues. In a parallel line of research, we employed antibodies which recognized CSF-1R and a novel antibody we prepared to recognize CSF-1R only when phosphorylated on TYR-721 in a study of 80 T1 and T2 breast cancer patients treated with tylectomy and primary radiation therapy. Strong staining with the generic anti-CSF-1R antibody correlated strongly with local relapse (P values <.03) in this patient cohort. Staining with the antibody specific for CSF-1R phosphorylated at TYR-721 was not associated with local relapse but did correlate with the development of distant metastases in this cohort of patients, particularly in axillary node-negative patients (P < .006). Conclusion: In summary, our observations demonstrate that CSF-1R activation and phosphorylation at specific tyrosines regulates invasiveness, anchorage, independent growth and tumorigenicity in vitro and in animal models and correlates with metastatic relapse in vivo. They also suggest that such intracellular signaling pathways--particularly those triggered by the phosphorylation of TYR-721 of CSF-1R--are logical targets for the development of a new class of anti-cancer agents which specifically block breast cancer cell metastatic potential without perturbing other normal cellular metabolic processes

  20. Biological macromolecules based targeted nanodrug delivery systems for the treatment of intracellular infections.

    Science.gov (United States)

    Aparna, V; Shiva, M; Biswas, Raja; Jayakumar, R

    2018-04-15

    Intracellular infections are tricky to treat, the reason being the poor penetration of antibiotics/antimycotics into the microbial niche (host cell). Macrophages are primary targets of facultative and obligate intracellular bacteria/fungi to be abused as host cells. The need for drugs with better intracellular penetration led to the development of endocytosable drug carriers, which can cross the cell membrane of the host cells (macrophages) by imitating the entry path of the pathogens. Therefore, the drugs can be targeted to macrophages ensuring enhanced therapeutic effect. This review discusses the exploitation of various nanocarriers for targeted delivery of drugs to the macrophages in the last two decades. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Effect of insulin resistance on intracellular signal transduction of vessels in diabetic

    International Nuclear Information System (INIS)

    Cen Rongguang; Wei Shaoying; Mo Xingju

    2003-01-01

    To investigate the relationship between the insulin resistance (IR) and the intracellular signal transduction of vessels, changes in fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), inositol triphosphate (IP 3 ), protein kinase C(PKC) and intracellular total calcium concentration in 31 diabetic patients were compared with those of 39 normal controls. The levels of FBG, FINS, TG and TC in diabetic patients were significantly higher than those of normal controls (P 3 and PKC in diabetic patients were significantly lower than those of normal controls (P<0.01). The results suggest that there is a causal relation between insulin resistance and abnormalities of cellular calcium metabolism and intracellular signal transduction of vessels

  2. Integration of intracellular telomerase monitoring by electrochemiluminescence technology and targeted cancer therapy by reactive oxygen species.

    Science.gov (United States)

    Zhang, Huairong; Li, Binxiao; Sun, Zhaomei; Zhou, Hong; Zhang, Shusheng

    2017-12-01

    Cancer therapies based on reactive oxygen species (ROS) have emerged as promising clinical treatments. Electrochemiluminescence (ECL) technology has also attracted considerable attention in the field of clinical diagnosis. However, studies about the integration of ECL diagnosis and ROS cancer therapy are very rare. Here we introduce a novel strategy that employs ECL technology and ROS to fill the above vacancy. Briefly, an ITO electrode was electrodeposited with polyluminol-Pt NPs composite films and modified with aptamer DNA to capture HL-60 cancer cells with high specificity. After that, mesoporous silica nanoparticles (MSNs) filled with phorbol 12-myristate 13-acetate (PMA) were closed by the telomerase primer DNA (T-primer DNA) and aptamer. After aptamer on MSN@PMA recognized and combined with the HL-60 cancer cells with high specificity, T-primer DNA on MSN@PMA could be moved away from the MSN@PMA surface after extension by telomerase in the HL-60 cancer cells and PMA was released to induce the production of ROS by the HL-60 cancer cells. After that, the polyluminol-Pt NPs composite films could react with hydrogen peroxide (a major ROS) and generate an ECL signal. Thus the intracellular telomerase activity of the HL-60 cancer cells could be detected in situ . Besides, ROS could induce apoptosis in the HL-60 cancer cells with high efficacy by causing oxidative damage to the lipids, protein, and DNA. Above all, the designed platform could not only detect intracellular telomerase activity instead of that of extracted telomerase, but could also kill targeted tumors by ECL technology and ROS.

  3. Abrin immunotoxin: targeted cytotoxicity and intracellular trafficking pathway.

    Directory of Open Access Journals (Sweden)

    Sudarshan Gadadhar

    Full Text Available BACKGROUND: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. METHODS: Protein synthesis assay using (3[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. RESULTS: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. CONCLUSIONS: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.

  4. The Molecular Basis of Toxins’ Interactions with Intracellular Signaling via Discrete Portals

    Directory of Open Access Journals (Sweden)

    Adi Lahiani

    2017-03-01

    Full Text Available An understanding of the molecular mechanisms by which microbial, plant or animal-secreted toxins exert their action provides the most important element for assessment of human health risks and opens new insights into therapies addressing a plethora of pathologies, ranging from neurological disorders to cancer, using toxinomimetic agents. Recently, molecular and cellular biology dissecting tools have provided a wealth of information on the action of these diverse toxins, yet, an integrated framework to explain their selective toxicity is still lacking. In this review, specific examples of different toxins are emphasized to illustrate the fundamental mechanisms of toxicity at different biochemical, molecular and cellular- levels with particular consideration for the nervous system. The target of primary action has been highlighted and operationally classified into 13 sub-categories. Selected examples of toxins were assigned to each target category, denominated as portal, and the modulation of the different portal’s signaling was featured. The first portal encompasses the plasma membrane lipid domains, which give rise to pores when challenged for example with pardaxin, a fish toxin, or is subject to degradation when enzymes of lipid metabolism such as phospholipases A2 (PLA2 or phospholipase C (PLC act upon it. Several major portals consist of ion channels, pumps, transporters and ligand gated ionotropic receptors which many toxins act on, disturbing the intracellular ion homeostasis. Another group of portals consists of G-protein-coupled and tyrosine kinase receptors that, upon interaction with discrete toxins, alter second messengers towards pathological levels. Lastly, subcellular organelles such as mitochondria, nucleus, protein- and RNA-synthesis machineries, cytoskeletal networks and exocytic vesicles are also portals targeted and deregulated by other diverse group of toxins. A fundamental concept can be drawn from these seemingly different

  5. A new type of intracellular retention signal identified in a pestivirus structural glycoprotein.

    Science.gov (United States)

    Burrack, Sandra; Aberle, Daniel; Bürck, Jochen; Ulrich, Anne S; Meyers, Gregor

    2012-08-01

    Sorting of membrane proteins into intracellular organelles is crucial for cell function. Viruses exploit intracellular transport and retention systems to concentrate envelope proteins at the site of virus budding. In pestiviruses, a group of important pathogens of pigs and ruminants closely related to human hepatitis C virus, the E(rns) protein translated from the viral RNA is secreted from the infected cells and found in the serum of infected animals. Secretion of the protein is regarded as crucial for its function as a viral virulence factor associated with its RNase activity. However, ∼95% of the E(rns) molecules are retained within the infected cell. Fusion of different E(rns) fragments to the C terminus of CD72 allowed identification of a retention signal within the C-terminal 65 aa of the viral protein. This C-terminal sequence represents its membrane anchor and folds into an amphipathic helix binding in-plane to the membrane surface. Residues L183, I190, and L208 are important for intracellular location of E(rns). Presentation of the retention signal on the cytoplasmic instead of the luminal face of the ER membrane in CD8α fusion proteins still led to retention. Thus, E(rns) contains in its C-terminal amphipathic helix an intracellular retention signal that is active on both faces of the membrane.

  6. Chemokine Signaling in Allergic Contact Dermatitis: Toward Targeted Therapies.

    Science.gov (United States)

    Smith, Jeffrey S; Rajagopal, Sudarshan; Atwater, Amber Reck

    2018-06-22

    Allergic contact dermatitis (ACD) is a common skin disease that results in significant cost and morbidity. Despite its high prevalence, therapeutic options are limited. Allergic contact dermatitis is regulated primarily by T cells within the adaptive immune system, but also by natural killer and innate lymphoid cells within the innate immune system. The chemokine receptor system, consisting of chemokine peptides and chemokine G protein-coupled receptors, is a critical regulator of inflammatory processes such as ACD. Specific chemokine signaling pathways are selectively up-regulated in ACD, most prominently CXCR3 and its endogenous chemokines CXCL9, CXCL10, and CXCL11. Recent research demonstrates that these 3 chemokines are not redundant and indeed activate distinct intracellular signaling profiles such as those activated by heterotrimeric G proteins and β-arrestin adapter proteins. Such differential signaling provides an attractive therapeutic target for novel ACD therapies and other inflammatory diseases.

  7. Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

    International Nuclear Information System (INIS)

    Akbari, Vajihe; Abedi, Daryoush; Pardakhty, Abbas; Sadeghi-Aliabadi, Hojjat

    2013-01-01

    In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300–600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

  8. Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Akbari, Vajihe; Abedi, Daryoush [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of); Pardakhty, Abbas [Kerman University of Medical Sciences, Pharmaceutics Research Center (Iran, Islamic Republic of); Sadeghi-Aliabadi, Hojjat, E-mail: sadeghi@pharm.mui.ac.ir [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of)

    2013-04-15

    In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300-600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

  9. Neurodegeneration in Alzheimer Disease: Role of Amyloid Precursor Protein and Presenilin 1 Intracellular Signaling

    Directory of Open Access Journals (Sweden)

    Mario Nizzari

    2012-01-01

    Full Text Available Alzheimer disease (AD is a heterogeneous neurodegenerative disorder characterized by (1 progressive loss of synapses and neurons, (2 intracellular neurofibrillary tangles, composed of hyperphosphorylated Tau protein, and (3 amyloid plaques. Genetically, AD is linked to mutations in few proteins amyloid precursor protein (APP and presenilin 1 and 2 (PS1 and PS2. The molecular mechanisms underlying neurodegeneration in AD as well as the physiological function of APP are not yet known. A recent theory has proposed that APP and PS1 modulate intracellular signals to induce cell-cycle abnormalities responsible for neuronal death and possibly amyloid deposition. This hypothesis is supported by the presence of a complex network of proteins, clearly involved in the regulation of signal transduction mechanisms that interact with both APP and PS1. In this review we discuss the significance of novel finding related to cell-signaling events modulated by APP and PS1 in the development of neurodegeneration.

  10. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Science.gov (United States)

    Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

    2011-01-01

    Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  11. Rare genomic variants link bipolar disorder to CREB regulated intracellular signaling pathways

    Directory of Open Access Journals (Sweden)

    Berit eKerner

    2013-11-01

    Full Text Available Bipolar disorder is a common, complex, and severe psychiatric disorder with cyclical disturbances of mood and a high suicide rate. Here, we describe a family with four siblings, three affected females and one unaffected male. The disease course was characterized by early-onset bipolar disorder and co-morbid anxiety spectrum disorders that followed the onset of bipolar disorder. Genetic risk factors were suggested by the early onset of the disease, the severe disease course, including multiple suicide attempts, and lack of adverse prenatal or early life events. In particular, drug and alcohol abuse did not contribute to the disease onset. Exome sequencing identified very rare, heterozygous, and likely protein-damaging variants in eight brain-expressed genes: IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, and FGGY. The variants were shared among all three affected family members but absent in the unaffected sibling and in more than 200 controls. The genes encode proteins with significant regulatory roles in the ERK/MAPK and CREB-regulated intracellular signaling pathways. These pathways are central to neuronal and synaptic plasticity, cognition, affect regulation and response to chronic stress. In addition, proteins in these pathways are the target of commonly used mood stabilizing drugs, such as tricyclic antidepressants, lithium and valproic acid. The combination of multiple rare, damaging mutations in these central pathways could lead to reduced resilience and increased vulnerability to stressful life events. Our results support a new model for psychiatric disorders, in which multiple rare, damaging mutations in genes functionally related to a common signaling pathway contribute to the manifestation of bipolar disorder.

  12. Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

    Science.gov (United States)

    Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

    2018-02-05

    During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

  13. A Bombesin-Shepherdin Radioconjugate Designed for Combined Extra- and Intracellular Targeting

    Directory of Open Access Journals (Sweden)

    Christiane A. Fischer

    2014-05-01

    Full Text Available Radiolabeled peptides which target tumor-specific membrane structures of cancer cells represent a promising class of targeted radiopharmaceuticals for the diagnosis and therapy of cancer. A potential drawback of a number of reported radiopeptides is the rapid washout of a substantial fraction of the initially delivered radioactivity from cancer cells and tumors. This renders the initial targeting effort in part futile and results in a lower imaging quality and efficacy of the radiotracer than achievable. We are investigating the combination of internalizing radiopeptides with molecular entities specific for an intracellular target. By enabling intracellular interactions of the radioconjugate, we aim at reducing/decelerating the externalization of radioactivity from cancer cells. Using the “click-to-chelate” approach, the 99mTc-tricarbonyl core as a reporter probe for single-photon emission computed tomography (SPECT was combined with the binding sequence of bombesin for extracellular targeting of the gastrin-releasing peptide receptor (GRP-r and peptidic inhibitors of the cytosolic heat shock 90 protein (Hsp90 for intracellular targeting. Receptor-specific uptake of the multifunctional radioconjugate could be confirmed, however, the cellular washout of radioactivity was not improved. We assume that either endosomal trapping or lysosomal degradation of the radioconjugate is accountable for these observations.

  14. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

    Science.gov (United States)

    Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

    2017-01-01

    Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

  15. Small things matter: Implications of APP intracellular domain AICD nuclear signaling in the progression and pathogenesis of Alzheimer's disease.

    Science.gov (United States)

    Bukhari, Hassan; Glotzbach, Annika; Kolbe, Katharina; Leonhardt, Gregor; Loosse, Christina; Müller, Thorsten

    2017-09-01

    Alzheimer's disease (AD) is the most common neurodegenerative disease with tens of millions of people affected worldwide. The pathogenesis is still poorly understood and various therapeutical approaches targeting the amyloid β (Aβ) peptide, a product of the amyloidogenic cleavage of the amyloid precursor protein (APP), failed. Moreover, a couple of studies critically questioned the relevance of Aβ in the pathogenesis of AD. Thus, new ideas need to be studied and one highly interesting hypothesis is the APP mediated signal transduction to the nucleus. As a consequence nuclear -potentially toxic- structures emerge, which were recently found to a high extent in human AD tissue and thus, may contribute to neurodegeneration. Relevant for the signaling machinery are modifications at the very C-terminal end of the precursor protein, the APP intracellular domain (AICD). In this review we update the knowledge on mechanisms on AICD referring to our 2008 article: The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeletal dynamics-Relevance for Alzheimer's disease (T. Muller, et al., 2008). We summarize how AICD is generated and degraded, we describe its intramolecular motifs, translational modifications, and how those as well as APP dimerization influence AICD generation and function. Moreover, we resume the AICD interactome and elucidate AICDs involvement in nuclear signaling, transcriptional regulation, cell death, DNA repair and cell cycle re-entry and we give insights in its physiological function. Results are summarized in the comprehensive poster "The world of AICD". Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Novel robust biomarkers for human bladder cancer based on activation of intracellular signaling pathways

    OpenAIRE

    Lezhnina, Ksenia; Kovalchuk, Olga; Zhavoronkov, Alexander A.; Korzinkin, Mikhail B.; Zabolotneva, Anastasia A.; Shegay, Peter V.; Sokov, Dmitry G.; Gaifullin, Nurshat M.; Rusakov, Igor G.; Aliper, Alexander M.; Roumiantsev, Sergey A.; Alekseev, Boris Y.; Borisov, Nikolay M.; Buzdin, Anton A.

    2014-01-01

    We recently proposed a new bioinformatic algorithm called OncoFinder for quantifying the activation of intracellular signaling pathways. It was proved advantageous for minimizing errors of high-throughput gene expression analyses and showed strong potential for identifying new biomarkers. Here, for the first time, we applied OncoFinder for normal and cancerous tissues of the human bladder to identify biomarkers of bladder cancer. Using Illumina HT12v4 microarrays, we profiled gene expression ...

  17. An enhanced functional interrogation/manipulation of intracellular signaling pathways with the peptide 'stapling' technology.

    Science.gov (United States)

    He, Y; Chen, D; Zheng, W

    2015-11-12

    Specific protein-protein interactions (PPIs) constitute a key underlying mechanism for the presence of a multitude of intracellular signaling pathways, which are essential for the survival of normal and cancer cells. Specific molecular blockers for a crucial PPI would therefore be invaluable tools for an enhanced functional interrogation of the signaling pathway harboring this particular PPI. On the other hand, if a particular PPI is essential for the survival of cancer cells but is absent in or dispensable for the survival of normal cells, its specific molecular blockers could potentially be developed into effective anticancer therapeutics. Due to the flat and extended PPI interface, it would be conceivably difficult for small molecules to achieve an effective blockade, a problem which could be potentially circumvented with peptides or proteins. However, the well-documented proteolytic instability and cellular impermeability of peptides and proteins in general would make their developing into effective intracellular PPI blockers quite a challenge. With the advent of the peptide 'stapling' technology which was demonstrated to be able to stabilize the α-helical conformation of a peptide via bridging two neighboring amino-acid side chains with a 'molecular staple', a linear parent peptide could be transformed into a stronger PPI blocker with enhanced proteolytic stability and cellular permeability. This review will furnish an account on the peptide 'stapling' technology and its exploitation in efforts to achieve an enhanced functional interrogation or manipulation of intracellular signaling pathways especially those that are cancer relevant.

  18. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Science.gov (United States)

    Wang, Jinli; Yang, Kun; Zhou, Lin; Minhaowu; Wu, Yongjian; Zhu, Min; Lai, Xiaomin; Chen, Tao; Feng, Lianqiang; Li, Meiyu; Huang, Chunyu; Zhong, Qiu; Huang, Xi

    2013-01-01

    Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  19. MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.

    Directory of Open Access Journals (Sweden)

    Jinli Wang

    Full Text Available Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7 reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3'-untranslated region of Ras homologue enriched in brain (Rheb, a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.

  20. The Rac1 hypervariable region in targeting and signaling

    Science.gov (United States)

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control subcellullar localization. The HVR encodes in many small GTPases a polybasic region (PBR) that permits charge-mediated association to the inner leaflet of the plasma membrane or to intracellular organelles. Over the past 15–20 years, evidence has accumulated for specific protein–protein interactions, mediated by the HVR, that control both targeting and signaling specificity of small GTPases. Using the RhoGTPase Rac1 as a paradigm we here review a series of protein partners that require the Rac1 HVR for association and that control various aspects of localized Rac1 signaling. Some of these proteins represent Rac1 activators, whereas others mediate Rac1 inactivation and degradation and yet others potentiate Rac1 downstream signaling. Finally, evidence is discussed which shows that the HVR of Rac1 also contributes to effector interactions, co-operating with the N-terminal effector domain. The complexity of localized Rac1 signaling, reviewed here, is most likely exemplary for many other small GTPases as well, representing a challenge to identify and define similar mechanisms controlling the specific signaling induced by small GTPases. PMID:23354415

  1. Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

    Directory of Open Access Journals (Sweden)

    Yu-Hsuan Tu

    Full Text Available Lactoferricin B (LfcinB is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP assays. Sixteen proteins were identified, and an E. coli interaction database (EcID analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

  2. Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

    Science.gov (United States)

    Stern, Javier E; Potapenko, Evgeniy S

    2013-08-15

    An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

  3. Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

    Science.gov (United States)

    Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

    2017-08-18

    Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

  4. Cell-autonomous intracellular androgen receptor signaling drives the growth of human prostate cancer initiating cells.

    Science.gov (United States)

    Vander Griend, Donald J; D'Antonio, Jason; Gurel, Bora; Antony, Lizamma; Demarzo, Angelo M; Isaacs, John T

    2010-01-01

    The lethality of prostate cancer is due to the continuous growth of cancer initiating cells (CICs) which are often stimulated by androgen receptor (AR) signaling. However, the underlying molecular mechanism(s) for such AR-mediated growth stimulation are not fully understood. Such mechanisms may involve cancer cell-dependent induction of tumor stromal cells to produce paracrine growth factors or could involve cancer cell autonomous autocrine and/or intracellular AR signaling pathways. We utilized clinical samples, animal models and a series of AR-positive human prostate cancer cell lines to evaluate AR-mediated growth stimulation of prostate CICs. The present studies document that stromal AR expression is not required for prostate cancer growth, since tumor stroma surrounding AR-positive human prostate cancer metastases (N = 127) are characteristically AR-negative. This lack of a requirement for AR expression in tumor stromal cells is also documented by the fact that human AR-positive prostate cancer cells grow equally well when xenografted in wild-type versus AR-null nude mice. AR-dependent growth stimulation was documented to involve secretion, extracellular binding, and signaling by autocrine growth factors. Orthotopic xenograft animal studies documented that the cellautonomous autocrine growth factors which stimulate prostate CIC growth are not the andromedins secreted by normal prostate stromal cells. Such cell autonomous and extracellular autocrine signaling is necessary but not sufficient for the optimal growth of prostate CICs based upon the response to anti-androgen plus/or minus preconditioned media. AR-induced growth stimulation of human prostate CICs requires AR-dependent intracellular pathways. The identification of such AR-dependent intracellular pathways offers new leads for the development of effective therapies for prostate cancer. (c) 2009 Wiley-Liss, Inc.

  5. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Science.gov (United States)

    Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

    2012-01-01

    Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

  6. Wave failure at strong coupling in intracellular C a2 + signaling system with clustered channels

    Science.gov (United States)

    Li, Xiang; Wu, Yuning; Gao, Xuejuan; Cai, Meichun; Shuai, Jianwei

    2018-01-01

    As an important intracellular signal, C a2 + ions control diverse cellular functions. In this paper, we discuss the C a2 + signaling with a two-dimensional model in which the inositol 1,4,5-trisphosphate (I P3 ) receptor channels are distributed in clusters on the endoplasmic reticulum membrane. The wave failure at large C a2 + diffusion coupling is discussed in detail in the model. We show that with varying model parameters the wave failure is a robust behavior with either deterministic or stochastic channel dynamics. We suggest that the wave failure should be a general behavior in inhomogeneous diffusing systems with clustered excitable regions and may occur in biological C a2 + signaling systems.

  7. Targeting Apoptosis Signaling in Pancreatic Cancer

    International Nuclear Information System (INIS)

    Fulda, Simone

    2011-01-01

    The ability to escape apoptosis or programmed cell death is a hallmark of human cancers, for example pancreatic cancer. This can promote tumorigenesis, since too little cell death by apoptosis disturbs tissue homeostasis. Additionally, defective apoptosis signaling is the underlying cause of failure to respond to current treatment approaches, since therapy-mediated antitumor activity requires the intactness of apoptosis signaling pathways in cancer cells. Thus, the elucidation of defects in the regulation of apoptosis in pancreatic carcinoma can result in the identification of novel targets for therapeutic interference and for exploitation for cancer drug discovery

  8. Targeting Apoptosis Signaling in Pancreatic Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Fulda, Simone [Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt (Germany)

    2011-01-11

    The ability to escape apoptosis or programmed cell death is a hallmark of human cancers, for example pancreatic cancer. This can promote tumorigenesis, since too little cell death by apoptosis disturbs tissue homeostasis. Additionally, defective apoptosis signaling is the underlying cause of failure to respond to current treatment approaches, since therapy-mediated antitumor activity requires the intactness of apoptosis signaling pathways in cancer cells. Thus, the elucidation of defects in the regulation of apoptosis in pancreatic carcinoma can result in the identification of novel targets for therapeutic interference and for exploitation for cancer drug discovery.

  9. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    International Nuclear Information System (INIS)

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.

    1987-01-01

    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size

  10. Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

    2017-08-29

    Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

  11. Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

    Science.gov (United States)

    Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

    2017-11-17

    With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

  12. Targeting Apoptosis Signaling Pathways for Anticancer Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Fulda, Simone, E-mail: simone.fulda@kgu.de [Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt (Germany)

    2011-08-29

    Treatment approaches for cancer, for example chemotherapy, radiotherapy, or immunotherapy, primarily act by inducing cell death in cancer cells. Consequently, the inability to trigger cell death pathways or alternatively, evasion of cancer cells to the induction of cell death pathways can result in resistance of cancers to current treatment protocols. Therefore, in order to overcome treatment resistance a better understanding of the underlying mechanisms that regulate cell death and survival pathways in cancers and in response to cancer therapy is necessary to develop molecular-targeted therapies. This strategy should lead to more effective and individualized treatment strategies that selectively target deregulated signaling pathways in a tumor type- and patient-specific manner.

  13. Targeting Apoptosis Signaling Pathways for Anticancer Therapy

    International Nuclear Information System (INIS)

    Fulda, Simone

    2011-01-01

    Treatment approaches for cancer, for example chemotherapy, radiotherapy, or immunotherapy, primarily act by inducing cell death in cancer cells. Consequently, the inability to trigger cell death pathways or alternatively, evasion of cancer cells to the induction of cell death pathways can result in resistance of cancers to current treatment protocols. Therefore, in order to overcome treatment resistance a better understanding of the underlying mechanisms that regulate cell death and survival pathways in cancers and in response to cancer therapy is necessary to develop molecular-targeted therapies. This strategy should lead to more effective and individualized treatment strategies that selectively target deregulated signaling pathways in a tumor type- and patient-specific manner.

  14. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

    Directory of Open Access Journals (Sweden)

    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  15. Intracellular Redox Compartmentation and ROS-Related Communication in Regulation and Signaling.

    Science.gov (United States)

    Noctor, Graham; Foyer, Christine H

    2016-07-01

    Recent years have witnessed enormous progress in understanding redox signaling related to reactive oxygen species (ROS) in plants. The consensus view is that such signaling is intrinsic to many developmental processes and responses to the environment. ROS-related redox signaling is tightly wedded to compartmentation. Because membranes function as barriers, highly redox-active powerhouses such as chloroplasts, peroxisomes, and mitochondria may elicit specific signaling responses. However, transporter functions allow membranes also to act as bridges between compartments, and so regulated capacity to transmit redox changes across membranes influences the outcome of triggers produced at different locations. As well as ROS and other oxidizing species, antioxidants are key players that determine the extent of ROS accumulation at different sites and that may themselves act as signal transmitters. Like ROS, antioxidants can be transported across membranes. In addition, the intracellular distribution of antioxidative enzymes may be modulated to regulate or facilitate redox signaling appropriate to the conditions. Finally, there is substantial plasticity in organellar shape, with extensions such as stromules, peroxules, and matrixules playing potentially crucial roles in organelle-organelle communication. We provide an overview of the advances in subcellular compartmentation, identifying the gaps in our knowledge and discussing future developments in the area. © 2016 American Society of Plant Biologists. All Rights Reserved.

  16. Intracellular calcium signals display an avalanche-like behavior over multiple lengthscales.

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    Lucía eLopez

    2012-09-01

    Full Text Available Many natural phenomena display "self-organized criticality'' (SOC. This refers to spatially extended systems for which patterns of activity characterized by different lengthscales can occur with a probability density that follows a power law with pattern size. Differently from power laws at phase transitions, systems displaying SOC do not need the tuning of an external parameter. Here we analyze intracellular calcium Ca2+ signals, a key component of the signaling toolkit of almost any cell type. Ca2+ signals can either be spatially restricted (local or propagate throughout the cell (global. Different models have suggested that the transition from local to global signals is similar to that of directed percolation. Directed percolation has been associated, in turn, to the appearance of self-organized criticality. In this paper we discuss these issues within the framework of simple models of Ca2+ signal propagation. We also analyze the size distribution of local signals ("puffs'' observed in immature Xenopus Laevis oocytes. The puff amplitude distribution obtained from observed local signals is not Gaussian with a noticeable fraction of large size events. The experimental distribution of puff areas in the spatio-temporal record of the image has a long tail that is approximately log-normal. The distribution can also be fitted with a power law relationship albeit with a smaller goodness of fit. The power law behavior is encountered within a simple model that includes some coupling among individual signals for a wide range of parameter values. An analysis of the model shows that a global elevation of the Ca2+ concentration plays a major role in determining whether the puff size distribution is long-tailed or not. This suggests that Ca2+-clearing from the cytosol is key to determine whether IP3-mediated Ca2+ signals can display a SOC-like behavior or not.

  17. Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

    Science.gov (United States)

    Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

    2017-06-26

    Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

  18. Spatiotemporal intracellular dynamics of neurotrophin and its receptors. Implications for neurotrophin signaling and neuronal function.

    Science.gov (United States)

    Bronfman, F C; Lazo, O M; Flores, C; Escudero, C A

    2014-01-01

    Neurons possess a polarized morphology specialized to contribute to neuronal networks, and this morphology imposes an important challenge for neuronal signaling and communication. The physiology of the network is regulated by neurotrophic factors that are secreted in an activity-dependent manner modulating neuronal connectivity. Neurotrophins are a well-known family of neurotrophic factors that, together with their cognate receptors, the Trks and the p75 neurotrophin receptor, regulate neuronal plasticity and survival and determine the neuronal phenotype in healthy and regenerating neurons. Is it now becoming clear that neurotrophin signaling and vesicular transport are coordinated to modify neuronal function because disturbances of vesicular transport mechanisms lead to disturbed neurotrophin signaling and to diseases of the nervous system. This chapter summarizes our current understanding of how the regulated secretion of neurotrophin, the distribution of neurotrophin receptors in different locations of neurons, and the intracellular transport of neurotrophin-induced signaling in distal processes are achieved to allow coordinated neurotrophin signaling in the cell body and axons.

  19. Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

    Directory of Open Access Journals (Sweden)

    Unzueta U

    2012-08-01

    Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

  20. β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

    Science.gov (United States)

    Galaz-Montoya, Monica; Wright, Sara J; Rodriguez, Gustavo J; Lichtarge, Olivier; Wensel, Theodore G

    2017-06-16

    Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Blockade of intracellular Zn2+ signaling in the basolateral amygdala affects object recognition memory via attenuation of dentate gyrus LTP.

    Science.gov (United States)

    Fujise, Yuki; Kubota, Mitsuyasu; Suzuki, Miki; Tamano, Haruna; Takeda, Atsushi

    2017-09-01

    Hippocampus-dependent memory is modulated by the amygdala. However, it is unknown whether intracellular Zn 2+ signaling in the amygdala is involved in hippocampus-dependent memory. On the basis of the evidence that intracellular Zn 2+ signaling in dentate granule cells (DGC) is necessary for object recognition memory via LTP at medial perforant pathway (PP)-DGC synapses, the present study examined whether intracellular Zn 2+ signaling in the amygdala influences object recognition memory via modulation of LTP at medial PP-DGC synapses. When ZnAF-2DA (100 μM, 2 μl) was injected into the basolateral amygdala (BLA), intracellular ZnAF-2 locally chelated intracellular Zn 2+ in the amygdala. Recognition memory was affected when training of object recognition test was performed 20 min after ZnAF-2DA injection into the BLA. Twenty minutes after injection of ZnAF-2DA into the BLA, LTP induction at medial PP-DGC synapses was attenuated, while LTP induction at PP-BLA synapses was potentiated and LTP induction at BLA-DGC synapses was attenuated. These results suggest that intracellular Zn 2+ signaling in the BLA is involved in BLA-associated LTP and modulates LTP at medial PP-DGC synapses, followed by modulation of object recognition memory. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Liraglutide, leptin and their combined effects on feeding: additive intake reduction through common intracellular signalling mechanisms.

    Science.gov (United States)

    Kanoski, S E; Ong, Z Y; Fortin, S M; Schlessinger, E S; Grill, H J

    2015-03-01

    To investigate the behavioural and intracellular mechanisms by which the glucagon like peptide-1 (GLP-1) receptor agonist, liraglutide, and leptin in combination enhance the food intake inhibitory and weight loss effects of either treatment alone. We examined the effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (s.c.) or to the third ventricle, respectively, on cumulative intake, meal patterns and hypothalamic expression of intracellular signalling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] in lean rats. A low-dose combination of liraglutide (25 µg/kg) and leptin (0.75 µg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75 µg/kg) and leptin (4 µg), examined separately, each reduced meal frequency, cumulative food intake and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot analysis showed elevated pSTAT3 in the hypothalamic tissue after liraglutide-leptin co-treatment, an effect which was greater than that of leptin treatment alone. In addition, s.c. liraglutide reduced the expression of PTP1B (a negative regulator of leptin receptor signalling), revealing a potential mechanism for the enhanced pSTAT3 response after liraglutide-leptin co-administration. Collectively, these results show novel behavioural and molecular mechanisms underlying the additive reduction in food intake and body weight after liraglutide-leptin combination treatment. © 2014 John Wiley & Sons Ltd.

  3. Rosiglitazone Inhibits Adrenocortical Cancer Cell Proliferation by Interfering with the IGF-IR Intracellular Signaling

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    Luconi Michaela

    2008-07-01

    Full Text Available Rosiglitazone (RGZ, a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR-γ, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R of human adrenocortical carcinoma (ACC and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR. We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K-Akt, and extracellular signal-regulated kinase (ERK1/2 cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.

  4. IMPDH2 Is an Intracellular Target of the Cyclophilin A and Sanglifehrin A Complex

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    Khian Hong Pua

    2017-01-01

    Full Text Available Summary: Natural products have demonstrated utility in the clinic and can also act as probes to understand complex cellular pathways. Sanglifehrin A (SFA is a mixed polyketide and non-ribosomal peptide synthase natural product with sub-nano-molar affinity for its receptor cyclophilin A (PPIA. It has been shown to behave in vitro as an immune suppressant. Here, we identify inosine-5′-monophosphate dehydrogenase 2 (IMPDH2 as an intracellular target of the PPIA-SFA binary complex. The formation of this ternary complex does not inhibit the enzymatic activity of IMPDH2. Rather, ternary complex formation modulates cell growth through interaction with the cystathionine-β-synthase (CBS domain of IMPDH2. We further demonstrate that the SFA complex is highly isoform selective for IMPDH2 (versus IMPDH1. This work reveals a role for the CBS domains of IMPDH2 in cellular proliferation, suggesting a more complex role than previously suspected for IMPDH2 in T cell activation and proliferation. : Pua et al. identify IMPDH2 as an intracellular target of the PPIA-SFA complex and show that the CBS domains of IMPDH2 are required for cellular proliferation. Keywords: cyclophilin A, sanglifehrin A, inosine-5′-monophosphate dehydrogenase, cystathionine-β-synthase domains, protein-protein interactions

  5. Targeting embryonic signaling pathways in cancer therapy.

    Science.gov (United States)

    Harris, Pamela Jo; Speranza, Giovanna; Dansky Ullmann, Claudio

    2012-01-01

    The embryonic signaling pathways (ESP), Hedgehog, Notch and Wnt, are critical for the regulation of normal stem cells and cellular development processes. They are also activated in the majority of cancers. ESP are operational in putative cancer stem cells (CSC), which drive initial tumorigenesis and sustain cancer progression and recurrence in non-CSC bulk subpopulations. ESP represent novel therapeutic targets. A variety of inhibitors and targeting strategies are being developed. This review discusses the rationale for targeting ESP for cancer treatment, as well as specific inhibitors under development; mainly focusing on those approaching clinical use and the challenges that lie ahead. The data sources utilized are several database search engines (PubMed, Google, Clinicaltrials.gov), and the authors' involvement in the field. CSC research is rapidly evolving. Expectations regarding their therapeutic targeting are rising quickly. Further definition of what constitutes a true CSC, proper validation of CSC markers, a better understanding of cross-talk among ESP and other pathways, and interactions with tumor non-CSC and the tumor microenvironment are needed. The appropriate patient population, the right clinical setting and combination strategies to test these therapies, as well as the proper pharmacodynamic markers to measure, need to be further established.

  6. Mitogen activated protein kinase signaling in the kidney: Target for intervention?

    NARCIS (Netherlands)

    de Borst, M.H.; Wassef, L.; Kelly, D.J.; van Goor, H.; Navis, Ger Jan

    2006-01-01

    Mitogen activated protein kinases (MAPKs) are intracellular signal transduction molecules, which connect cell-surface receptor signals to intracellular processes. MAPKs regulate a range of cellular activities including cell proliferation, gene expression, apoptosis, cell differentiation and cytokine

  7. The Yeast Retrograde Response as a Model of Intracellular Signaling of Mitochondrial Dysfunction

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    S. Michal eJazwinski

    2012-05-01

    Full Text Available Mitochondrial dysfunction activates intracellular signaling pathways that impact yeast longevity, and the best known of these pathways is the retrograde response. More recently, similar responses have been discerned in other systems, from invertebrates to human cells. However, the identity of the signal transducers is either unknown or apparently diverse, contrasting with the well-established signaling module of the yeast retrograde response. On the other hand, it has become equally clear that several other pathways and processes interact with the retrograde response, embedding it in a network responsive to a variety of cellular states. An examination of this network supports the notion that the master regulator NFkB aggregated a variety of mitochondria-related cellular responses at some point in evolution and has become the retrograde transcription factor. This has significant consequences for how we view some of the deficits associated with aging, such as inflammation. The support for NFkB as the retrograde response transcription factor is not only based on functional analyses. It is bolstered by the fact that NFkB can regulate Myc-Max, which is activated in human cells with dysfunctional mitochondria and impacts cellular metabolism. Myc-Max is homologous to the yeast retrograde response transcription factor Rtg1-Rtg3. Further research will be needed to disentangle the pro-aging from the anti-aging effects of NFkB. Interestingly, this is also a challenge for the complete understanding of the yeast retrograde response.

  8. An odor-specific threshold deficit implicates abnormal intracellular cyclic AMP signaling in schizophrenia.

    Science.gov (United States)

    Turetsky, Bruce I; Moberg, Paul J

    2009-02-01

    Although olfactory deficits are common in schizophrenia, their underlying pathophysiology remains unknown. Recent evidence has suggested that cAMP signaling may be disrupted in schizophrenia. Since cAMP mediates signal transduction in olfactory receptor neurons, this could contribute to the etiology of observed olfactory deficits. This study was designed to test this hypothesis by determining odor detection threshold sensitivities to two odorants that differ in their relative activations of this intracellular cAMP signaling cascade. Thirty schizophrenia patients, 25 healthy comparison subjects, and 19 unaffected first-degree relatives of schizophrenia patients were studied. Odor detection threshold sensitivities were measured for the two odorants citralva and lyral. Although both have fruity/floral scents, citralva strongly activates adenylyl cyclase to increase cAMP levels, while lyral is a very weak activator of adenylyl cyclase. There was a significant group-by-odor interaction. Both schizophrenia patients and unaffected first-degree relatives were impaired in their ability to detect lyral versus citralva. Comparison subjects were equally sensitive to both odorants. This selective deficit could not be explained by differences in age, sex, smoking, clinical symptom profile, or medication use. This study establishes the presence of an odor-specific hyposmia that may denote a disruption of cAMP-mediated signal transduction in schizophrenia. The presence of a parallel deficit in the patients' unaffected first-degree relatives suggests that this deficit is genetically mediated. Although additional physiological studies are needed to confirm the underlying mechanism, these results offer strong inferential support for the hypothesis that cAMP signaling is dysregulated in schizophrenia.

  9. Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

    Directory of Open Access Journals (Sweden)

    Chao Xie

    Full Text Available Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin and in HEK293 cells (hr-irisin for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work

  10. Irisin Controls Growth, Intracellular Ca2+ Signals, and Mitochondrial Thermogenesis in Cardiomyoblasts.

    Science.gov (United States)

    Xie, Chao; Zhang, Yuan; Tran, Tran D N; Wang, Hai; Li, Shiwu; George, Eva Vertes; Zhuang, Haoyang; Zhang, Peilan; Kandel, Avi; Lai, Yimu; Tang, Dongqi; Reeves, Westley H; Cheng, Henrique; Ding, Yousong; Yang, Li-Jun

    2015-01-01

    Exercise offers short-term and long-term health benefits, including an increased metabolic rate and energy expenditure in myocardium. The newly-discovered exercise-induced myokine, irisin, stimulates conversion of white into brown adipocytes as well as increased mitochondrial biogenesis and energy expenditure. Remarkably, irisin is highly expressed in myocardium, but its physiological effects in the heart are unknown. The objective of this work is to investigate irisin's potential multifaceted effects on cardiomyoblasts and myocardium. For this purpose, H9C2 cells were treated with recombinant irisin produced in yeast cells (r-irisin) and in HEK293 cells (hr-irisin) for examining its effects on cell proliferation by MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and on gene transcription profiles by qRT-PCR. R-irisin and hr-irisin both inhibited cell proliferation and activated genes related to cardiomyocyte metabolic function and differentiation, including myocardin, follistatin, smooth muscle actin, and nuclear respiratory factor-1. Signal transduction pathways affected by r-irisin in H9C2 cells and C57BL/6 mice were examined by detecting phosphorylation of PI3K-AKT, p38, ERK or STAT3. We also measured intracellular Ca2+ signaling and mitochondrial thermogenesis and energy expenditure in r-irisin-treated H9C2 cells. The results showed that r-irisin, in a certain concentration rage, could activate PI3K-AKT and intracellular Ca2+ signaling and increase cellular oxygen consumption in H9C2 cells. Our study also suggests the existence of irisin-specific receptor on the membrane of H9C2 cells. In conclusion, irisin in a certain concentration rage increased myocardial cell metabolism, inhibited cell proliferation and promoted cell differentiation. These effects might be mediated through PI3K-AKT and Ca2+ signaling, which are known to activate expression of exercise-related genes such as follistatin and myocardin. This work supports the value

  11. The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

    Science.gov (United States)

    Wei, Jian; Zhang, Ying-Yu; Luo, Jie; Wang, Ju-Qiong; Zhou, Yu-Xia; Miao, Hong-Hua; Shi, Xiong-Jie; Qu, Yu-Xiu; Xu, Jie; Li, Bo-Liang; Song, Bao-Liang

    2017-06-27

    Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Essential role of flotillin-1 palmitoylation in the intracellular localization and signaling function of IGF-1 receptor.

    Science.gov (United States)

    Jang, Donghwan; Kwon, Hayeong; Jeong, Kyuho; Lee, Jaewoong; Pak, Yunbae

    2015-06-01

    Here, we explored flotillin-1-mediated regulation of insulin-like growth factor-1 (IGF-1) signaling. Flotillin-1-deficient cells exhibited a reduction in the activation of IGF-1 receptor (IGF-1R), ERK1/2 and Akt pathways, and the transcriptional activation of Elk-1 and the proliferation in response to IGF-1 were reduced in these cells. We found that IGF-1-independent flotillin-1 palmitoylation at Cys34 in the endoplasmic reticulum (ER) was required for the ER exit and the plasma membrane localization of flotillin-1 and IGF-1R. IGF-1-dependent depalmitoylation and repalmitoylation of flotillin-1 sustained tyrosine kinase activation of the plasma-membrane-targeted IGF-1R. Dysfunction and blocking the turnover of flotillin-1 palmitoylation abrogated cancer cell proliferation after IGF-1R signaling activation. Our data show that flotillin-1 palmitoylation is a new mechanism by which the intracellular localization and activation of IGF-1R are controlled. © 2015. Published by The Company of Biologists Ltd.

  13. A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.

    Science.gov (United States)

    Lin, Hong-Yu; Haegele, Joseph A; Disare, Michael T; Lin, Qishan; Aye, Yimon

    2015-05-20

    Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

  14. Blockade of intracellular Zn2+ signaling in the dentate gyrus erases recognition memory via impairment of maintained LTP.

    Science.gov (United States)

    Tamano, Haruna; Minamino, Tatsuya; Fujii, Hiroaki; Takada, Shunsuke; Nakamura, Masatoshi; Ando, Masaki; Takeda, Atsushi

    2015-08-01

    There is no evidence on the precise role of synaptic Zn2+ signaling on the retention and recall of recognition memory. On the basis of the findings that intracellular Zn2+ signaling in the dentate gyrus is required for object recognition, short-term memory, the present study deals with the effect of spatiotemporally blocking Zn2+ signaling in the dentate gyrus after LTP induction and learning. Three-day-maintained LTP was impaired 1 day after injection of clioquinol into the dentate gyrus, which transiently reduced intracellular Zn2+ signaling in the dentate gyrus. The irreversible impairment was rescued not only by co-injection of ZnCl2 , which ameliorated the loss of Zn2+ signaling, but also by pre-injection of Jasplakinolide, a stabilizer of F-actin, prior to clioquinol injection. Simultaneously, 3-day-old space recognition memory was impaired 1 day after injection of clioquinol into the dentate gyrus, but not by pre-injection of Jasplakinolide. Jasplakinolide also rescued both impairments of 3-day-maintained LTP and 3-day-old memory after injection of ZnAF-2DA into the dentate gyrus, which blocked intracellular Zn2+ signaling in the dentate gyrus. The present paper indicates that the blockade and/or loss of intracellular Zn2+ signaling in the dentate gyrus coincidently impair maintained LTP and recognition memory. The mechanism maintaining LTP via intracellular Zn2+ signaling in dentate granule cells, which may be involved in the formation of F-actin, may retain space recognition memory. © 2015 Wiley Periodicals, Inc.

  15. The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

    Science.gov (United States)

    Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

    2018-05-15

    Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies

  16. Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Ichijo, Yuta; Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Satou, Kazuhiro; Negishi, Jun [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Itabashi-Ku, Tokyo 173-8605 (Japan); Oshima, Natsuki [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Mogi, Chihiro; Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2016-01-01

    Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the G{sub s}-protein/cAMP/CRE, G{sub 12/13}-protein/Rho/SRE, and G{sub q}-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174{sup th} position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. - Highlights: • Zebrafish two G2A homologs are proton

  17. Anabolic Androgenic Steroids and Intracellular Calcium Signaling: A Mini Review on Mechanisms and Physiological Implications

    Science.gov (United States)

    Vicencio, J.M.; Estrada, M.; Galvis, D.; Bravo, R.; Contreras, A.E.; Rotter, D.; Szabadkai, G.; Hill, J.A.; Rothermel, B.A.; Jaimovich, E.; Lavandero, S.

    2015-01-01

    Increasing evidence suggests that nongenomic effects of testosterone and anabolic androgenic steroids (AAS) operate concertedly with genomic effects. Classically, these responses have been viewed as separate and independent processes, primarily because nongenomic responses are faster and appear to be mediated by membrane androgen receptors, whereas long-term genomic effects are mediated through cytosolic androgen receptors regulating transcriptional activity. Numerous studies have demonstrated increases in intracellular Ca2+ in response to AAS. These Ca2+ mediated responses have been seen in a diversity of cell types, including osteoblasts, platelets, skeletal muscle cells, cardiac myocytes and neurons. The versatility of Ca2+ as a second messenger provides these responses with a vast number of pathophysiological implications. In cardiac cells, testosterone elicits voltage-dependent Ca2+ oscillations and IP3R-mediated Ca2+ release from internal stores, leading to activation of MAPK and mTOR signaling that promotes cardiac hypertrophy. In neurons, depending upon concentration, testosterone can provoke either physiological Ca2+ oscillations, essential for synaptic plasticity, or sustained, pathological Ca2+ transients that lead to neuronal apoptosis. We propose therefore, that Ca2+ acts as an important point of crosstalk between nongenomic and genomic AAS signaling, representing a central regulator that bridges these previously thought to be divergent responses. PMID:21443511

  18. Role of α7 nicotinic receptor in the immune system and intracellular signaling pathways.

    Science.gov (United States)

    Zdanowski, Robert; Krzyżowska, Małgorzata; Ujazdowska, Dominika; Lewicka, Aneta; Lewicki, Sławomir

    2015-01-01

    Acetylcholine has been well known as one of the most exemplary neurotransmitters. In humans, this versatile molecule and its synthesizing enzyme, choline acetyltransferase, have been found in various non-neural tissues such as the epithelium, endothelium, mesothelium muscle, blood cells and immune cells. The non-neuronal acetylcholine is accompanied by the expression of acetylcholinesterase and nicotinic/muscarinic acetylcholine receptors. Increasing evidence of the non-neuronal acetylcholine system found throughout the last few years has indicated this neurotransmitter as one of the major cellular signaling molecules (associated e.g. with kinases and transcription factors activity). This system is responsible for maintenance and optimization of the cellular function, such as proliferation, differentiation, adhesion, migration, intercellular contact and apoptosis. Additionally, it controls proper activity of immune cells and affects differentiation, antigen presentation or cytokine production (both pro- and anti-inflammatory). The present article reviews recent findings about the non-neuronal cholinergic system in the field of immune system and intracellular signaling pathways.

  19. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

    Science.gov (United States)

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-07-17

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

  20. Intracellular signaling entropy can be a biomarker for predicting the development of cervical intraepithelial neoplasia.

    Directory of Open Access Journals (Sweden)

    Masakazu Sato

    Full Text Available While the mortality rates for cervical cancer have been drastically reduced after the introduction of the Pap smear test, it still is one of the leading causes of death in women worldwide. Additionally, studies that appropriately evaluate the risk of developing cervical lesions are needed. Therefore, we investigated whether intracellular signaling entropy, which is measured with microarray data, could be useful for predicting the risks of developing cervical lesions. We used three datasets, GSE63514 (histology, GSE27678 (cytology and GSE75132 (cytology, a prospective study. From the data in GSE63514, the entropy rate was significantly increased with disease progression (normal < cervical intraepithelial neoplasia, CIN < cancer (Kruskal-Wallis test, p < 0.0001. From the data in GSE27678, similar results (normal < low-grade squamous intraepithelial lesions, LSILs < high-grade squamous intraepithelial lesions, HSILs ≤ cancer were obtained (Kruskal-Wallis test, p < 0.001. From the data in GSE75132, the entropy rate tended to be higher in the HPV-persistent groups than the HPV-negative group. The group that was destined to progress to CIN 3 or higher had a tendency to have a higher entropy rate than the HPV16-positive without progression group. In conclusion, signaling entropy was suggested to be different for different lesion statuses and could be a useful biomarker for predicting the development of cervical intraepithelial neoplasia.

  1. Genus-specific PCR Primers Targeting Intracellular Parasite Euduboscquella (Dinoflagellata: Syndinea)

    Science.gov (United States)

    Jung, Jae-Ho; Choi, Jung Min; Kim, Young-Ok

    2018-03-01

    We designed a genus-specific primer pair targeting the intracellular parasite Euduboscquella. To increase target specificity and inhibit untargeted PCR, two nucleotides were added at the 3' end of the reverse primer, one being a complementary nucleotide to the Euduboscquella-specific SNP (single-nucleotide polymorphism) and the other a deliberately mismatched nucleotide. Target specificity of the primer set was verified experimentally using PCR of two Euduboscquella species (positive controls) and 15 related species (negative controls composed of ciliates, diatoms and dinoflagellates), and analytical comparison with SILVA SSU rRNA gene database (release 119) in silico. In addition, we applied the Euduboscquella-specific primer set to four environmental samples previously determined by cytological staining to be either positive or negative for Euduboscquella. As expected, only positive controls and environmental samples known to contain Euduboscquella were successfully amplified by the primer set. An inferred SSU rRNA gene phylogeny placed environmental samples containing aloricate ciliates infected by Euduboscquella in a cluster discrete from Euduboscquella groups a-d previously reported from loricate, tintinnid ciliates.

  2. Involvement of intracellular Zn2+ signaling in LTP at perforant pathway-CA1 pyramidal cell synapse.

    Science.gov (United States)

    Tamano, Haruna; Nishio, Ryusuke; Takeda, Atsushi

    2017-07-01

    Physiological significance of synaptic Zn 2+ signaling was examined at perforant pathway-CA1 pyramidal cell synapses. In vivo long-term potentiation (LTP) at perforant pathway-CA1 pyramidal cell synapses was induced using a recording electrode attached to a microdialysis probe and the recording region was locally perfused with artificial cerebrospinal fluid (ACSF) via the microdialysis probe. Perforant pathway LTP was not attenuated under perfusion with CaEDTA (10 mM), an extracellular Zn 2+ chelator, but attenuated under perfusion with ZnAF-2DA (50 μM), an intracellular Zn 2+ chelator, suggesting that intracellular Zn 2+ signaling is required for perforant pathway LTP. Even in rat brain slices bathed in CaEDTA in ACSF, intracellular Zn 2+ level, which was measured with intracellular ZnAF-2, was increased in the stratum lacunosum-moleculare where perforant pathway-CA1 pyramidal cell synapses were contained after tetanic stimulation. These results suggest that intracellular Zn 2+ signaling, which originates in internal stores/proteins, is involved in LTP at perforant pathway-CA1 pyramidal cell synapses. Because the influx of extracellular Zn 2+ , which originates in presynaptic Zn 2+ release, is involved in LTP at Schaffer collateral-CA1 pyramidal cell synapses, synapse-dependent Zn 2+ dynamics may be involved in plasticity of postsynaptic CA1 pyramidal cells. © 2017 Wiley Periodicals, Inc.

  3. Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Aseem Pandey

    2018-04-01

    Full Text Available Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR sensor IRE1α (inositol-requiring enzyme 1 and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1 conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

  4. Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles

    DEFF Research Database (Denmark)

    Ragelle, Héloïse; Colombo, Stefano; Pourcelle, Vincent

    2015-01-01

    chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse...... that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards...

  5. Folate mediated self-assembled phytosterol-alginate nanoparticles for targeted intracellular anticancer drug delivery.

    Science.gov (United States)

    Wang, Jianting; Wang, Ming; Zheng, Mingming; Guo, Qiong; Wang, Yafan; Wang, Heqing; Xie, Xiangrong; Huang, Fenghong; Gong, Renmin

    2015-05-01

    Self-assembled core/shell nanoparticles (NPs) were synthesized from water-soluble alginate substituted by hydrophobic phytosterols. Folate, a cancer-cell-specific ligand, was conjugated to the phytosterol-alginate (PA) NPs for targeting folate-receptor-overexpressing cancer cells. The physicochemical properties of folate-phytosterol-alginate (FPA) NPs were characterized by nuclear magnetic resonance, transmission electron microscopy, dynamic light scattering, electrophoretic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug, was entrapped inside prepared NPs by dialysis method. The identification of prepared FPA NPs to folate-receptor-overexpressing cancer cells (KB cells) was confirmed by cytotoxicity and folate competition assays. Compared to the pure DOX and DOX/PA NPs, the DOX/FPA NPs had lower IC50 value to KB cells because of folate-receptor-mediated endocytosis process and the cytotoxicity of DOX/FPA NPs to KB cells could be competitively inhibited by free folate. The cellular uptake and internalization of pure DOX and DOX/FPA NPs was confirmed by confocal laser scanning microscopy image and the higher intracellular uptake of drug for DOX/FPA NPs over pure DOX was observed. The FPA NPs had the potential as a promising carrier to target drugs to cancer cells overexpressing folate receptors and avoid cytotoxicity to normal tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Targeting NRF2 signaling for cancer chemoprevention

    International Nuclear Information System (INIS)

    Kwak, Mi-Kyoung; Kensler, Thomas W.

    2010-01-01

    Modulation of the metabolism and disposition of carcinogens through induction of cytoprotective enzymes is one of several promising strategies to prevent cancer. Chemopreventive efficacies of inducers such as dithiolethiones and sulforaphane have been extensively studied in animals as well as in humans. The KEAP1-NRF2 system is a key, but not unilateral, molecular target for these chemopreventive agents. The transcription factor NRF2 (NF-E2-related factor 2) is a master regulator of the expression of a subset of genes, which produce proteins responsible for the detoxication of electrophiles and reactive oxygen species as well as the removal or repair of some of their damage products. It is believed that chemopreventive enzyme inducers affect the interaction between KEAP1 and NRF2 through either mediating conformational changes of the KEAP1 protein or activating phosphorylation cascades targeting the KEAP1-NRF2 complex. These events in turn affect NRF2 stability and trafficking. Recent advances elucidating the underlying structural biology of KEAP1-NRF2 signaling and identification of the gene clusters under the transcriptional control of NRF2 are facilitating understanding of the potential pleiotropic effects of NRF2 activators and discovery of novel classes of potent chemopreventive agents such as the triterpenoids. Although there is appropriately a concern regarding a deleterious role of the KEAP1-NRF2 system in cancer cell biology, especially as the pathway affects cell survival and drug resistance, the development and the use of NRF2 activators as chemopreventive agents still holds a great promise for protection of normal cells from a diversity of environmental stresses that contribute to the burden of cancer and other chronic, degenerative diseases.

  7. Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function.

    Science.gov (United States)

    Clemente, M G; De Virgiliis, S; Kang, J S; Macatagney, R; Musu, M P; Di Pierro, M R; Drago, S; Congia, M; Fasano, A

    2003-02-01

    Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

  8. Methoxychlor and Vinclozolin Induce Rapid Changes in Intercellular and Intracellular Signaling in Liver Progenitor Cells.

    Science.gov (United States)

    Babica, Pavel; Zurabian, Rimma; Kumar, Esha R; Chopra, Rajus; Mianecki, Maxwell J; Park, Joon-Suk; Jaša, Libor; Trosko, James E; Upham, Brad L

    2016-09-01

    Methoxychlor (MXC) and vinclozolin (VIN) are well-recognized endocrine disrupting chemicals known to alter epigenetic regulations and transgenerational inheritance; however, non-endocrine disruption endpoints are also important. Thus, we determined the effects of MXC and VIN on the dysregulation of gap junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells. Both chemicals induced a rapid dysregulation of GJIC at non-cytotoxic doses, with 30 min EC50 values for GJIC inhibition being 10 µM for MXC and 126 µM for VIN. MXC inhibited GJIC for at least 24 h, while VIN effects were transient and GJIC recovered after 4 h. VIN induced rapid hyperphosphorylation and internalization of gap junction protein connexin43, and both chemicals also activated MAPK ERK1/2 and p38. Effects on GJIC were not prevented by MEK1/2 inhibitor, but by an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), resveratrol, and in the case of VIN, also, by a p38 inhibitor. Estrogen (ER) and androgen receptor (AR) modulators (estradiol, ICI 182,780, HPTE, testosterone, flutamide, VIN M2) did not attenuate MXC or VIN effects on GJIC. Our data also indicate that the effects were elicited by the parental compounds of MXC and VIN. Our study provides new evidence that MXC and VIN dysregulate GJIC via mechanisms involving rapid activation of PC-PLC occurring independently of ER- or AR-dependent genomic signaling. Such alterations of rapid intercellular and intracellular signaling events involved in regulations of gene expression, tissue development, function and homeostasis, could also contribute to transgenerational epigenetic effects of endocrine disruptors. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Targeting AMPK Signaling as a Neuroprotective Strategy in Parkinson's Disease.

    Science.gov (United States)

    Curry, Daniel W; Stutz, Bernardo; Andrews, Zane B; Elsworth, John D

    2018-03-26

    Parkinson's disease (PD) is the second most common neurodegenerative disorder. It is characterized by the accumulation of intracellular α-synuclein aggregates and the degeneration of nigrostriatal dopaminergic neurons. While no treatment strategy has been proven to slow or halt the progression of the disease, there is mounting evidence from preclinical PD models that activation of 5'-AMP-activated protein kinase (AMPK) may have broad neuroprotective effects. Numerous dietary supplements and pharmaceuticals (e.g., metformin) that increase AMPK activity are available for use in humans, but clinical studies of their effects in PD patients are limited. AMPK is an evolutionarily conserved serine/threonine kinase that is activated by falling energy levels and functions to restore cellular energy balance. However, in response to certain cellular stressors, AMPK activation may exacerbate neuronal atrophy and cell death. This review describes the regulation and functions of AMPK, evaluates the controversies in the field, and assesses the potential of targeting AMPK signaling as a neuroprotective treatment for PD.

  10. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

    International Nuclear Information System (INIS)

    Jung, Kyung oh; Youn, Hyewon; Kim, Seung Hoo; Kim, Young-Hwa; Kang, Keon Wook; Chung, June-Key

    2016-01-01

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and "6"4Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

  11. A new fluorescence/PET probe for targeting intracellular human telomerase reverse transcriptase (hTERT) using Tat peptide-conjugated IgM

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Kyung oh [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Youn, Hyewon, E-mail: hwyoun@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of); Cancer Imaging Center, Seoul National University Hospital, Seoul (Korea, Republic of); Kim, Seung Hoo [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kim, Young-Hwa [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Kang, Keon Wook [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Chung, June-Key, E-mail: jkchung@snu.ac.kr [Department of Nuclear Medicine, Seoul National University College of Medicine (Korea, Republic of); Biomedical Sciences, Seoul National University College of Medicine (Korea, Republic of); Cancer Research Institute, Seoul National University College of Medicine (Korea, Republic of); Tumor Microenvironment Global Core Research Center, Seoul National University (Korea, Republic of)

    2016-08-26

    Despite an increasing need for methods to visualize intracellular proteins in vivo, the majority of antibody-based imaging methods available can only detect membrane proteins. The human telomerase reverse transcriptase (hTERT) is an intracellular target of great interest because of its high expression in several types of cancer. In this study, we developed a new probe for hTERT using the Tat peptide. An hTERT antibody (IgG or IgM) was conjugated with the Tat peptide, a fluorescence dye and {sup 64}Cu. HT29 (hTERT+) and U2OS (hTERT−) were used to visualize the intracellular hTERT. The hTERT was detected by RT-PCR and western blot. Fluorescence signals for hTERT were obtained by confocal microscopy, live cell imaging, and analyzed by Tissue-FAXS. In nude mice, tumors were visualized using the fluorescence imaging devices Maestro™ and PETBOX. In RT-PCR and western blot, the expression of hTERT was detected in HT29 cells, but not in U2OS cells. Fluorescence signals were clearly observed in HT29 cells and in U2OS cells after 1 h of treatment, but signals were only detected in HT29 cells after 24 h. Confocal microscopy showed that 9.65% of U2OS and 78.54% of HT29 cells had positive hTERT signals. 3D animation images showed that the probe could target intranuclear hTERT in the nucleus. In mice models, fluorescence and PET imaging showed that hTERT in HT29 tumors could be efficiently visualized. In summary, we developed a new method to visualize intracellular and intranuclear proteins both in vitro and in vivo. - Highlights: • We developed new probes for imaging hTERT using Tat-conjugated IgM antibodies labeled with a fluorescent dye and radioisotope. • This probes could be used to overcome limitation of conventional antibody imaging system in live cell imaging. • This system could be applicable to monitor intracellular and intranuclear proteins in vitro and in vivo.

  12. Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

    Science.gov (United States)

    Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Schwartz, Simó; Villaverde, Antonio; Vazquez, Esther

    2014-10-01

    CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. [Cell signaling pathways interaction in cellular proliferation: Potential target for therapeutic interventionism].

    Science.gov (United States)

    Valdespino-Gómez, Víctor Manuel; Valdespino-Castillo, Patricia Margarita; Valdespino-Castillo, Víctor Edmundo

    2015-01-01

    Nowadays, cellular physiology is best understood by analysing their interacting molecular components. Proteins are the major components of the cells. Different proteins are organised in the form of functional clusters, pathways or networks. These molecules are ordered in clusters of receptor molecules of extracellular signals, transducers, sensors and biological response effectors. The identification of these intracellular signaling pathways in different cellular types has required a long journey of experimental work. More than 300 intracellular signaling pathways have been identified in human cells. They participate in cell homeostasis processes for structural and functional maintenance. Some of them participate simultaneously or in a nearly-consecutive progression to generate a cellular phenotypic change. In this review, an analysis is performed on the main intracellular signaling pathways that take part in the cellular proliferation process, and the potential use of some components of these pathways as target for therapeutic interventionism are also underlined. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

  14. Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.

    Science.gov (United States)

    Lang, Bethan; Makuch, Mateusz; Moloney, Teresa; Dettmann, Inga; Mindorf, Swantje; Probst, Christian; Stoecker, Winfried; Buckley, Camilla; Newton, Charles R; Leite, M Isabel; Maddison, Paul; Komorowski, Lars; Adcock, Jane; Vincent, Angela; Waters, Patrick; Irani, Sarosh R

    2017-04-01

    Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined. Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin ( 125 I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of 125 I-αDTX and 125 I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2. VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated 125 I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response. Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against

  15. Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

    Science.gov (United States)

    Eid, Islam; Elsebaei, Mohamed M; Mohammad, Haroon; Hagras, Mohamed; Peters, Christine E; Hegazy, Youssef A; Cooper, Bruce; Pogliano, Joe; Pogliano, Kit; Abulkhair, Hamada S; Seleem, Mohamed N; Mayhoub, Abdelrahman S

    2017-10-20

    The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

    Science.gov (United States)

    Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

    2018-03-01

    The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

  17. Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

    Science.gov (United States)

    Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

    2016-01-01

    The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

  18. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Laura Picas

    2016-03-01

    Full Text Available Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction.

  19. Targeting Signaling Pathways in Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Johannes Haybaeck

    2013-05-01

    Full Text Available Ovarian carcinoma (OC is the most lethal gynecological malignancy. Response to platinum-based chemotherapy is poor in some patients and, thus, current research is focusing on new therapy options. The various histological types of OC are characterized by distinctive molecular genetic alterations that are relevant for ovarian tumorigenesis. The understanding of these molecular pathways is essential for the development of novel therapeutic strategies. Purpose: We want to give an overview on the molecular genetic changes of the histopathological types of OC and their role as putative therapeutic targets. In Depth Review of Existing Data: In 2012, the vascular endothelial growth factor (VEGF inhibitor, bevacizumab, was approved for OC treatment. Bevacizumab has shown promising results as single agent and in combination with conventional chemotherapy, but its target is not distinctive when analyzed before treatment. At present, mammalian target of rapamycin (mTOR inhibitors, poly-ADP-ribose polymerase (PARP inhibitors and components of the EGFR pathway are in the focus of clinical research. Interestingly, some phytochemical substances show good synergistic effects when used in combination with chemotherapy. Conclusion: Ongoing studies of targeted agents in conjunction with chemotherapy will show whether there are alternative options to bevacizumab available for OC patients. Novel targets which can be assessed before therapy to predict efficacy are needed. The assessment of therapeutic targets is continuously improved by molecular pathological analyses on tumor tissue. A careful selection of patients for personalized treatment will help to reduce putative side effects and toxicity.

  20. Targeting and localized signalling by small GTPases

    NARCIS (Netherlands)

    ten Klooster, Jean Paul; Hordijk, Peter L.

    2007-01-01

    Polarized cellular responses, for example, cell migration, require the co-ordinated assembly of signalling complexes at a particular subcellular location, such as the leading edge of cells. Small GTPases of the Ras superfamily play central roles in many (polarized) responses to growth factors,

  1. Intracellular pH is a tightly controlled signal in yeast

    NARCIS (Netherlands)

    Orij, R.; Brul, S.; Smits, G.J.

    2011-01-01

    Background: Nearly all processes in living cells are pH dependent, which is why intracellular pH (pHi) is a tightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pHi responds to extracellular conditions such as the availability of nutrients. This

  2. A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts.

    Science.gov (United States)

    Galperin, Michael Y

    2005-06-14

    Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. This paper presents results of a comprehensive census of signal transduction proteins--histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases--encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes

  3. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the

  4. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Karen K Y Lam

    Full Text Available Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  5. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis.

    Science.gov (United States)

    Lam, Karen K Y; Zheng, Xingji; Forestieri, Roberto; Balgi, Aruna D; Nodwell, Matt; Vollett, Sarah; Anderson, Hilary J; Andersen, Raymond J; Av-Gay, Yossef; Roberge, Michel

    2012-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment.

  6. Synthesis and Characterization of a Gd-DOTA-D-Permeation Peptide for Magnetic Resonance Relaxation Enhancement of Intracellular Targets

    Directory of Open Access Journals (Sweden)

    Andrew M. Prantner

    2003-10-01

    Full Text Available Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 ± 0.11 mM−1 sec−1 at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.

  7. The impact of the amount of intracellular SPIO on MR signal intensity during in vivo tracking of macrophage homing

    International Nuclear Information System (INIS)

    Kim, Dae Yoon; Lee, Jin Seong; Kang, Ju Hee; Sohn, Jin Young; Kim, Sang Tae; Woo, Chul Woong

    2008-01-01

    To determine whether the amount of intracellular superparamagnetic iron oxide (SPIO) in macrophages influences MR signal intensity during in vivo celluar tracking. Peritoneal macrophages harvested from thioglycolate-treated mice were labeled with SPIO using concentrations of 112, 56, and 28 μ gFe/ml, and different incubation times of 3h, 6h, 12h, 24h and 48h, respectively. The iron concentration was quantified with the use of absorption spectrophotometry. Each group of macrophages labeled with different concentrations of SPIO was intravenously injected into 18 mice, after inoculation with S. aureus to the thigh. The relative signal intensity (SI) of the abscess wall (SI of the abscess wall/SI of muscle) was measured on MR and was analyzed by the use of the Kruskal-Wallis test. A higher concentration of SPIO in the labeling solution and a longer incubation time resulted in a higher concentration of SPIO in the macrophages. The relative SI of the abscess wall (0.63 for 112 μ gFe/mL; 0.67 for 56 μ gFe/ml; 0.89 for 28 μ gFe/mL) significantly decreased with an increase of SPIO concentration (κ 2 = 10.53, ρ < 0.005). The amount of intracellular SPIO influences the MR signal intensity by the susceptibility effect and it is recommended to use sufficient iron-oxide label as long as it dose not affect cellular function and viability

  8. Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells.

    Science.gov (United States)

    Rimpelä, Anna-Kaisa; Hagström, Marja; Kidron, Heidi; Urtti, Arto

    2018-05-31

    Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016-0.73 and in non-pigmented cells 0.017-1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, F ic , were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3-4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity. Copyright © 2017. Published by Elsevier B.V.

  9. Dextran sulfate sodium upregulates MAPK signaling for the uptake and subsequent intracellular survival of Brucella abortus in murine macrophages.

    Science.gov (United States)

    Reyes, Alisha Wehdnesday Bernardo; Arayan, Lauren Togonon; Simborio, Hannah Leah Tadeja; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2016-02-01

    Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

    Science.gov (United States)

    Floss, D M; Schönberg, M; Franke, M; Horstmeier, F C; Engelowski, E; Schneider, A; Rosenfeldt, E M; Scheller, J

    2017-11-09

    IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

  11. In Vivo Characterization of Intracellular Signaling Pathways Activated by the Nerve Agent Sarin

    National Research Council Canada - National Science Library

    Shih, Tsung-Ming A; Snyder, Gretchen L; Hendrick, Joseph P; Fienberg, Allen A; McDonough, John H

    2004-01-01

    ..., an excessive stimulation of nicotinic and muscarinic receptors. Preliminary evidence using diverse OPs indicates that the DARPP-32/PP-1 signaling pathway is activated by nicotinic receptor stimulation...

  12. A T4SS Effector Targets Host Cell Alpha-Enolase Contributing to Brucella abortus Intracellular Lifestyle.

    Science.gov (United States)

    Marchesini, María I; Morrone Seijo, Susana M; Guaimas, Francisco F; Comerci, Diego J

    2016-01-01

    Brucella abortus , the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus , ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus .

  13. Delineating neurotrophin-3 dependent signaling pathways underlying sympathetic axon growth along intermediate targets.

    Science.gov (United States)

    Keeler, Austin B; Suo, Dong; Park, Juyeon; Deppmann, Christopher D

    2017-07-01

    Postganglionic sympathetic neurons detect vascular derived neurotrophin 3 (NT3) via the axonally expressed receptor tyrosine kinase, TrkA, to promote chemo-attraction along intermediate targets. Once axons arrive to their final target, a structurally related neurotrophic factor, nerve growth factor (NGF), also acts through TrkA to promote final target innervation. Does TrkA signal differently at these different locales? We previously found that Coronin-1 is upregulated in sympathetic neurons upon exposure to NGF, thereby endowing the NGF-TrkA complex with new signaling capabilities (i.e. calcium signaling), which dampens axon growth and branching. Based on the notion that axons do not express functional levels of Coronin-1 prior to final target innervation, we developed an in vitro model for axon growth and branching along intermediate targets using Coro1a -/- neurons grown in NT3. We found that, similar to NGF-TrkA, NT3-TrkA is capable of inducing MAPK and PI3K in the presence or absence of Coronin-1. However, unlike NGF, NT3 does not induce calcium release from intracellular stores. Using a combination of pharmacology, knockout neurons and in vitro functional assays, we suggest that the NT3-TrkA complex uses Ras/MAPK and/or PI3K-AKT signaling to induce axon growth and inhibit axon branching along intermediate targets. However, in the presence of Coronin-1, these signaling pathways lose their ability to impact NT3 dependent axon growth or branching. This is consistent with a role for Coronin-1 as a molecular switch for axon behavior and suggests that Coronin-1 suppresses NT3 dependent axon behavior. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. pH-Switch Nanoprecipitation of Polymeric Nanoparticles for Multimodal Cancer Targeting and Intracellular Triggered Delivery of Doxorubicin.

    Science.gov (United States)

    Herranz-Blanco, Bárbara; Shahbazi, Mohammad-Ali; Correia, Alexandra R; Balasubramanian, Vimalkumar; Kohout, Tomáš; Hirvonen, Jouni; Santos, Hélder A

    2016-08-01

    Theranostic nanoparticles are emerging as potent tools for noninvasive diagnosis, treatment, and monitoring of solid tumors. Herein, an advanced targeted and multistimuli responsive theranostic platform is presented for the intracellular triggered delivery of doxorubicin. The system consists of a polymeric-drug conjugate solid nanoparticle containing encapsulated superparamagnetic iron oxide nanoparticles (IO@PNP) and decorated with a tumor homing peptide, iRGD. The production of this nanosystem is based on a pH-switch nanoprecipitation method in organic-free solvents, making it ideal for biomedical applications. The nanosystem shows sufficient magnetization saturation for magnetically guided therapy along with reduced cytotoxicity and hemolytic effects. IO@PNP are largely internalized by endothelial and metastatic cancer cells and iRGD decorated IO@PNP moderately enhance their internalization into endothelial cells, while no enhancement is found for the metastatic cancer cells. Poly(ethylene glycol)-block-poly(histidine) with pH-responsive and proton-sponge properties promotes prompt lysosomal escape once the nanoparticles are endocyted. In addition, the polymer-doxorubicin conjugate solid nanoparticles show both intracellular lysosomal escape and efficient translocation of doxorubicin to the nuclei of the cells via cleavage of the amide bond. Overall, IO@PNP-doxorubicin and the iRGD decorated counterpart demonstrate to enhance the toxicity of doxorubicin in cancer cells by improving the intracellular delivery of the drug carried in the IO@PNP. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    Science.gov (United States)

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-05

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  16. Use of multiple singular value decompositions to analyze complex intracellular calcium ion signals

    KAUST Repository

    Martinez, Josue G.

    2009-12-01

    We compare calcium ion signaling (Ca(2+)) between two exposures; the data are present as movies, or, more prosaically, time series of images. This paper describes novel uses of singular value decompositions (SVD) and weighted versions of them (WSVD) to extract the signals from such movies, in a way that is semi-automatic and tuned closely to the actual data and their many complexities. These complexities include the following. First, the images themselves are of no interest: all interest focuses on the behavior of individual cells across time, and thus, the cells need to be segmented in an automated manner. Second, the cells themselves have 100+ pixels, so that they form 100+ curves measured over time, so that data compression is required to extract the features of these curves. Third, some of the pixels in some of the cells are subject to image saturation due to bit depth limits, and this saturation needs to be accounted for if one is to normalize the images in a reasonably un-biased manner. Finally, the Ca(2+) signals have oscillations or waves that vary with time and these signals need to be extracted. Thus, our aim is to show how to use multiple weighted and standard singular value decompositions to detect, extract and clarify the Ca(2+) signals. Our signal extraction methods then lead to simple although finely focused statistical methods to compare Ca(2+) signals across experimental conditions.

  17. Extremely low frequency electromagnetic fields promote mesenchymal stem cell migration by increasing intracellular Ca2+ and activating the FAK/Rho GTPases signaling pathways in vitro.

    Science.gov (United States)

    Zhang, Yingchi; Yan, Jiyuan; Xu, Haoran; Yang, Yong; Li, Wenkai; Wu, Hua; Liu, Chaoxu

    2018-05-21

    The ability of mesenchymal stem cells (MSCs) to migrate to the desired tissues or lesions is crucial for stem cell-based regenerative medicine and tissue engineering. Optimal therapeutics for promoting MSC migration are expected to become an effective means for tissue regeneration. Electromagnetic fields (EMF), as a noninvasive therapy, can cause a lot of biological changes in MSCs. However, whether EMF can promote MSC migration has not yet been reported. We evaluated the effects of EMF on cell migration in human bone marrow-derived MSCs. With the use of Helmholtz coils and an EMF stimulator, 7.5, 15, 30, 50, and 70 Hz/1 mT EMF was generated. Additionally, we employed the L-type calcium channel blocker verapamil and the focal adhesion kinase (FAK) inhibitor PF-573228 to investigate the role of intracellular calcium content, cell adhesion proteins, and the Rho GTPase protein family (RhoA, Rac1, and Cdc42) in EMF-mediated MSC migration. Cell adhesion proteins (FAK, talin, and vinculin) were detected by Western blot analysis. The Rho GTPase protein family activities were assessed by G-LISA, and F-actin levels, which reflect actin cytoskeletal organization, were detected using immunofluorescence. All the 7.5, 15, 30, 50, and 70 Hz/1 mT EMF promoted MSC migration. EMF increased MSC migration in an intracellular calcium-dependent manner. Notably, EMF-enhanced migration was mediated by FAK activation, which was critical for the formation of focal contacts, as evidenced by increased talin and vinculin expression. Moreover, RhoA, Rac1, and Cdc42 were activated by FAK to increase cytoskeletal organization, thus promoting cell contraction. EMF promoted MSC migration by increasing intracellular calcium and activating the FAK/Rho GTPase signaling pathways. This study provides insights into the mechanisms of MSC migration and will enable the rational design of targeted therapies to improve MSC engraftment.

  18. Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

    Directory of Open Access Journals (Sweden)

    Kyle T Beggs

    Full Text Available BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor

  19. Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells

    DEFF Research Database (Denmark)

    Ayesh Hafez Ali, Dalia; Abuelreich, Sarah; Alkeraishan, Nora

    2018-01-01

    during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor......Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling...... signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand...

  20. MPD model for radar echo signal of hypersonic targets

    Directory of Open Access Journals (Sweden)

    Xu Xuefei

    2014-08-01

    Full Text Available The stop-and-go (SAG model is typically used for echo signal received by the radar using linear frequency modulation pulse compression. In this study, the authors demonstrate that this model is not applicable to hypersonic targets. Instead of SAG model, they present a more realistic echo signal model (moving-in-pulse duration (MPD for hypersonic targets. Following that, they evaluate the performances of pulse compression under the SAG and MPD models by theoretical analysis and simulations. They found that the pulse compression gain has an increase of 3 dB by using the MPD model compared with the SAG model in typical cases.

  1. Neurotrophin-3 Regulates Synapse Development by Modulating TrkC-PTPσ Synaptic Adhesion and Intracellular Signaling Pathways.

    Science.gov (United States)

    Han, Kyung Ah; Woo, Doyeon; Kim, Seungjoon; Choii, Gayoung; Jeon, Sangmin; Won, Seoung Youn; Kim, Ho Min; Heo, Won Do; Um, Ji Won; Ko, Jaewon

    2016-04-27

    Neurotrophin-3 (NT-3) is a secreted neurotrophic factor that binds neurotrophin receptor tyrosine kinase C (TrkC), which in turn binds to presynaptic protein tyrosine phosphatase σ (PTPσ) to govern excitatory synapse development. However, whether and how NT-3 cooperates with the TrkC-PTPσ synaptic adhesion pathway and TrkC-mediated intracellular signaling pathways in rat cultured neurons has remained unclear. Here, we report that NT-3 enhances TrkC binding affinity for PTPσ. Strikingly, NT-3 treatment bidirectionally regulates the synaptogenic activity of TrkC: at concentrations of 10-25 ng/ml, NT-3 further enhanced the increase in synapse density induced by TrkC overexpression, whereas at higher concentrations, NT-3 abrogated TrkC-induced increases in synapse density. Semiquantitative immunoblotting and optogenetics-based imaging showed that 25 ng/ml NT-3 or light stimulation at a power that produced a comparable level of NT-3 (6.25 μW) activated only extracellular signal-regulated kinase (ERK) and Akt, whereas 100 ng/ml NT-3 (light intensity, 25 μW) further triggered the activation of phospholipase C-γ1 and CREB independently of PTPσ. Notably, disruption of TrkC intracellular signaling pathways, extracellular ligand binding, or kinase activity by point mutations compromised TrkC-induced increases in synapse density. Furthermore, only sparse, but not global, TrkC knock-down in cultured rat neurons significantly decreased synapse density, suggesting that intercellular differences in TrkC expression level are critical for its synapse-promoting action. Together, our data demonstrate that NT-3 is a key factor in excitatory synapse development that may direct higher-order assembly of the TrkC/PTPσ complex and activate distinct intracellular signaling cascades in a concentration-dependent manner to promote competition-based synapse development processes. In this study, we present several lines of experimental evidences to support the conclusion that

  2. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    International Nuclear Information System (INIS)

    Kodavanti, Prasada Rao S.; Osorio, Cristina; Royland, Joyce E.; Ramabhadran, Ram; Alzate, Oscar

    2011-01-01

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca 2+ -mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit β (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: ► We performed brain proteomic analysis of rats exposed to the neurotoxicant, Aroclor 1254. ► Cerebellum and

  3. Use of multiple singular value decompositions to analyze complex intracellular calcium ion signals

    KAUST Repository

    Martinez, Josue G.; Huang, Jianhua Z.; Burghardt, Robert C.; Barhoumi, Rola; Carroll, Raymond J.

    2009-01-01

    ) to extract the signals from such movies, in a way that is semi-automatic and tuned closely to the actual data and their many complexities. These complexities include the following. First, the images themselves are of no interest: all interest focuses

  4. A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

    Science.gov (United States)

    Jing, Chaoran

    2013-01-01

    Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR) tagged protein displaces the quencher. Thus, we began by building a non-fluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins, but also less abundant, more dynamic cytoplasmic proteins. These results suggest that fluorogenic TMP-tag can significantly impact highresolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering. PMID:23745575

  5. Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

    Science.gov (United States)

    Qureshi, Farhan M; Dejene, Eden A; Corbin, Kathryn L; Nunemaker, Craig S

    2015-05-01

    In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

    Science.gov (United States)

    Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

    1998-01-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

  7. Multiple cues on the physiochemical, mesenchymal, and intracellular trafficking interactions with nanocarriers to maximize tumor target efficiency

    Directory of Open Access Journals (Sweden)

    Kim SW

    2015-06-01

    Full Text Available Sang-Woo Kim, Dongwoo Khang Nanomedicine Laboratory, Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, South Korea Abstract: Over the past 60 years, numerous medical strategies have been employed to overcome neoplasms. In fact, with the exception of lung, bronchial, and pancreatic cancers, the 5-year survival rate of most cancers currently exceeds 70%. However, the quality of life of patients during chemotherapy remains unsatisfactory despite the increase in survival rate. The side effects of current chemotherapies stem from poor target efficiency at tumor sites due to the uncontrolled biodistribution of anticancer agents (ie, conventional or current approved nanodrugs. This review discusses the effective physiochemical factors for determining biodistribution of nanocarriers and, ultimately, increasing tumor-targeting probability by avoiding the reticuloendothelial system. Second, stem cell-conjugated nanotherapeutics was addressed to maximize the tumor searching ability and to inhibit tumor growth. Lastly, physicochemical material properties of anticancer nanodrugs were discussed for targeting cellular organelles with modulation of drug-release time. A better understanding of suggested topics will increase the tumor-targeting ability of anticancer drugs and, ultimately, promote the quality of life of cancer patients during chemotherapy. Keywords: cancer, anticancer nanodrugs, mesenchymal stem cell, intracellular trafficking

  8. Intracellular Signaling Defects Contribute to TH17 Dysregulation during HIV Infection

    Science.gov (United States)

    2014-05-16

    review of biochemistry 62:543-85 353. Xu H, Wang X, Liu DX, Moroney-Rasmussen T, Lackner AA, Veazey RS. 2012. IL-17-producing innate lymphoid cells ...maximum, CD4+ cell counts (blue) decline sharply at first because of trapping in lymphoid tissues but then rise again to a moderately subnormal level...then disseminates to draining lymph nodes and other lymphoid tissues, where it infects CD4+ target cells (42; 206; 241). Dissemination coincides

  9. Targeting the intracellular environment in cystic fibrosis: restoring autophagy as a novel strategy to circumvent the CFTR defect

    Directory of Open Access Journals (Sweden)

    Valeria Rachela Villella

    2013-01-01

    Full Text Available Cystic fibrosis (CF patients harboring the most common deletion mutation of the cystic fibrosis transmembrane conductance regulator (CFTR, F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the plasma membrane even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1, a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the plasma membrane. We focus on the relationship between the improvement of peripheral proteostasis and CFTR plasma membrane stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent preclinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation.

  10. Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect

    Science.gov (United States)

    Villella, Valeria Rachela; Esposito, Speranza; Bruscia, Emanuela M.; Maiuri, Maria Chiara; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

    2013-01-01

    Cystic fibrosis (CF) patients harboring the most common deletion mutation of the CF transmembrane conductance regulator (CFTR), F508del, are poor responders to potentiators of CFTR channel activity which can be used to treat a small subset of CF patients who genetically carry plasma membrane (PM)-resident CFTR mutants. The misfolded F508del-CFTR protein is unstable in the PM even if rescued by pharmacological agents that prevent its intracellular retention and degradation. CF is a conformational disease in which defective CFTR induces an impressive derangement of general proteostasis resulting from disabled autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of autophagosome formation, either by means of direct gene transfer or indirectly by administration of proteostasis regulators, could stabilize F508del-CFTR at the PM. We focus on the relationship between the improvement of peripheral proteostasis and CFTR PM stability in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article reviews recent pre-clinical evidence indicating that targeting the intracellular environment surrounding the misfolded mutant CFTR instead of protein itself could constitute an attractive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the beneficial action of CFTR potentiators on lung inflammation. PMID:23346057

  11. Efficient intracellular delivery of 5-fluorodeoxyuridine into colon cancer cells by targeted immunoliposomes

    NARCIS (Netherlands)

    Koning, GA; Kamps, JAAM; Scherphof, GL

    2002-01-01

    Immunoliposomes, liposomes with monoclonal antibodies attached, are being developed for targeting the anti-cancer drug 5-fluoro-2'-deoxyuridine (FUdR) to colon cancer cells. A monoclonal antibody against the rat colon carcinoma CC531 was covalently coupled to liposomes containing a dipalmitoylated

  12. Fluorescent protein pair emit intracellular FRET signal suitable for FACS screening

    International Nuclear Information System (INIS)

    Johansson, Daniel X.; Brismar, Hjalmar; Persson, Mats A.A.

    2007-01-01

    The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 ± 1.5% (mean ± SEM) and 8.0 ± 0.7% in ECFP fluorescence for the specific antibodies reacting with gp120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 ± 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 ± 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals

  13. Crosstalk between intracellular and extracellular signals regulating interneuron production migration and integration into the cortex

    Directory of Open Access Journals (Sweden)

    Elise ePeyre

    2015-04-01

    Full Text Available During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: 1/ Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; 2/ Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; 3/ Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex.

  14. Crosstalk between intracellular and extracellular signals regulating interneuron production, migration and integration into the cortex.

    Science.gov (United States)

    Peyre, Elise; Silva, Carla G; Nguyen, Laurent

    2015-01-01

    During embryogenesis, cortical interneurons are generated by ventral progenitors located in the ganglionic eminences of the telencephalon. They travel along multiple tangential paths to populate the cortical wall. As they reach this structure they undergo intracortical dispersion to settle in their final destination. At the cellular level, migrating interneurons are highly polarized cells that extend and retract processes using dynamic remodeling of microtubule and actin cytoskeleton. Different levels of molecular regulation contribute to interneuron migration. These include: (1) Extrinsic guidance cues distributed along migratory streams that are sensed and integrated by migrating interneurons; (2) Intrinsic genetic programs driven by specific transcription factors that grant specification and set the timing of migration for different subtypes of interneurons; (3) Adhesion molecules and cytoskeletal elements/regulators that transduce molecular signalings into coherent movement. These levels of molecular regulation must be properly integrated by interneurons to allow their migration in the cortex. The aim of this review is to summarize our current knowledge of the interplay between microenvironmental signals and cell autonomous programs that drive cortical interneuron porduction, tangential migration, and intergration in the developing cerebral cortex.

  15. Intercellular and intracellular signaling pathways mediating ionizing radiation-induced bystander effects

    International Nuclear Information System (INIS)

    Hamada, Nobuyuki; Hara, Takamitsu; Kobayashi, Yasuhiko; Matsumoto, Hideki

    2007-01-01

    A rapidly growing body of experimental evidence indicates that ionizing radiation induces biological effects in non-irradiated bystander cells that have received signals from adjacent or distant irradiated cells. This phenomenon, which has been termed the ionizing radiation-induced bystander effect, challenges the long-standing paradigm that radiation traversal through the nucleus of a cell is a prerequisite to elicit genetic damage or a biological response. Bystander effects have been observed in a number of experimental systems, and cells whose nucleus or cytoplasm is irradiated exert bystander responses. Bystander cells manifest a multitude of biological consequences, such as genetic and epigenetic changes, alterations in gene expression, activation of signal transduction pathways, and delayed effects in their progeny. Several mediating mechanisms have been proposed. These involve gap junction-mediated intercellular communication, secreted soluble factors, oxidative metabolism, plasma membrane-bound lipid rafts, and calcium fluxes. This paper reviews briefly the current knowledge of the bystander effect with a focus on proposed mechanisms. The potential benefit of bystander effects to cancer radiotherapy will also be discussed. (author)

  16. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    International Nuclear Information System (INIS)

    Wang Chan; Tao Shengyang; Hu Tao; Yang Jingbang; Meng Changgong; Lv Piping; Wei Wei

    2011-01-01

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N 2 adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m 2 g -1 , a pore size of 1.91 nm and a saturation magnetization of 32 emu g -1 . Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  17. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    Science.gov (United States)

    Wang, Chan; Lv, Piping; Wei, Wei; Tao, Shengyang; Hu, Tao; Yang, Jingbang; Meng, Changgong

    2011-10-01

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N2 adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m2 g - 1, a pore size of 1.91 nm and a saturation magnetization of 32 emu g - 1. Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  18. A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

    Energy Technology Data Exchange (ETDEWEB)

    Wang Chan; Tao Shengyang; Hu Tao; Yang Jingbang; Meng Changgong [School of Chemical Engineering, Dalian University of Technology, Dalian, Liaoning (China); Lv Piping; Wei Wei, E-mail: taosy@dlut.edu.cn [National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing (China)

    2011-10-14

    A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N{sub 2} adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m{sup 2} g{sup -1}, a pore size of 1.91 nm and a saturation magnetization of 32 emu g{sup -1}. Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.

  19. p120-catenin differentially regulates cell migration by Rho-dependent intracellular and secreted signals

    DEFF Research Database (Denmark)

    Epifano, Carolina; Megias, Diego; Perez-Moreno, Mirna

    2014-01-01

    The adherens junction protein p120-catenin is implicated in the regulation of cadherin stability, cell migration and inflammatory responses in mammalian epithelial tissues. How these events are coordinated to promote wound repair is not understood. We show that p120 catenin regulates the intrinsic...... migratory properties of primary mouse keratinocytes, but also influences the migratory behavior of neighboring cells by secreted signals. These events are rooted in the ability of p120-catenin to regulate RhoA GTPase activity, which leads to a two-tiered control of cell migration. One restrains cell...... motility via an increase in actin stress fibers, reduction in integrin turnover and an increase in the robustness of focal adhesions. The other is coupled to the secretion of inflammatory cytokines including interleukin-24, which causally enhances randomized cell movements. Taken together, our results...

  20. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    International Nuclear Information System (INIS)

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken.

    2007-01-01

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences

  1. Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

    Science.gov (United States)

    Mizejewski, G J

    2015-12-01

    The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer.

  2. Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Kodavanti, Prasada Rao S., E-mail: kodavanti.prasada@epa.gov [Neurotoxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Osorio, Cristina [Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States); Royland, Joyce E.; Ramabhadran, Ram [Genetic and Cellular Toxicology Branch, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina (United States); Alzate, Oscar [Department of Cellular and Developmental Biology, University of North Carolina at Chapel Hill, North Carolina (United States); Systems Proteomics Center, University of North Carolina at Chapel Hill, North Carolina (United States); Program on Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, North Carolina (United States)

    2011-11-15

    The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca{sup 2+}-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0 mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit {beta} (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity. -- Highlights: Black-Right-Pointing-Pointer We performed brain proteomic analysis of rats exposed to the neurotoxicant

  3. Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

    Science.gov (United States)

    Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Vazquez-Martinez, Rafael; Malagon, Maria M

    2013-02-01

    Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

  4. GPR Signal Denoising and Target Extraction With the CEEMD Method

    KAUST Repository

    Li, Jing

    2015-04-17

    In this letter, we apply a time and frequency analysis method based on the complete ensemble empirical mode decomposition (CEEMD) method in ground-penetrating radar (GPR) signal processing. It decomposes the GPR signal into a sum of oscillatory components, with guaranteed positive and smoothly varying instantaneous frequencies. The key idea of this method relies on averaging the modes obtained by empirical mode decomposition (EMD) applied to several realizations of Gaussian white noise added to the original signal. It can solve the mode-mixing problem in the EMD method and improve the resolution of ensemble EMD (EEMD) when the signal has a low signal-to-noise ratio. First, we analyze the difference between the basic theory of EMD, EEMD, and CEEMD. Then, we compare the time and frequency analysis with Hilbert-Huang transform to test the results of different methods. The synthetic and real GPR data demonstrate that CEEMD promises higher spectral-spatial resolution than the other two EMD methods in GPR signal denoising and target extraction. Its decomposition is complete, with a numerically negligible error.

  5. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  6. mTOR Signaling Confers Resistance to Targeted Cancer Drugs.

    Science.gov (United States)

    Guri, Yakir; Hall, Michael N

    2016-11-01

    Cancer is a complex disease and a leading cause of death worldwide. Extensive research over decades has led to the development of therapies that target cancer-specific signaling pathways. However, the clinical benefits of such drugs are at best transient due to tumors displaying intrinsic or adaptive resistance. The underlying compensatory pathways that allow cancer cells to circumvent a drug blockade are poorly understood. We review here recent studies suggesting that mammalian TOR (mTOR) signaling is a major compensatory pathway conferring resistance to many cancer drugs. mTOR-mediated resistance can be cell-autonomous or non-cell-autonomous. These findings suggest that mTOR signaling should be monitored routinely in tumors and that an mTOR inhibitor should be considered as a co-therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    Science.gov (United States)

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

  8. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2012-07-15

    Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

  9. Moderate elevation of intracellular creatine by targeting the creatine transporter protects mice from acute myocardial infarction

    Science.gov (United States)

    Lygate, Craig A.; Bohl, Steffen; ten Hove, Michiel; Faller, Kiterie M.E.; Ostrowski, Philip J.; Zervou, Sevasti; Medway, Debra J.; Aksentijevic, Dunja; Sebag-Montefiore, Liam; Wallis, Julie; Clarke, Kieran; Watkins, Hugh; Schneider, Jürgen E.; Neubauer, Stefan

    2012-01-01

    Aims Increasing energy storage capacity by elevating creatine and phosphocreatine (PCr) levels to increase ATP availability is an attractive concept for protecting against ischaemia and heart failure. However, testing this hypothesis has not been possible since oral creatine supplementation is ineffectual at elevating myocardial creatine levels. We therefore used mice overexpressing creatine transporter in the heart (CrT-OE) to test for the first time whether elevated creatine is beneficial in clinically relevant disease models of heart failure and ischaemia/reperfusion (I/R) injury. Methods and results CrT-OE mice were selected for left ventricular (LV) creatine 20–100% above wild-type values and subjected to acute and chronic coronary artery ligation. Increasing myocardial creatine up to 100% was not detrimental even in ageing CrT-OE. In chronic heart failure, creatine elevation was neither beneficial nor detrimental, with no effect on survival, LV remodelling or dysfunction. However, CrT-OE hearts were protected against I/R injury in vivo in a dose-dependent manner (average 27% less myocardial necrosis) and exhibited greatly improved functional recovery following ex vivo I/R (59% of baseline vs. 29%). Mechanisms contributing to ischaemic protection in CrT-OE hearts include elevated PCr and glycogen levels and improved energy reserve. Furthermore, creatine loading in HL-1 cells did not alter antioxidant defences, but delayed mitochondrial permeability transition pore opening in response to oxidative stress, suggesting an additional mechanism to prevent reperfusion injury. Conclusion Elevation of myocardial creatine by 20–100% reduced myocardial stunning and I/R injury via pleiotropic mechanisms, suggesting CrT activation as a novel, potentially translatable target for cardiac protection from ischaemia. PMID:22915766

  10. Intracellular calcium overloading and oxidative stress in cardiomyocyte necrosis via a mitochondriocentric signal-transducer-effector pathway

    Science.gov (United States)

    Shaheen, Mazen; Cheema, Yaser; Shahbaz, Atta U; Bhattacharya, Syamal K; Weber, Karl T

    2011-01-01

    Congestive heart failure (CHF), a common clinical syndrome, has reached epidemic proportions. Its disabling symptoms account for frequent hospitalizations and readmissions. Pathophysiological mechanisms that lead to CHF and account for its progressive nature are of considerable interest. Important scientific observations obtained from Dr Pawan K Singal’s laboratory in Winnipeg, Manitoba, have provided crucial insights to our understanding of the pathophysiological factors that contribute to cardiomyocyte necrosis (the heart is a postmitotic organ incapable of tolerating an ongoing loss of these cells without adverse functional consequences). This increment in knowledge and the mechanistic insights afforded by Dr Singal and his colleagues have highlighted the role of excessive intracellular calcium accumulation and the appearance of oxidative stress in CHF, in which the rate of reactive oxygen species generation overwhelms their rate of detoxification by antioxidant defenses. They have shown that this common pathophysiological scenario applies to diverse entities such as ischemia/reperfusion and hypoxia/reoxygenation forms of injury, myocardial infarction and the cardiomyopathies that accompany diabetes and excess levels of catecholamines and adriamycin. The authors are honoured to be invited to contribute to the present focus issue of Experimental & Clinical Cardiology in recognizing Dr Singal’s numerous scholarly accomplishments. The present article reviews the authors’ recent work on a mitochondriocentric signal-transducer-effector pathway to cardiomyocyte necrosis found in rats with either an acute stressor state that accompanies isoproterenol administration or a chronic stressor state manifested after four weeks of aldosterone/salt treatment. PMID:22131852

  11. TRPC1, STIM1, and ORAI influence signal-regulated intracellular and endoplasmic reticulum calcium dynamics in human myometrial cells.

    Science.gov (United States)

    Murtazina, Dilyara A; Chung, Daesuk; Ulloa, Aida; Bryan, Emily; Galan, Henry L; Sanborn, Barbara M

    2011-08-01

    To explore the relationship between signal-stimulated increases in intracellular calcium ([Ca(2+)](i)) and depletion and refilling of the endoplasmic reticulum (ER) Ca(2+) stores ([Ca(2+)](L)) in human myometrial cells, we measured simultaneous changes in [Ca(2+)](i) and [Ca(2+)](L) using Fura-2 and Mag-fluo-4, respectively, in PHM1-41 immortalized and primary cells derived from pregnant myometrium and in primary cells derived from nonpregnant tissue. Signal- and extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and ER refilling stimulated by oxytocin and cyclopiazonic acid were not inhibited by voltage-operated channel blocker nifedipine or mibefradil, inhibition of Na(+)/Ca(2+) exchange with KB-R7943, or zero extracellular Na(+) in PHM1-41 cells. Gadolinium-inhibited oxytocin- and cyclopiazonic acid-induced SRCE and slowed ER store refilling. TRPC1 mRNA knockdown specifically inhibited oxytocin-stimulated SRCE but had no statistically significant effect on ER store refilling and no effect on either parameter following cyclopiazonic acid treatment. Dominant negative STIMΔERM expression attenuated oxytocin- and thapsigargin-stimulated SRCE. Both STIM1 and ORAI1-ORAI3 mRNA knockdowns significantly attenuated oxytocin- and cyclopiazonic acid-stimulated SRCE. The data also suggest that reduction in STIM1 or ORAI1-ORAI3 mRNA can impede the rate of ER store refilling following removal of SERCA inhibition. These data provide evidence for both distinct and overlapping influences of TRPC1, STIM1, and ORAI1-ORAI3 on SRCE and ER store refilling in human myometrial cells that may contribute to the regulation of myometrial Ca(2+) dynamics. These findings have important implications for understanding the control of myometrial Ca(2+) dynamics in relation to myometrial contractile function.

  12. Target acquisition performance : Effects of target aspect angle, dynamic imaging and signal processing

    NARCIS (Netherlands)

    Beintema, J.A.; Bijl, P.; Hogervorst, M.A.; Dijk, J.

    2008-01-01

    In an extensive Target Acquisition (TA) performance study, we recorded static and dynamic imagery of a set of military and civilian two-handheld objects at a range of distances and aspect angles with an under-sampled uncooled thermal imager. Next, we applied signal processing techniques including

  13. Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

    Directory of Open Access Journals (Sweden)

    Pablo J Sáez

    Full Text Available Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻ showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

  14. Disruption in connexin-based communication is associated with intracellular Ca²⁺ signal alterations in astrocytes from Niemann-Pick type C mice.

    Science.gov (United States)

    Sáez, Pablo J; Orellana, Juan A; Vega-Riveros, Natalia; Figueroa, Vania A; Hernández, Diego E; Castro, Juan F; Klein, Andrés D; Jiang, Jean X; Zanlungo, Silvana; Sáez, Juan C

    2013-01-01

    Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1⁻/⁻) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1⁻/⁻ hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1⁻/⁻ astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1⁻/⁻ astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.

  15. HER3 signaling and targeted therapy in cancer

    Directory of Open Access Journals (Sweden)

    Rosalin Mishra

    2018-05-01

    Full Text Available ERBB family members including epidermal growth factor receptor (EGFR also known as HER1, ERBB2/HER2/Neu, ERBB3/HER3 and ERBB4/HER4 are aberrantly activated in multiple cancers and hence serve as drug targets and biomarkers in modern precision therapy. The therapeutic potential of HER3 has long been underappreciated, due to impaired kinase activity and relatively low expression in tumors. However, HER3 has received attention in recent years as it is a crucial heterodimeric partner for other EGFR family members and has the potential to regulate EGFR/HER2-mediated resistance. Upregulation of HER3 is associated with several malignancies where it fosters tumor progression via interaction with different receptor tyrosine kinases (RTKs. Studies also implicate HER3 contributing significantly to treatment failure, mostly through the activation of PI3K/AKT, MAPK/ERK and JAK/STAT pathways. Moreover, activating mutations in HER3 have highlighted the role of HER3 as a direct therapeutic target. Therapeutic targeting of HER3 includes abrogating its dimerization partners’ kinase activity using small molecule inhibitors (lapatinib, erlotinib, gefitinib, afatinib, neratinib or direct targeting of its extracellular domain. In this review, we focus on HER3-mediated signaling, its role in drug resistance and discuss the latest advances to overcome resistance by targeting HER3 using mono- and bispecific antibodies and small molecule inhibitors.

  16. Intracellular and extracellular microtubule associated protein tau as a therapeutic target in Alzheimer disease and other tauopathies.

    Science.gov (United States)

    Avila, Jesús; Pallas, Noemí; Bolós, Marta; Sayas, C Laura; Hernandez, Felix

    2016-06-01

    Microtubule associated protein tau, a protein mainly expressed in neurons, plays an important role in several diseases related to dementia, named tauopathies. Alzheimer disease is the most relevant tauopathy. The role of tau protein in dementia is now a topic under discussion, and is the focus of this review. We have covered two major areas: tau pathology and tau as a therapeutic target. Tau pathology is mainly related to a gain of toxic function due to an abnormal accumulation, aberrant modifications (such as hyperphosphorylation and truncation, among others) and self-aggregation of tau into oligomers or larger structures. Also, tau can be found extracellularly in a toxic form. Tau-based therapy is mainly centered on avoiding the gain of these toxic functions of tau. Tau therapies are focused on lowering tau levels, mainly of modified tau species that could be toxic for neurons (phosphorylated, truncated or aggregated tau), in intracellular or extracellular form. Decreasing the levels of those toxic species is a possible therapeutic strategy.

  17. Dopamine elevates intracellular zinc concentration in cultured rat embryonic cortical neurons through the cAMP-nitric oxide signaling cascade.

    Science.gov (United States)

    Hung, Hui-Hsing; Kao, Lung-Sen; Liu, Pei-Shan; Huang, Chien-Chang; Yang, De-Ming; Pan, Chien-Yuan

    2017-07-01

    Zinc ion (Zn 2+ ), the second most abundant transition metal after iron in the body, is essential for neuronal activity and also induces toxicity if the concentration is abnormally high. Our previous results show that exposure of cultured cortical neurons to dopamine elevates intracellular Zn 2+ concentrations ([Zn 2+ ] i ) and induces autophagosome formation but the mechanism is not clear. In this study, we characterized the signaling pathway responsible for the dopamine-induced elevation of [Zn 2+ ] i and the effect of [Zn 2+ ] i in modulating the autophagy in cultured rat embryonic cortical neurons. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a membrane-permeable Zn 2+ chelator, could rescue the cell death and suppress the autophagosome puncta number induced by dopamine. Dopamine treatment increased the lipidation level of the endogenous microtubule-associated protein 1A/1B-light chain 3 (LC3 II), an autophagosome marker. TPEN added 1h before, but not after, dopamine treatment suppressed the dopamine-induced elevation of LC3 II level. Inhibitors of the dopamine D1-like receptor, protein kinase A (PKA), and NOS suppressed the dopamine-induced elevation of [Zn 2+ ] i . PKA activators and NO generators directly increased [Zn 2+ ] i in cultured neurons. Through cell fractionation, proteins with m.w. values between 5 and 10kD were found to release Zn 2+ following NO stimulation. In addition, TPEN pretreatment and an inhibitor against PKA could suppress the LC3 II level increased by NO and dopamine, respectively. Therefore, our results demonstrate that dopamine-induced elevation of [Zn 2+ ] i is mediated by the D1-like receptor-PKA-NO pathway and is important in modulating the cell death and autophagy. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Ganoderma lucidum targeting lung cancer signaling: A review.

    Science.gov (United States)

    Gill, Balraj Singh; Navgeet; Kumar, Sanjeev

    2017-06-01

    Lung cancer causes huge mortality to population, and pharmaceutical companies require new drugs as an alternative either synthetic or natural targeting lung cancer. The conventional therapies cause side effects, and therefore, natural products are used as a therapeutic candidate in lung cancer. Chemical diversity among natural products highlights the impact of evolution and survival of fittest. One such neglected natural product is Ganoderma lucidum used for promoting health and longevity for a longer time. The major bioconstituents of G. lucidum are mainly terpenes, polysaccharides, and proteins, which were explored for various activities ranging from apoptosis to autophagy. The bioconstituents of G. lucidum activate plasma membrane receptors and initiate various downstream signaling leading to nuclear factor-κB, phosphoinositide 3-kinase, Akt, and mammalian target of rapamycin in cancer. The bioconstituents regulate the expression of various genes involved in cell cycle, immune response, apoptosis, and autophagy in lung cancer. This review highlights the inextricable role of G. lucidum and its bioconstituents in lung cancer signaling for the first time.

  19. The effects of red ginseng saponin fraction-A (RGSF-A) on phagocytosis and intracellular signaling in Brucella abortus infected RAW 264.7 cells.

    Science.gov (United States)

    Arayan, Lauren Togonon; Simborio, Hannah Leah; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

    2015-06-01

    This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Therapeutically targeting mitochondrial redox signalling alleviates endothelial dysfunction in preeclampsia.

    Science.gov (United States)

    McCarthy, Cathal; Kenny, Louise C

    2016-09-08

    Aberrant placentation generating placental oxidative stress is proposed to play a critical role in the pathophysiology of preeclampsia. Unfortunately, therapeutic trials of antioxidants have been uniformly disappointing. There is provisional evidence implicating mitochondrial dysfunction as a source of oxidative stress in preeclampsia. Here we provide evidence that mitochondrial reactive oxygen species mediates endothelial dysfunction and establish that directly targeting mitochondrial scavenging may provide a protective role. Human umbilical vein endothelial cells exposed to 3% plasma from women with pregnancies complicated by preeclampsia resulted in a significant decrease in mitochondrial function with a subsequent significant increase in mitochondrial superoxide generation compared to cells exposed to plasma from women with uncomplicated pregnancies. Real-time PCR analysis showed increased expression of inflammatory markers TNF-α, TLR-9 and ICAM-1 respectively in endothelial cells treated with preeclampsia plasma. MitoTempo is a mitochondrial-targeted antioxidant, pre-treatment of cells with MitoTempo protected against hydrogen peroxide-induced cell death. Furthermore MitoTempo significantly reduced mitochondrial superoxide production in cells exposed to preeclampsia plasma by normalising mitochondrial metabolism. MitoTempo significantly altered the inflammatory profile of plasma treated cells. These novel data support a functional role for mitochondrial redox signaling in modulating the pathogenesis of preeclampsia and identifies mitochondrial-targeted antioxidants as potential therapeutic candidates.

  1. The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: Implications in targeted cancer therapies

    Science.gov (United States)

    Yang, Ke; Wang, Xin; Zhang, Hongmei; Wang, Zhongliang; Nan, Guoxin; Li, Yasha; Zhang, Fugui; Mohammed, Maryam K.; Haydon, Rex C.; Luu, Hue H.; Bi, Yang; He, Tong-Chuan

    2015-01-01

    The canonical WNT/β-catenin signaling pathway governs a myriad of biological processes underlying development and maintenance of adult tissue homeostasis, including regulation of stem cell self-renewal, cell proliferation, differentiation, and apoptosis. WNTs are secreted lipid-modified glycoproteins that act as short-range ligands to activate receptor-mediated signaling pathways. The hallmark of the canonical pathway is the activation of β-catenin mediated transcriptional activity. Canonical WNTs control the β-catenin dynamics as the cytoplasmic level of β-catenin is tightly regulated via phosphorylation by the ‘destruction complex’, consisting of glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), the scaffold protein AXIN, and the tumor suppressor adenomatous polyposis coli (APC). Aberrant regulation of this signaling cascade is associated with varieties of human diseases, especially cancers. Over the past decade, significant progress has been made in understanding the mechanisms of canonical WNT signaling. In this review, we focus on the current understanding of WNT signaling at the extracellular, cytoplasmic membrane, and intracellular/nuclear levels, including the emerging knowledge of crosstalk with other pathways. Recent progresses in developing novel WNT pathway-targeted therapies will also be reviewed. Thus, this review is intended to serve as a refresher of the current understanding about the physiologic and pathogenic roles of WNT/β-catenin signaling pathway, and to outline potential therapeutic opportunities by targeting the canonical WNT pathway. PMID:26618721

  2. The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

    Science.gov (United States)

    Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

    2016-04-01

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. © 2016 Authors; published by Portland Press Limited.

  3. MGAT1 is a novel transcriptional target of Wnt/β-catenin signaling pathway.

    Science.gov (United States)

    Akiva, Izzet; Birgül Iyison, Necla

    2018-01-08

    The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway, which has important functions in vertebrate early development, axis formation, cellular proliferation and morphogenesis. Additionally, Wnt/β-catenin signaling pathway is one of the most important intracellular pathways that controls cancer progression. To date most of the identified targets of this pathway are shown to harbor tumorigenic properties. We previously showed that Mannosyl glycoprotein acetylglucosaminyl-transferase (MGAT1) enzyme is among the Wnt/β-catenin signaling putative target genes in hepatocellular carcinoma cell lines (Huh7). MGAT1 protein levels were determined by Western Blotting from Huh7 cell lines in which Wnt/β-catenin pathway was activated by means of different approaches such as LiCl treatment and mutant β-catenin overexpression. Luciferase reporter assay was used to analyze the promoter activity of MGAT1. The mRNA levels of MGAT1 were determined by quantitative real-time PCR from Huh7 cells that were treated with either Wnt agonist or GSK-3β inhibitor. Wound healing and XTT cell proliferation assays were performed in order to determine the proliferation and migration capacities of MGAT1 overexpressing stable Huh7 cells. Finally, xenograft experiments were carried out to measure the tumor formation capacities in vivo. In this study we showed that the activation of Wnt/β-catenin pathway culminates in the upregulation of MGAT1 enzyme both at transcriptional and post-transcriptional levels. We also showed that overexpression of the β-catenin gene (CTNNB1) increased the promoter activity of MGAT1. We applied a set of complementary approaches to elucidate the functional importance of MGAT1 as a vital target of Wnt/β-catenin signaling in Huh7 cells. Our analyses related to cell proliferation and migration assays showed that in comparison to the control cells, MGAT1 expressing Huh7 cells have greater proliferative and invasive capabilities. Furthermore, the

  4. Leptin signaling molecular actions and drug target in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Jiang N

    2014-11-01

    Full Text Available Nan Jiang,1,* Rongtong Sun,2,* Qing Sun3 1Shandong University School of Medicine, Jinan, Shandong Province, People’s Republic of China; 2Weihai Municipal Hospital, Weihai, Shandong Province, People’s Republic of China; 3Department of Pathology, QianFoShan Hospital Affiliated to Shandong University, Jinan, Shandong Province, People’s Republic of China *These authors contributed equally to this work Abstract: Previous reports indicate that over 13 different tumors, including hepatocellular carcinoma (HCC, are related to obesity. Obesity-associated inflammatory, metabolic, and endocrine mediators, as well as the functioning of the gut microbiota, are suspected to contribute to tumorigenesis. In obese people, proinflammatory cytokines/chemokines including tumor necrosis factor-alpha, interleukin (IL-1 and IL-6, insulin and insulin-like growth factors, adipokines, plasminogen activator inhibitor-1, adiponectin, and leptin are found to play crucial roles in the initiation and development of cancer. The cytokines induced by leptin in adipose tissue or tumor cells have been intensely studied. Leptin-induced signaling pathways are critical for biological functions such as adiposity, energy balance, endocrine function, immune reaction, and angiogenesis as well as oncogenesis. Leptin is an activator of cell proliferation and anti-apoptosis in several cell types, and an inducer of cancer stem cells; its critical roles in tumorigenesis are based on its oncogenic, mitogenic, proinflammatory, and pro-angiogenic actions. This review provides an update of the pathological effects of leptin signaling with special emphasis on potential molecular mechanisms and therapeutic targeting, which could potentially be used in future clinical settings. In addition, leptin-induced angiogenic ability and molecular mechanisms in HCC are discussed. The stringent binding affinity of leptin and its receptor Ob-R, as well as the highly upregulated expression of both

  5. Intracellular co-delivery of Sr ion and phenamil drug through mesoporous bioglass nanocarriers synergizes BMP signaling and tissue mineralization.

    Science.gov (United States)

    Lee, Jung-Hwan; Mandakhbayar, Nandin; El-Fiqi, Ahmed; Kim, Hae-Won

    2017-09-15

    Inducing differentiation and maturation of resident multipotent stem cells (MSCs) is an important strategy to regenerate hard tissues in mal-calcification conditions. Here we explore a co-delivery approach of therapeutic molecules comprised of ion and drug through a mesoporous bioglass nanoparticle (MBN) for this purpose. Recently, MBN has offered unique potential as a nanocarrier for hard tissues, in terms of high mesoporosity, bone bioactivity (and possibly degradability), tunable delivery of biomolecules, and ionic modification. Herein Sr ion is structurally doped to MBN while drug Phenamil is externally loaded as a small molecule activator of BMP signaling, for the stimulation of osteo/odontogenesis and mineralization of human MSCs derived from dental pulp. The Sr-doped MBN (85Si:10Ca:5Sr) sol-gel processed presents a high mesoporosity with a pore size of ∼6nm. In particular, Sr ion is released slowly at a daily rate of ∼3ppm per mg nanoparticles for up to 7days, a level therapeutically effective for cellular stimulation. The Sr-MBN is internalized to most MSCs via an ATP dependent macropinocytosis within hours, increasing the intracellular levels of Sr, Ca and Si ions. Phenamil is loaded maximally ∼30% into Sr-MBN and then released slowly for up to 7days. The co-delivered molecules (Sr ion and Phenamil drug) have profound effects on the differentiation and maturation of cells, i.e., significantly enhancing expression of osteo/odontogenic genes, alkaline phosphatase activity, and mineralization of cells. Of note, the stimulation is a result of a synergism of Sr and Phenamil, through a Trb3-dependent BMP signaling pathway. This biological synergism is further evidenced in vivo in a mal-calcification condition involving an extracted tooth implantation in dorsal subcutaneous tissues of rats. Six weeks post operation evidences the osseous-dentinal hard tissue formation, which is significantly stimulated by the Sr/Phenamil delivery, based on histomorphometric

  6. β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+ release by tentacle extract from the jellyfish Cyanea capillata.

    Science.gov (United States)

    Wang, Qianqian; Zhang, Hui; Wang, Bo; Wang, Chao; Xiao, Liang; Zhang, Liming

    2017-07-25

    Intracellular Ca 2+ overload induced by extracellular Ca 2+ entry has previously been confirmed to be an important mechanism for the cardiotoxicity as well as the acute heart dysfunction induced by jellyfish venom, while the underlying mechanism remains to be elucidated. Under extracellular Ca 2+ -free or Ca 2+ -containing conditions, the Ca 2+ fluorescence in isolated adult mouse cardiomyocytes pre-incubated with tentacle extract (TE) from the jellyfish Cyanea capillata and β blockers was scanned by laser scanning confocal microscope. Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats. The increase of intracellular Ca 2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca 2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca 2+ in the presence of 1.8 mM extracellular Ca 2+ and completely abolished the Ca 2+ increase under an extracellular Ca 2+ -free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats. Our findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca 2+ overload through intracellular Ca 2+ release by TE from the jellyfish C. capillata.

  7. Sensing miRNA: Signal Amplification by Cognate RISC for Intracellular Detection of miRNA in Live Cells.

    Science.gov (United States)

    Kavishwar, Amol; Medarova, Zdravka

    2016-01-01

    The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence "turn on." Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecific cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.

  8. Romidepsin targets multiple survival signaling pathways in malignant T cells

    International Nuclear Information System (INIS)

    Valdez, B C; Brammer, J E; Li, Y; Murray, D; Liu, Y; Hosing, C; Nieto, Y; Champlin, R E; Andersson, B S

    2015-01-01

    Romidepsin is a cyclic molecule that inhibits histone deacetylases. It is Food and Drug Administration-approved for treatment of cutaneous and peripheral T-cell lymphoma, but its precise mechanism of action against malignant T cells is unknown. To better understand the biological effects of romidepsin in these cells, we exposed PEER and SUPT1 T-cell lines, and a primary sample from T-cell lymphoma patient (Patient J) to romidepsin. We then examined the consequences in some key oncogenic signaling pathways. Romidepsin displayed IC 50 values of 10.8, 7.9 and 7.0 nm in PEER, SUPT1 and Patient J cells, respectively. Strong inhibition of histone deacetylases and demethylases, increased production of reactive oxygen species and decreased mitochondrial membrane potential were observed, which may contribute to the observed DNA-damage response and apoptosis. The stress-activated protein kinase/c-Jun N-terminal kinase signaling pathway and unfolded protein response in the endoplasmic reticulum were activated, whereas the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and β-catenin pro-survival pathways were inhibited. The decreased level of β-catenin correlated with the upregulation of its inhibitor SFRP1 through romidepsin-mediated hypomethylation of its gene promoter. Our results provide new insights into how romidepsin invokes malignant T-cell killing, show evidence of its associated DNA hypomethylating activity and offer a rationale for the development of romidepsin-containing combination therapies

  9. Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-05-05

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  10. Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-06-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  11. Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

    Science.gov (United States)

    Hancock, Melissa L.; Canetta, Sarah E.; Role, Lorna W.; Talmage, David A.

    2008-01-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function. PMID:18458158

  12. Signal Transduction and Molecular Targets of Selected Flavonoids

    Science.gov (United States)

    Bode, Ann M.

    2013-01-01

    Abstract Significance: Diet exerts a major influence on the risk for developing cancer and heart disease. Food factors such as flavonoids are alleged to protect cells from premature aging and disease by shielding DNA, proteins, and lipids from oxidative damage. Recent Advances: Our work has focused on clarifying the effects of dietary components on cancer cell proliferation and tumor growth, discovering mechanisms to explain the effects, and identifying the specific molecular targets of these compounds. Our strategy for identifying specific molecular targets of phytochemicals involves the use of supercomputer technology combined with protein crystallography, molecular biology, and experimental laboratory verification. Critical Issues: One of the greatest challenges for scientists is to reduce the accumulation of distortion and half truths reported in the popular media regarding the health benefits of certain foods or food supplements. The use of these is not new, but interest has increased dramatically because of perceived health benefits that are presumably acquired without unpleasant side effects. Flavonoids are touted to exert many beneficial effects in vitro. However, whether they can produce these effects in vivo is disputed. Future Directions: The World Health Organization indicates that one third of all cancer deaths are preventable and that diet is closely linked to prevention. Based on this idea and epidemiological findings, attention has centered on dietary phytochemicals as an effective intervention in cancer development. However, an unequivocal link between diet and cancer has not been established. Thus, identifying cancer preventive dietary agents with specific molecular targets is essential to move forward toward successful cancer prevention. Antioxid. Redox Signal. 19, 163–180. PMID:23458437

  13. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer.

    Science.gov (United States)

    Priceman, Saul J; Gerdts, Ethan A; Tilakawardane, Dileshni; Kennewick, Kelly T; Murad, John P; Park, Anthony K; Jeang, Brook; Yamaguchi, Yukiko; Yang, Xin; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E; Wu, Anna M; Brown, Christine E; Forman, Stephen J

    2018-01-01

    Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with "on-target off-tumor" activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

  14. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

    Science.gov (United States)

    Priceman, Saul J.; Gerdts, Ethan A.; Tilakawardane, Dileshni; Kennewick, Kelly T.; Murad, John P.; Park, Anthony K.; Jeang, Brook; Yamaguchi, Yukiko; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E.; Brown, Christine E.; Forman, Stephen J.

    2018-01-01

    ABSTRACT Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. PMID:29308300

  15. Time course of hydrogen peroxide-thioredoxin balance and its influence on the intracellular signalling in myocardial infarction.

    Science.gov (United States)

    Schenkel, Paulo Cavalheiro; Tavares, Angela Maria Vicente; Fernandes, Rafael Oliveira; Diniz, Gabriela Placoná; Ludke, Ana Raquel Lehenbauer; Ribeiro, Maria Flavia Marques; Araujo, Alex Sander da Rosa; Barreto-Chaves, Maria Luiza; Belló-Klein, Adriane

    2012-06-01

    We investigated the myocardial thioredoxin-1 and hydrogen peroxide concentrations and their association with some prosurvival and pro-apoptotic proteins, during the transition from myocardial infarction (MI) to heart failure in rats. Male Wistar rats were divided into the following six groups: three sham-operated groups and three MI groups, each at at 2, 7 and 28 days postsurgery. Cardiac function was analysed by echocardiography; the concentration of H(2)O(2) and the ratio of reduced to oxidized glutathione were measured spectrophotometrically, while the myocardial immunocontent of thioredoxin-1, angiotensin II, angiotensin II type 1 and type 2 receptors, p-JNK/JNK, p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK3β/GSK3β was evaluated by Western blot. Our results show that thioredoxin-1 appears to make an important contribution to the reduced H(2)O(2) concentration. It was associated with lower JNK expression in the early period post-MI (2 days). However, thioredoxin-1 decreased, while renin-angiotensin system markers and levels of H(2)O(2) increased, over 28 days post-MI, in parallel with some signalling proteins involved in maladaptative cardiac remodelling and ventricular dysfunction. These findings provide insight into the time course profile of endogenous antioxidant adaptation to ischaemic injury, which may be useful for the design of therapeutical strategies targeting oxidative stress post-MI.

  16. The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-γ.

    Science.gov (United States)

    Ma, Feng; Xu, Sheng; Liu, Xingguang; Zhang, Qian; Xu, Xiongfei; Liu, Mofang; Hua, Minmin; Li, Nan; Yao, Hangping; Cao, Xuetao

    2011-07-24

    Interferon-γ (IFN-γ) has a critical role in immune responses to intracellular bacterial infection. MicroRNAs (miRNAs) are important in the regulation of innate and adaptive immunity. However, whether miRNAs can directly target IFN-γ and regulate IFN-γ production post-transcriptionally remains unknown. Here we show that infection of mice with Listeria monocytogenes or Mycobacterium bovis bacillus Calmette-Guérin (BCG) downregulated miR-29 expression in IFN-γ-producing natural killer cells, CD4(+) T cells and CD8(+) T cells. Moreover, miR-29 suppressed IFN-γ production by directly targeting IFN-γ mRNA. We developed mice with transgenic expression of a 'sponge' target to compete with endogenous miR-29 targets (GS29 mice). We found higher serum concentrations of IFN-γ and lower L. monocytogenes burdens in L. monocytogenes-infected GS29 mice than in their littermates. GS29 mice had enhanced T helper type 1 (T(H)1) responses and greater resistance to infection with BCG or Mycobacterium tuberculosis. Therefore, miR-29 suppresses immune responses to intracellular pathogens by targeting IFN-γ.

  17. The flavonoid fisetin as an anticancer agent targeting the growth signaling pathways.

    Science.gov (United States)

    Rengarajan, Thamaraiselvan; Yaacob, Nik Soriani

    2016-10-15

    Epidemiological studies show that consumption of diets rich in fruits and vegetables is associated with lower risks of cancer. This evidence has kindled interest into research on bioactive food components and has till date resulted in the identification of many compounds with cancer preventive and therapeutic potential. Among such compounds is fisetin (3,7,3,4-tetrahydroxyflavone), a flavonol that is commonly found in many fruits and vegetables such as apples, persimmons, grapes, kiwis, strawberries, onions and cucumbers. Fisetin has been shown to inhibit or retard the growth of various cancer cells in culture and implanted tumors in vivo. Fisetin targets many components of intracellular signaling pathways including regulators of cell survival and apoptosis, tumor angiogenic and metastatic switches by modulating a distinct set of upstream kinases, transcription factors and their regulators. Current evidence supports the idea that fisetin is a promising agent for cancer treatment. This review summarizes reported anticancer effects of fisetin, and re-emphasizes its potential therapeutic role in the treatment of cancer. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Participation of intracellular signal transduction in the radio-adaptive response induced by low-dose X-irradiation in human embryonic cells

    International Nuclear Information System (INIS)

    Ishii, Keiichiro; Hoshi, Yuko; Iwasaki, Toshiyasu; Watanabe, Masami.

    1996-01-01

    To elucidate the induction mechanism of radio-adaptive response in normal cells, we searched the literatures of the intracellular signal transduction. Furthermore, we examined the induction of radio-adaptive response with or without inhibitors of several kinds of protein kinase. The major results obtained were as follows; (1) According to the literature survey it is revealed that there are 4 intracellular signal transduction pathways which are possibly involved in the induction of radio-adaptive response: pathways depending on cAMP, calcium, cGMP, or protein-tyrosine kinase. (2) Addition of either inhibitor of protein-tyrosine kinase or protein kinase C to the cell culture medium during the low-dose X-irradiation inhibited the induction of radio-adaptive response. However, the addition of inhibitor of cAMP-dependent protein kinase, cGMP-dependent protein kinase, or Ca 2+ -calmodulin kinase II failed to inhibit the induction of radio-adaptive response. (3) These results suggest that the signal induced in cells by low-dose X-irradiation was transduced from protein-tyrosine kinase to protein kinase C via either pathway of phosphatidylinositol 3-kinase or splitting of profilin binding phosphatidylinositol 4,5-bisphosphate. (author)

  19. Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

    Science.gov (United States)

    Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

    2016-08-31

    Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

  20. Impact of adrenaline and metabolic stress on exercise-induced intracellular signaling and PGC-1α mRNA response in human skeletal muscle

    DEFF Research Database (Denmark)

    Brandt, Nina; Gunnarsson, Thomas Gunnar Petursson; Hostrup, Morten

    2016-01-01

    This study tested the hypothesis that elevated plasma adrenaline or metabolic stress enhances exercise-induced PGC-1α mRNA and intracellular signaling in human muscle. Trained (VO2-max: 53.8 ± 1.8 mL min(-1) kg(-1)) male subjects completed four different exercise protocols (work load of the legs...... exercise than at rest in all protocols, and higher (P adrenaline nor muscle metabolic stress determines the magnitude of PGC-1α mRNA response in human muscle. Furthermore, higher exercise-induced changes in AMPK, p38, and CREB...

  1. Systemic Responses of Multidrug-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii Following Exposure to the Antimicrobial Peptide Cathelicidin-BF Imply Multiple Intracellular Targets

    Directory of Open Access Journals (Sweden)

    Cunbao Liu

    2017-11-01

    Full Text Available Cathelicidin-BF, derived from the banded krait (Bungarus fasciatus, is a typically cationic, amphiphilic and α-helical antimicrobial peptide (AMP with 30 amino acids that exerts powerful effects on multidrug-resistant (MDR clinical isolates, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, but whether it targets plasma membranes or intracellular targets to kill bacteria is still controversial. In the present study, we demonstrated that the disruption of bacterial membranes with high concentrations of cathelicidin-BF was the cause of bacterial death, as with conventional antibiotics at high concentrations. At lower concentrations, cathelicidin-BF did not cause bacterial plasma membrane disruption, but it was able to cross the membrane and aggregate at the nucleoid regions. Functional proteins of the transcription processes of P. aeruginosa and A. baumannii were affected by sublethal doses of cathelicidin-BF, as demonstrated by comparative proteomics using isobaric tags for relative and absolute quantification and subsequent gene ontology (GO analysis. Analysis using the Kyoto Encyclopedia of Genes and Genomes showed that cathelicidin-BF mainly interferes with metabolic pathways related to amino acid synthesis, metabolism of cofactors and vitamins, metabolism of purine and energy supply, and other processes. Although specific targets of cathelicidin-BF must still be validated, our study offers strong evidence that cathelicidin-BF may act upon intracellular targets to kill superbugs, which may be helpful for further efforts to discover novel antibiotics to fight against them.

  2. Human T cell lymphotropic virus type I genomic expression and impact on intracellular signaling pathways during neurodegenerative disease and leukemia.

    Science.gov (United States)

    Yao, J; Wigdahl, B

    2000-01-01

    HTLV-I has been identified as the etiologic agent of neoplasia within the human peripheral blood T lymphocyte population, and a progressive neurologic disorder based primarily within the central nervous system. We have examined the role of HTLV-I in these two distinctly different clinical syndromes by examining the life cycle of the virus, with emphasis on the regulation of viral gene expression within relevant target cell populations. In particular, we have examined the impact of specific viral gene products, particularly Tax, on cellular metabolic function. Tax is a highly promiscuous and pleiotropic viral oncoprotein, and is the most important factor contributing to the initial stages of viral-mediated transformation of T cells after HTLV-I infection. Tax, which weakly binds to Tax response element 1 (TRE-1) in the viral long terminal repeat (LTR), can dramatically trans-activate viral gene expression by interacting with cellular transcription factors, such as activated transcription factors and cyclic AMP response element binding proteins (ATF/CREB), CREB binding protein (CBP/p300), and factors involved with the basic transcription apparatus. At the same time, Tax alters cellular gene expression by directly or indirectly interacting with a variety of cellular transcription factors, cell cycle control elements, and cellular signal transduction molecules ultimately resulting in dysregulated cell proliferation. The mechanisms associated with HTLV-I infection, leading to tropical spastic paraparesis (TSP) are not as clearly resolved. Possible explanations of viral-induced neurologic disease range from central nervous system (CNS) damage caused by direct viral invasion of the CNS to bystander CNS damage caused by the immune response to HTLV-I infection. It is interesting to note that it is very rare for an HTLV-I infected individual to develop both adult T cell leukemia (ATL) and TSP in his/her life time, suggesting that the mechanisms governing development of these

  3. Signal Detection, Target Tracking and Differential Geometry Applications to Statistical Inference

    National Research Council Canada - National Science Library

    Rao, C

    1997-01-01

    Signal detection and target tracking. A novel method known as polynomial rooting approach is proposed to obtain estimates of frequencies, amplitudes and noise variance of two-dimensional exponential signals...

  4. Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling

    Directory of Open Access Journals (Sweden)

    Anat Elmann

    2016-03-01

    Full Text Available Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures was determined by lactate dehydrogenase (LDH activity, intracellular ROS levels were detected using 2′,7′-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS. These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1 and p44/42 mitogen-activated protein kinases (MAPK, and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

  5. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

    Science.gov (United States)

    Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

    2015-01-01

    Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

  6. Noradrenaline represses PPAR (peroxisome-proliferator-activated receptor) gamma2 gene expression in brown adipocytes: intracellular signalling and effects on PPARgamma2 and PPARgamma1 protein levels

    DEFF Research Database (Denmark)

    Lindgren, Eva M; Nielsen, Ronni; Petrovic, Natasa

    2004-01-01

    phases, with the highest mRNA levels being found at the time of transition between the phases. PPARgamma2 mRNA levels were downregulated by noradrenaline treatment (EC50, 0.1 microM) in both proliferative and differentiating cells, with a lagtime of 1 h and lasting up to 4 h, after which expression...... was thus to investigate the influence of noradrenaline on PPARgamma gene expression in brown adipocytes. In primary cultures of brown adipocytes, PPARgamma2 mRNA levels were 20-fold higher than PPARgamma1 mRNA levels. PPARgamma expression occurred during both the proliferation and the differentiation...... gradually recovered. The down-regulation was beta-adrenoceptor-induced and intracellularly mediated via cAMP and protein kinase A; the signalling pathway did not involve phosphoinositide 3-kinase, Src, p38 mitogen-activated protein kinase or extracellular-signal-regulated kinases 1 and 2. Treatment...

  7. New method to extract radial acceleration of target from short-duration signal at low SNR

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to extract target radial acceleration from radar echo signal at low SNR (signal-to-noise), this paper employed FRFT (fractional Fourier transformation) to analyze short-duration radar echo and studied the relations between signal convergence peaks in matched transformation domain and signal duration and modu- lated frequency of signal. When signal duration is specified, the method of multi- plying sampled signal by the known frequency modulated signal to alter modulated frequency was presented, which generated the new signal with larger convergence peaks than the initial signal in matched transformation domain. Thus, it could successfully estimate the radial acceleration of radar target at low SNR. Simulations were conducted to show the feasibility and effectiveness of the method.

  8. Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline-Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays.

    Science.gov (United States)

    Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

    2016-06-01

    Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular

  9. Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays*

    Science.gov (United States)

    Ho, Yu-Hsuan; Shah, Pramod; Chen, Yi-Wen; Chen, Chien-Sheng

    2016-01-01

    Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular

  10. Discovering Small Molecule Inhibitors Targeted to Ligand-Stimulated RAGE-DIAPH1 Signaling Transduction

    Science.gov (United States)

    Pan, Jinhong

    The receptor of advanced glycation end product (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules, which plays an important role in immune responses. Full-length RAGE includes three extracellular immunoglobulin domains, a transmembrane domain and an intracellular domain. It is a pattern recognition receptor that can bind diverse ligands. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. It is found that calgranulin binding to the C1C2 domain or AGEs binding to the V domain activates extracellular signaling, which triggers interactions of the RAGE cytoplasmic tail (ctRAGE) with intracellular effector, such as diaphanous 1 (DIAPH1), to initiate signal transduction cascades. ctRAGE is essential for RAGE-ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE is over-expressed in diseased tissues of most RAGE-associated pathogenic conditions, such as complications of Alzheimer's diseases, diabetes, vascular diseases, inflammation, cancers and neurodegeneration. They are the major diseases affecting a large population worldwide. RAGE can function as a biomarker or drug target for these diseases. The cytoplasmic tail of RAGE can be used as a drug target to inhibit RAGE-induced intracellular signaling by small molecule inhibitors to treat RAGE-associated diseases. We developed a high throughput screening assay with which we probed a small molecule library of 58,000 compounds to find that 777 small molecules displayed 50% inhibition and 97 compounds demonstrated dose-dependent inhibition of the binding of ctRAGE-DIAPH1. Eventually, there were 13 compounds which displayed dose-dependent inhibition of ctRAGE binding to DIAPH1 and direct binding to ctRAGE analyzed by 15N HSQC-NMR and native tryptophan fluorescence titration experiments; thus, they were

  11. Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation.

    Science.gov (United States)

    Roehrich, A Dorothea; Guillossou, Enora; Blocker, Ariel J; Martinez-Argudo, Isabel

    2013-02-01

    Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation. © 2013 Blackwell Publishing Ltd.

  12. Analysis and Simulation of Multi-target Echo Signals from a Phased Array Radar

    OpenAIRE

    Jia Zhen; Zhou Rui

    2017-01-01

    The construction of digital radar simulation systems has been a research hotspot of the radar field. This paper focuses on theoretical analysis and simulation of multi-target echo signals produced in a phased array radar system, and constructs an array antenna element and a signal generation environment. The antenna element is able to simulate planar arrays and optimizes these arrays by adding window functions. And the signal environment can model and simulate radar transmission signals, rada...

  13. AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells.

    Directory of Open Access Journals (Sweden)

    Elisabeth Panther

    Full Text Available The nucleotide adenosine-5'-monophosphate (AMP can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1 and A(2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC function. AMP increased intracellular Ca(2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+CD45RA(+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene diphosphate (APCP. Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

  14. Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

    Science.gov (United States)

    Ruwanpura, Saleela M; McLeod, Louise; Dousha, Lovisa F; Seow, Huei J; Alhayyani, Sultan; Tate, Michelle D; Deswaerte, Virginie; Brooks, Gavin D; Bozinovski, Steven; MacDonald, Martin; Garbers, Christoph; King, Paul T; Bardin, Philip G; Vlahos, Ross; Rose-John, Stefan; Anderson, Gary P; Jenkins, Brendan J

    2016-12-15

    The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. We used the gp130 F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

  15. Targeting Signaling to YAP for the Therapy of NF2

    Science.gov (United States)

    2016-12-01

    nucleus, thus activating YAP-driven transcription and oncogenesis. Genetic epistasis experiments provided evidence that this oncogenic pathway...which loss of Merlin activates mitogenic signaling by using somatic cell genetics and biochemistry. In addition, we have initiated high-throughput...which represents the ultimate long term goal of this grant. Although our studies have identified a critical role for nuclear Merlin in inhibition of

  16. Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

    Energy Technology Data Exchange (ETDEWEB)

    Velho, Albertina M. [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); Jarvis, Simon M., E-mail: S.M.Jarvis@westminster.ac.uk [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); University of Westminster, School of Biosciences, London W1W 6UW (United Kingdom)

    2009-08-01

    The Na{sup +}-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 ({approx} 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated ({approx} 68-90 kDa) with only 31-65% of WT L-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered ({approx} 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at {approx} 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 {sup o}C or 37 {sup o}C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

  17. G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

    Science.gov (United States)

    O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

    2012-12-18

    Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

  18. Intracellular Wnt/Beta-Catenin Signaling Underlying 17beta-Estradiol-Induced Matrix Metalloproteinase 9 Expression in Human Endometriosis.

    Science.gov (United States)

    Zhang, Ling; Xiong, Wenqian; Xiong, Yao; Liu, Hengwei; Li, Na; Du, Yu; Liu, Yi

    2016-03-01

    Extracellular matrix remodeling is necessary for ectopic endometrium implantation. Many studies have shown an increased expression of matrix metalloproteinase 9 (MMP9) in the ectopic endometrium of endometriosis. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. The objective of our study was to investigate the association between MMP9 and the Wnt signaling pathway under the regulation of 17beta-estradiol (E2) in endometrial stromal cells. We found that MMP9 was elevated in tissues from women with endometriosis compared with normal women. Furthermore, MMP9 and beta-catenin increased concurrently in a time- and dose-dependent manner after E2 treatment. To clarify the relationship between MMP9 and beta-catenin, we performed luciferase promoter reporter and chromatin immunoprecipitation assays. A beta-catenin/TCF3/LEF1 complex bound to a specific site on the MMP9 promoter that promoted MMP9 gene and protein expression. The promotion of MMP9 by the Wnt signaling pathway under the regulation of E2 may contribute to the pathophysiology of this disease. © 2016 by the Society for the Study of Reproduction, Inc.

  19. Rac1 in human diseases: The therapeutic potential of targeting Rac1 signaling regulatory mechanisms.

    Science.gov (United States)

    Marei, Hadir; Malliri, Angeliki

    2017-07-03

    Abnormal Rac1 signaling is linked to a number of debilitating human diseases, including cancer, cardiovascular diseases and neurodegenerative disorders. As such, Rac1 represents an attractive therapeutic target, yet the search for effective Rac1 inhibitors is still underway. Given the adverse effects associated with Rac1 signaling perturbation, cells have evolved several mechanisms to ensure the tight regulation of Rac1 signaling. Thus, characterizing these mechanisms can provide invaluable information regarding major cellular events that lead to aberrant Rac1 signaling. Importantly, this information can be utilized to further facilitate the development of effective pharmacological modulators that can restore normal Rac1 signaling. In this review, we focus on the pathological role of Rac1 signaling, highlighting the benefits and potential drawbacks of targeting Rac1 in a clinical setting. Additionally, we provide an overview of available compounds that target key Rac1 regulatory mechanisms and discuss future therapeutic avenues arising from our understanding of these mechanisms.

  20. TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING.

    Directory of Open Access Journals (Sweden)

    Yanming Wang

    2015-06-01

    Full Text Available Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α was induced by herpes simplex virus type 1 (HSV-1 infection in dendritic cells (DCs. Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING, which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.

  1. Targeting Signaling to YAP for the Therapy of NF2

    Science.gov (United States)

    2015-10-01

    which encodes the FERM domain-containing protein Merlin. Children and young adults, who inherit an NF2 mutation, develop Schwannomas, usually of the...targeted therapy in the same way Chronic Myelogenous Leukemia is cured by Gleevec. The prospect of resistance is minimal, as Schwannoma cells do not seem to... treatment with Verteporfin, suggesting that this reporter system is not sensitive enough in physiopathologically relevant cell types. In parallel, it

  2. Direct targets of pSTAT5 signalling in erythropoiesis.

    Directory of Open Access Journals (Sweden)

    Kevin R Gillinder

    Full Text Available Erythropoietin (EPO acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25, epigenetic regulation (Suv420h2, and EpoR turnover (Clint1/EpsinR. Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

  3. AMP Affects Intracellular Ca2+ Signaling, Migration, Cytokine Secretion and T Cell Priming Capacity of Dendritic Cells

    Science.gov (United States)

    Panther, Elisabeth; Dürk, Thorsten; Ferrari, Davide; Di Virgilio, Francesco; Grimm, Melanie; Sorichter, Stephan; Cicko, Sanja; Herouy, Yared; Norgauer, Johannes; Idzko, Marco; Müller, Tobias

    2012-01-01

    The nucleotide adenosine-5′-monophosphate (AMP) can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A1 and A2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC) function. AMP increased intracellular Ca2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4+CD45RA+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5′-(alpha,beta-methylene) diphosphate (APCP). Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA) was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders. PMID:22624049

  4. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    OpenAIRE

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian; McBride, Jere W.

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E.?chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E.?chaffeensis, via the TRP120 effector, activat...

  5. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

    Science.gov (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

    2017-01-01

    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  6. Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

    DEFF Research Database (Denmark)

    Argetsinger, L S; Norstedt, G; Billestrup, Nils

    1996-01-01

    In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately...... for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner...

  7. Cancer Cell Signaling Pathways Targeted by Spice-Derived Nutraceuticals

    Science.gov (United States)

    Sung, Bokyung; Prasad, Sahdeo; Yadav, Vivek R.; Aggarwal, Bharat B.

    2012-01-01

    Extensive research within the last half a century has revealed that cancer is caused by dysregulation of as many as 500 different gene products. Most natural products target multiple gene products and thus are ideally suited for prevention and treatment of various chronic diseases, including cancer. Dietary agents such as spices have been used extensively in the Eastern world for a variety of ailments for millennia, and five centuries ago they took a golden journey to the Western world. Various spice-derived nutraceuticals, including 1′-acetoxychavicol acetate, anethole, capsaicin, car-damonin, curcumin, dibenzoylmethane, diosgenin, eugenol, gambogic acid, gingerol, thymoquinone, ursolic acid, xanthohumol, and zerumbone derived from galangal, anise, red chili, black cardamom, turmeric, licorice, fenugreek, clove, kokum, ginger, black cumin, rosemary, hop, and pinecone ginger, respectively, are the focus of this review. The modulation of various transcription factors, growth factors, protein kinases, and inflammatory mediators by these spice-derived nutraceuticals are described. The anticancer potential through the modulation of various targets is also the subject of this review. Although they have always been used to improve taste and color and as a preservative, they are now also used for prevention and treatment of a wide variety of chronic inflammatory diseases, including cancer. PMID:22149093

  8. MicroRNA 128a increases intracellular ROS level by targeting Bmi-1 and inhibits medulloblastoma cancer cell growth by promoting senescence.

    Directory of Open Access Journals (Sweden)

    Sujatha Venkataraman

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes, and thereby can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the expression of microRNAs in medulloblastoma and then investigated the functional role of one specific one, miR-128a, in regulating medulloblastoma cell growth. We found that many microRNAs associated with normal neuronal differentiation are significantly down regulated in medulloblastoma. One of these, miR-128a, inhibits growth of medulloblastoma cells by targeting the Bmi-1 oncogene. In addition, miR-128a alters the intracellular redox state of the tumor cells and promotes cellular senescence. CONCLUSIONS AND SIGNIFICANCE: Here we report the novel regulation of reactive oxygen species (ROS by microRNA 128a via the specific inhibition of the Bmi-1 oncogene. We demonstrate that miR-128a has growth suppressive activity in medulloblastoma and that this activity is partially mediated by targeting Bmi-1. This data has implications for the modulation of redox states in cancer stem cells, which are thought to be resistant to therapy due to their low ROS states.

  9. Target sites for chemical regulation of strigolactone signaling

    Directory of Open Access Journals (Sweden)

    Hidemitsu eNakamura

    2014-11-01

    Full Text Available Demands for plant growth regulators (chemicals that control plant growth are increasing globally, especially in developing countries. Both positive and negative plant growth regulators are widely used to enhance crop production and to suppress unwanted shoot growth, respectively. Strigolactones (SLs are multifunctional molecules that function as phytohormones, inhibiting shoot branching and also functioning in the rhizospheric communication with symbiotic fungi and parasitic weeds. Therefore, it is anticipated that chemicals that regulate the functions of SLs will be widely used in agricultural applications. Although the SL biosynthetic pathway is not fully understood, it has been demonstrated that beta-carotene isomerases, carotenoid cleavage dioxygenases (CCDs, and a cytochrome P450 monooxygenase are involved in strigolactone biosynthesis. A CCD inhibitor, abamine, which is also an inhibitor of abscisic acid biosynthesis, reduces the levels of SL in several plant species and reduces the germination rate of Orobanche minor seeds grown with tobacco. On the basis of the structure of abamine, several chemicals have been designed to specifically inhibit CCDs during SL synthesis. Cytochrome P450 monooxygenase is another target enzyme in the development of SL biosynthesis inhibitors, and the triazole-derived TIS series of chemicals is known to include SL biosynthesis inhibitors, although their target enzyme has not been identified. Recently, DWARF14 (D14 has been shown to be a receptor for SLs, and the D-ring moiety of SL is essential for its recognition by D14. A variety of SL agonists are currently under development and most agonists commonly contain the D-ring or a D-ring-like moiety. Several research groups have also resolved the crystal structure of D14 in the last two years. It is expected that this information on the D14 structure will be invaluable not only for developing SL agonists with novel structures but also in the design of inhibitors

  10. Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

    Energy Technology Data Exchange (ETDEWEB)

    Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

    2015-06-12

    The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

  11. Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

    Directory of Open Access Journals (Sweden)

    Li Wang

    2016-12-01

    Full Text Available Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR, detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular

  12. Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

    Science.gov (United States)

    Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

  13. Targeting FLT3 Signaling in Childhood Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Amy N. Sexauer

    2017-11-01

    Full Text Available Acute myeloid leukemia (AML is the second most common leukemia of childhood and is associated with high rates of chemotherapy resistance and relapse. Clinical outcomes for children with AML treated with maximally intensive multi-agent chemotherapy lag far behind those of children with the more common acute lymphoblastic leukemia, demonstrating continued need for new therapeutic approaches to decrease relapse risk and improve long-term survival. Mutations in the FMS-like tyrosine kinase-3 receptor gene (FLT3 occur in approximately 25% of children and adults with AML and are associated with particularly poor prognoses. Identification and development of targeted FLT3 inhibitors represents a major precision medicine paradigm shift in the treatment of patients with AML. While further development of many first-generation FLT3 inhibitors was hampered by limited potency and significant toxicity due to effects upon other kinases, the more selective second- and third-generation FLT3 inhibitors have demonstrated excellent tolerability and remarkable efficacy in the relapsed/refractory and now de novo FLT3-mutated AML settings. While these newest and most promising inhibitors have largely been studied in the adult population, pediatric investigation of FLT3 inhibitors with chemotherapy is relatively recently ongoing or planned. Successful development of FLT3 inhibitor-based therapies will be essential to improve outcomes in children with this high-risk subtype of AML.

  14. Participation of intercellular communication and intracellular signal transduction in the radio-adaptive response of human fibroblastic cells

    International Nuclear Information System (INIS)

    Ishii, Keiichiro; Hoshi, Yuko; Iwasaki, Toshiyasu; Watanabe, Masami

    1997-01-01

    To investigate the radio-adaptive response of normal cells to low-dose radiation, we irradiated human embryonic cells with low-dose X-rays and examined the changes in sensitivity to subsequent high-dose X-irradiation. When the cells were irradiated by 200 cGy, the growth ratio of the viable cells five days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a conditioning irradiation of 10 to 20 cGy four hours before the irradiation of 200 cGy, the growth ratio increased significantly to 45-53%, and a peak was reached at a conditioning dose of 13 cGy. Cells blocked off intercellular communication either in Ca 2+ ion-free medium or in TPA added medium during the conditioning irradiation of 13 cGy did not show the improvement of growth ratio. Addition of H-7, as an inhibitor of PKC, to the medium during the conditioning irradiation inhibited the induction of the radio-adaptive response. However, addition of either inhibitor of A kinase, H-89, or inhibitor of G kinase, H-8, failed to inhibit the induction of the radio-adaptive response. These results suggest that: (1) normal cells show an adaptive response to low-dose radiation, (2) intercellular communication may play a role in radio-adaptive responses, (3) the transduction of the signal induced in cells by low-dose X-irradiation via protein kinase C was involved in radio-adaptive responses, not via A kinase nor G kinase. (author)

  15. Zinc Signals and Immunity.

    Science.gov (United States)

    Maywald, Martina; Wessels, Inga; Rink, Lothar

    2017-10-24

    Zinc homeostasis is crucial for an adequate function of the immune system. Zinc deficiency as well as zinc excess result in severe disturbances in immune cell numbers and activities, which can result in increased susceptibility to infections and development of especially inflammatory diseases. This review focuses on the role of zinc in regulating intracellular signaling pathways in innate as well as adaptive immune cells. Main underlying molecular mechanisms and targets affected by altered zinc homeostasis, including kinases, caspases, phosphatases, and phosphodiesterases, will be highlighted in this article. In addition, the interplay of zinc homeostasis and the redox metabolism in affecting intracellular signaling will be emphasized. Key signaling pathways will be described in detail for the different cell types of the immune system. In this, effects of fast zinc flux, taking place within a few seconds to minutes will be distinguish from slower types of zinc signals, also designated as "zinc waves", and late homeostatic zinc signals regarding prolonged changes in intracellular zinc.

  16. Multifunctional pH-sensitive magnetic nanoparticles for simultaneous imaging, sensing and targeted intracellular anticancer drug delivery

    International Nuclear Information System (INIS)

    Banerjee, Shashwat S; Chen, D-H

    2008-01-01

    A novel multifunctional magnetic nanocarrier was fabricated for synchronous cancer therapy and sensing. The nanocarrier, programed to display a response to environmental stimuli (pH value), was synthesized by coupling doxorubicin (DOX) to adipic dihydrazide-grafted gum arabic modified magnetic nanoparticles (ADH-GAMNP) via the hydrolytically degradable pH-sensitive hydrazone bond. The resultant nanocarrier, DOX-ADH-GAMNP, had a mean diameter of 13.8 nm and the amount of DOX coupled was about 6.52 mg g -1 . Also, it exhibited pH triggered release of DOX in an acidic environment (pH 5.0) but was relatively stable at physiological pH (pH 7.4). Furthermore, both GAMNP and DOX were found to possess fluorescence properties when excited in the near-infrared region due to the two-photon absorption mechanism. The coupling of DOX to GAMNP resulted in a reversible self-quenching of fluorescence through the fluorescence resonant energy transfer (FRET) between the donor GAMNP and acceptor DOX. The release of DOX from DOX-ADH-GAMNP when exposed to acidic media indicated the recovery of fluorescence from both GAMNP and DOX. The change in the fluorescence intensity of DOX-ADH-GAMNP on the release of DOX can act as a potential sensor to sense the delivery of the drug. The analysis of zeta potential and plasmon absorbance in different pH conditions also confirmed the pH sensitivity of the product. This multifunctional nanocarrier is a significant breakthrough in developing a drug delivery vehicle that combines drug targeting as well as sensing and therapy at the same time.

  17. Intracellular targeting of mercaptoundecahydrododecaborate (BSH) to malignant glioma by transferrin-PEG liposomes for boron neutron capture therapy (BNCT)

    International Nuclear Information System (INIS)

    Doi, Atsushi; Miyatake, Shin-ichi; Iida, Kyouko

    2006-01-01

    Malignant glioma is one of the most difficult tumor to control with usual therapies. In our institute, we select boron neutron capture therapy (BNCT) as an adjuvant radiation therapy after surgical resection. This therapy requires the selective delivery of high concentration of 10 B to malignant tumor tissue. In this study, we focused on a tumor-targeting 10 B delivery system (BDS) for BNCT that uses transferrin-conjugated polyethylene-glycol liposome encapsulating BSH (TF-PEG liposome-BSH) and compared 10 B uptake of the tumor among BSH, PEG liposome-BSH and TF-PEG liposome-BSH. In vitro, we analyzed 10 B concentration of the cultured human U87Δ glioma cells incubated in medium containing 20 μg 10 B/ml derived from each BDS by inductively coupled plasma atomic emission spectrometry (ICP-AES). In vivo, human U87Δ glioma-bearing nude mice were administered with each BDS (35mg 10 B/kg) intravenously. We analyzed 10 B concentration of tumor, normal brain and blood by ICP-AES. The TF-PEG liposome-BSH showed higher absolute concentration more than the other BDS. Moreover, TF-PEG liposome-BSH decreased 10 B concentration in blood and normal tissue while it maintained high 10 B concentration in tumor tissue for a couple of days. This showed the TF-PEG liposome-BSH caused the selective delivery of high concentration of 10 B to malignant tumor tissue. The TF-PEG liposome-BSH is more potent BDS for BNCT to obtain absolute high 10 B concentration and good contrast between tumor and normal tissue than BSH and PEG liposome-BSH. (author)

  18. The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is important for pathogenesis and a target for vaccine development.

    Directory of Open Access Journals (Sweden)

    R van der Geize

    2011-08-01

    Full Text Available Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551, ipdB (rv3552, fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD and 3aα-H-4α(3'-propionic acid-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP. Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.

  19. Synergistic target combination prediction from curated signaling networks: Machine learning meets systems biology and pharmacology.

    Science.gov (United States)

    Chua, Huey Eng; Bhowmick, Sourav S; Tucker-Kellogg, Lisa

    2017-10-01

    Given a signaling network, the target combination prediction problem aims to predict efficacious and safe target combinations for combination therapy. State-of-the-art in silico methods use Monte Carlo simulated annealing (mcsa) to modify a candidate solution stochastically, and use the Metropolis criterion to accept or reject the proposed modifications. However, such stochastic modifications ignore the impact of the choice of targets and their activities on the combination's therapeutic effect and off-target effects, which directly affect the solution quality. In this paper, we present mascot, a method that addresses this limitation by leveraging two additional heuristic criteria to minimize off-target effects and achieve synergy for candidate modification. Specifically, off-target effects measure the unintended response of a signaling network to the target combination and is often associated with toxicity. Synergy occurs when a pair of targets exerts effects that are greater than the sum of their individual effects, and is generally a beneficial strategy for maximizing effect while minimizing toxicity. mascot leverages on a machine learning-based target prioritization method which prioritizes potential targets in a given disease-associated network to select more effective targets (better therapeutic effect and/or lower off-target effects); and on Loewe additivity theory from pharmacology which assesses the non-additive effects in a combination drug treatment to select synergistic target activities. Our experimental study on two disease-related signaling networks demonstrates the superiority of mascot in comparison to existing approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. In vivo optical microprobe imaging for intracellular Ca2+ dynamics in response to dopaminergic signaling in deep brain evoked by cocaine

    Science.gov (United States)

    Luo, Zhongchi; Pan, Yingtian; Du, Congwu

    2012-02-01

    Ca2+ plays a vital role as second messenger in signal transduction and the intracellular Ca2+ ([Ca2+]i) change is an important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum at >3mm under the brain surface) and image [Ca2+]i changes with cellular resolutions. Here, we present two micro-probe approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of neuronal [Ca2+]i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca2+]i dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca2+]i changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their different mean [Ca2+]i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition, microlens images can characterize the different [Ca2+]i dynamics of D1 and D2 neurons in response to cocaine, including new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.

  1. The effect of pulsed electric fields on the electrotactic migration of human neural progenitor cells through the involvement of intracellular calcium signaling.

    Science.gov (United States)

    Hayashi, Hisamitsu; Edin, Fredrik; Li, Hao; Liu, Wei; Rask-Andersen, Helge

    2016-12-01

    Endogenous electric fields (EFs) are required for the physiological control of the central nervous system development. Application of the direct current EFs to neural stem cells has been studied for the possibility of stem cell transplantation as one of the therapies for brain injury. EFs generated within the nervous system are often associated with action potentials and synaptic activity, apparently resulting in a pulsed current in nature. The aim of this study is to investigate the effect of pulsed EF, which can reduce the cytotoxicity, on the migration of human neural progenitor cells (hNPCs). We applied the mono-directional pulsed EF with a strength of 250mV/mm to hNPCs for 6h. The migration distance of the hNPCs exposed to pulsed EF was significantly greater compared with the control not exposed to the EF. Pulsed EFs, however, had less of an effect on the migration of the differentiated hNPCs. There was no significant change in the survival of hNPCs after exposure to the pulsed EF. To investigate the role of Ca 2+ signaling in electrotactic migration of hNPCs, pharmacological inhibition of Ca 2+ channels in the EF-exposed cells revealed that the electrotactic migration of hNPCs exposed to Ca 2+ channel blockers was significantly lower compared to the control group. The findings suggest that the pulsed EF induced migration of hNPCs is partly influenced by intracellular Ca 2+ signaling. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics

    Directory of Open Access Journals (Sweden)

    Krakstad Camilla

    2010-06-01

    Full Text Available Abstract Glioblastoma multiforme (GBM is the most common primary brain tumour in adults and one of the most aggressive cancers in man. Despite technological advances in surgical management, combined regimens of radiotherapy with new generation chemotherapy, the median survival for these patients is 14.6 months. This is largely due to a highly deregulated tumour genome with opportunistic deletion of tumour suppressor genes, amplification and/or mutational hyper-activation of receptor tyrosine kinase receptors. The net result of these genetic changes is augmented survival pathways and systematic defects in the apoptosis signalling machinery. The only randomised, controlled phase II trial conducted targeting the epidermal growth factor receptor (EGFR signalling with the small molecule inhibitor, erlotinib, has showed no therapeutic benefit. Survival signalling and apoptosis resistance in GBMs can be viewed as two sides of the same coin. Targeting increased survival is unlikely to be efficacious without at the same time targeting apoptosis resistance. We have critically reviewed the literature regarding survival and apoptosis signalling in GBM, and highlighted experimental, preclinical and recent clinical trials attempting to target these pathways. Combined therapies simultaneously targeting apoptosis and survival signalling defects might shift the balance from tumour growth stasis to cytotoxic therapeutic responses that might be associated with greater therapeutic benefits.

  3. Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique

    International Nuclear Information System (INIS)

    Lamprecht, C; Unterauer, B; Plochberger, B; Brameshuber, M; Hinterdorfer, P; Ebner, A; Gierlinger, N; Hild, S; Heister, E

    2012-01-01

    The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs’ G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized. (paper)

  4. Intracellular Signalling in Retinal Ischemia

    Science.gov (United States)

    1990-07-01

    spatial buffering capacity of the Miller cells of axolotl retina indicated that gap junctional coupling can increase the capacity of the Muller cell to...1974. 27 33. Mobbs P, Brew H, and Atwell D: A quantitative analysis of glial cell coupling in the retina of the axolotl (Ambystoma mexicanum). Brain

  5. Fractal properties of background noise and target signal enhancement using CSEM data

    Science.gov (United States)

    Benavides, Alfonso; Everett, Mark E.; Pierce, Carl; Nguyen, Cam

    2003-09-01

    Controlled-source electromagnetic (CSEM) spatial profiles and 2-D conductivity maps were obtained on the Brazos Valley, TX floodplain to study the fractal statistics of geological signals and effects of man-made conductive targets using Geonics EM34, EM31 and EM63. Using target-free areas, a consistent power-law power spectrum (|A(k)| ~ k ^-β) for the profiles was found with β values typical of fractional Brownian motion (fBm). This means that the spatial variation of conductivity does not correspond to Gaussian statistics, where there are spatial correlations at different scales. The presence of targets tends to flatten the power-law power spectrum (PS) at small wavenumbers. Detection and localization of targets can be achieved using short-time Fourier transform (STFT). The presence of targets is enhanced because the signal energy is spread to higher wavenumbers (small scale numbers) in the positions occupied by the targets. In the case of poor spatial sampling or small amount of data, the information available from the power spectrum is not enough to separate spatial correlations from target signatures. Advantages are gained by using the spatial correlations of the fBm in order to reject the background response, and to enhance the signals from highly conductive targets. This approach was tested for the EM31 using a pre-processing step that combines apparent conductivity readings from two perpendicular transmitter-receiver orientations at each station. The response obtained using time-domain CSEM is influence to a lesser degree by geological noise and the target response can be processed to recover target features. The homotopy method is proposed to solve the inverse problem using a set of possible target models and a dynamic library of responses used to optimize the starting model.

  6. Intracellular Targeting of CEA Results in Th1-Type Antibody Responses Following Intradermal Genetic Vaccination by a Needle-Free Jet Injection Device

    Directory of Open Access Journals (Sweden)

    Susanne Johansson

    2007-01-01

    Full Text Available The route and method of immunization, as well as the cellular localization of the antigen, can influence the generation of an immune response. In general, intramuscular immunization results in Th1 responses, whereas intradermal delivery of DNA by gene gun immunization often results in more Th2 responses. Here we investigate how altering the cellular localization of the tumor antigen CEA (carcinoembryonic antigen affects the quality and amplitude of DNA vaccine-induced antibody responses in mice following intradermal delivery of DNA by a needle-free jet injection device (Biojector. CEA was expressed either in a membrane-bound form (wild-type CEA or in two truncated forms (CEA6 and CEA66 with cytoplasmic localization, where CEA66 was fused to a promiscuous T-helper epitope from tetanus toxin. Repeated intradermal immunization of BALB/c mice with DNA encoding wild-type CEA produced high antibody titers of a mixed IgG1/IgG2a ratio. In contrast, utilizing the DNA construct that resulted in intracellular targeting of CEA led to a reduced capacity to induce CEA-specific antibodies, but instead induced a Th1-biased immune response.

  7. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    Science.gov (United States)

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  9. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

    2014-02-26

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

  10. Targeting GPCR-Gβγ-GRK2 signaling as a novel strategy for treating cardiorenal pathologies.

    Science.gov (United States)

    Rudomanova, Valeria; Blaxall, Burns C

    2017-08-01

    The pathologic crosstalk between the heart and kidney is known as cardiorenal syndrome (CRS). While the specific mechanisms underlying this crosstalk remain poorly understood, CRS is associated with exacerbated dysfunction of either or both organs and reduced survival. Maladaptive fibrotic remodeling is a key component of both heart and kidney failure pathogenesis and progression. G-protein coupled receptor (GPCR) signaling is a crucial regulator of cardiovascular and renal function. Chronic/pathologic GPCR signaling elicits the interaction of the G-protein Gβγ subunit with GPCR kinase 2 (GRK2), targeting the receptor for internalization, scaffolding to pathologic signals, and receptor degradation. Targeting this pathologic Gβγ-GRK2 interaction has been suggested as a possible strategy for the treatment of HF. In the current review, we discuss recent updates in understanding the role of GPCR-Gβγ-GRK2 signaling as a crucial mediator of maladaptive organ remodeling detected in HF and kidney dysfunction, with specific attention to small molecule-mediated inhibition of pathologic Gβγ-GRK2 interactions. Further, we explore the potential of GPCR-Gβγ-GRK2 signaling as a possible therapeutic target for cardiorenal pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Overlapping activities of TGF-β and Hedgehog signaling in cancer: therapeutic targets for cancer treatment.

    Science.gov (United States)

    Perrot, Carole Y; Javelaud, Delphine; Mauviel, Alain

    2013-02-01

    Recent advances in the field of cancer therapeutics come from the development of drugs that specifically recognize validated oncogenic or pro-metastatic targets. The latter may be mutated proteins with altered function, such as kinases that become constitutively active, or critical components of growth factor signaling pathways, whose deregulation leads to aberrant malignant cell proliferation and dissemination to metastatic sites. We herein focus on the description of the overlapping activities of two important developmental pathways often exacerbated in cancer, namely Transforming Growth Factor-β (TGF-β) and Hedgehog (HH) signaling, with a special emphasis on the unifying oncogenic role played by GLI1/2 transcription factors. The latter are the main effectors of the canonical HH pathway, yet are direct target genes of TGF-β/SMAD signal transduction. While tumor-suppressor in healthy and pre-malignant tissues, TGF-β is often expressed at high levels in tumors and contributes to tumor growth, escape from immune surveillance, invasion and metastasis. HH signaling regulates cell proliferation, differentiation and apoptosis, and aberrant HH signaling is found in a variety of cancers. We discuss the current knowledge on HH and TGF-β implication in cancer including cancer stem cell biology, as well as the current state, both successes and failures, of targeted therapeutics aimed at blocking either of these pathways in the pre-clinical and clinical settings. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yelin [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Hu, Chen; Cheng, Jun [Department of Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Chen, Binquan [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Ke, Qinghong; Lv, Zhen; Wu, Jian [Department of Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China); Zhou, Yanfeng, E-mail: zyfhdj@yahoo.com [Department of Anesthesiology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou (China)

    2014-04-18

    Highlights: • MiR-145 expression is down-regulated in HCC tissues and inversely related with IRS1 levels. • MiR-145 directly targets IRS1 in HCC cells. • Restored expression of miR-145 suppressed HCC cell proliferation and growth. • MiR-145 induced IRS1 under-expression potentially reduced downstream AKT signaling. - Abstract: Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

  13. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B

    2013-01-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We...

  14. Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p

    NARCIS (Netherlands)

    Boteva, R.; Koek, A.; Visser, N.V.; Visser, A.J.W.G.; Krieger, E.; Zlateva, T.; Veenhuis, M.; Klei, van der I.

    2003-01-01

    Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We

  15. Homogeneous electrochemical aptamer-based ATP assay with signal amplification by exonuclease III assisted target recycling.

    Science.gov (United States)

    Liu, Shufeng; Wang, Ying; Zhang, Chengxin; Lin, Ying; Li, Feng

    2013-03-21

    A novel and homogeneous electrochemical aptamer-based adenosine triphosphate (ATP) assay was demonstrated with signal amplification by exonuclease III-assisted target recycling. A superior detection limit of 1 nM toward ATP with an excellent selectivity could be achieved.

  16. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

    Directory of Open Access Journals (Sweden)

    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  17. Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family

    Science.gov (United States)

    Shepard, H. Michael; Brdlik, Cathleen M.; Schreiber, Hans

    2008-01-01

    The human EGFR (HER) family is essential for communication between many epithelial cancer cell types and the tumor microenvironment. Therapeutics targeting the HER family have demonstrated clinical success in the treatment of diverse epithelial cancers. Here we propose that the success of HER family–targeted monoclonal antibodies in cancer results from their ability to interfere with HER family consolidation of signals initiated by a multitude of other receptor systems. Ligand/receptor systems that initiate these signals include cytokine receptors, chemokine receptors, TLRs, GPCRs, and integrins. We further extrapolate that improvements in cancer therapeutics targeting the HER family are likely to incorporate mechanisms that block or reverse stromal support of malignant progression by isolating the HER family from autocrine and stromal influences. PMID:18982164

  18. Identification of the feedforward component in manual control with predictable target signals.

    Science.gov (United States)

    Drop, Frank M; Pool, Daan M; Damveld, Herman J; van Paassen, Marinus M; Mulder, Max

    2013-12-01

    In the manual control of a dynamic system, the human controller (HC) often follows a visible and predictable reference path. Compared with a purely feedback control strategy, performance can be improved by making use of this knowledge of the reference. The operator could effectively introduce feedforward control in conjunction with a feedback path to compensate for errors, as hypothesized in literature. However, feedforward behavior has never been identified from experimental data, nor have the hypothesized models been validated. This paper investigates human control behavior in pursuit tracking of a predictable reference signal while being perturbed by a quasi-random multisine disturbance signal. An experiment was done in which the relative strength of the target and disturbance signals were systematically varied. The anticipated changes in control behavior were studied by means of an ARX model analysis and by fitting three parametric HC models: two different feedback models and a combined feedforward and feedback model. The ARX analysis shows that the experiment participants employed control action on both the error and the target signal. The control action on the target was similar to the inverse of the system dynamics. Model fits show that this behavior can be modeled best by the combined feedforward and feedback model.

  19. Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats.

    Science.gov (United States)

    Hu, Min; Zhang, Yuehui; Feng, Jiaxing; Xu, Xue; Zhang, Jiao; Zhao, Wei; Guo, Xiaozhu; Li, Juan; Vestin, Edvin; Cui, Peng; Li, Xin; Wu, Xiao-Ke; Brännström, Mats; Shao, Linus R; Billig, Håkan

    2018-05-01

    Impaired progesterone (P4) signaling is linked to endometrial dysfunction and infertility in women with polycystic ovary syndrome (PCOS). Here, we report for the first time that elevated expression of progesterone receptor (PGR) isoforms A and B parallels increased estrogen receptor (ER) expression in PCOS-like rat uteri. The aberrant PGR-targeted gene expression in PCOS-like rats before and after implantation overlaps with dysregulated expression of Fkbp52 and Ncoa2 , two genes that contribute to the development of uterine P4 resistance. In vivo and in vitro studies of the effects of metformin on the regulation of the uterine P4 signaling pathway under PCOS conditions showed that metformin directly inhibits the expression of PGR and ER along with the regulation of several genes that are targeted dependently or independently of PGR-mediated uterine implantation. Functionally, metformin treatment corrected the abnormal expression of cell-specific PGR and ER and some PGR-target genes in PCOS-like rats with implantation. Additionally, we documented how metformin contributes to the regulation of the PGR-associated MAPK/ERK/p38 signaling pathway in the PCOS-like rat uterus. Our data provide novel insights into how metformin therapy regulates uterine P4 signaling molecules under PCOS conditions. © 2018 Society for Endocrinology.

  20. To fingolimod and beyond: The rich pipeline of drug candidates that target S1P signaling.

    Science.gov (United States)

    Chew, Wee Siong; Wang, Wei; Herr, Deron R

    2016-11-01

    Sphingosine 1-phosphate (S1P) is an extracellular lipid signaling molecule that acts as a selective, high-affinity ligand for a family of five G protein-coupled receptors. This signaling system was first identified twenty years ago, and has since been shown to regulate a diverse range of physiological processes and disease states, such as cardiovascular development, immune function, hypoxic responses, and cancer. The therapeutic potential of targeting this system took center stage when it was demonstrated that the immune modulator, fingolimod (FTY720/Gilenya), exerts it lymphopenic effect by acting on S1P receptors, primarily on S1P receptor 1 (S1P 1 ). In 2010, fingolimod became the first oral medication approved for the treatment of multiple sclerosis (MS). Since then, second-generation S1P receptor modulators have been under development in an effort to provide improved safety and efficacy profiles for MS, and to broaden their use to other autoimmune indications. Beyond the development of S1P 1 -modulators, there has been considerable effort in targeting other components of the S1P signaling pathway for the treatment of other diseases, such as cardiovascular disease, sepsis, and cancer. This manuscript provides an overview of the clinical and preclinical development of drugs targeting S1P signaling. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

    Science.gov (United States)

    Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

    2014-01-01

    The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

  2. GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation

    Directory of Open Access Journals (Sweden)

    Li Wang

    2018-01-01

    Full Text Available Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF signaling pathway in early embryoid bodies (EBs. Gcn5−/− EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.

  3. cGMP signaling as a target for the prevention and treatment of breast cancer.

    Science.gov (United States)

    Windham, Perrin F; Tinsley, Heather N

    2015-04-01

    One in eight women in the United States will be diagnosed with invasive breast cancer in her lifetime. Advances in therapeutic strategies, diagnosis, and improved awareness have resulted in a significant reduction in breast cancer related mortality. However, there is a continued need for more effective and less toxic drugs for both the prevention and the treatment of breast cancer in order to see a continued decline in the morbidity and mortality associated with this disease. Recent studies suggest that the cGMP signaling pathway may be aberrantly regulated in breast cancer. As such, this pathway may serve as a source of novel targets for future breast cancer drug discovery efforts. This review provides an overview of cGMP signaling in normal physiology and in breast cancer as well as current strategies being investigated for targeting this pathway in breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Lithium inhibits tumorigenic potential of PDA cells through targeting hedgehog-GLI signaling pathway.

    Directory of Open Access Journals (Sweden)

    Zhonglu Peng

    Full Text Available Hedgehog signaling pathway plays a critical role in the initiation and development of pancreatic ductal adenocarcinoma (PDA and represents an attractive target for PDA treatment. Lithium, a clinical mood stabilizer for mental disorders, potently inhibits the activity of glycogen synthase kinase 3β (GSK3β that promotes the ubiquitin-dependent proteasome degradation of GLI1, an important downstream component of hedgehog signaling. Herein, we report that lithium inhibits cell proliferation, blocks G1/S cell-cycle progression, induces cell apoptosis and suppresses tumorigenic potential of PDA cells through down-regulation of the expression and activity of GLI1. Moreover, lithium synergistically enhances the anti-cancer effect of gemcitabine. These findings further our knowledge of mechanisms of action for lithium and provide a potentially new therapeutic strategy for PDA through targeting GLI1.

  5. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    Science.gov (United States)

    Tolmachov, Oleg E

    2015-01-01

    Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

  6. Automatic detection of the unknown number point targets in FMICW radar signals

    Czech Academy of Sciences Publication Activity Database

    Rejfek, L.; Mošna, Zbyšek; Beran, L.; Fišer, O.; Dobrovolný, M.

    2017-01-01

    Roč. 4, č. 11 (2017), s. 116-120 ISSN 2313-626X R&D Projects: GA ČR(CZ) GA15-24688S Institutional support: RVO:68378289 Keywords : FMICW radar * 2D FFT * signal filtration * taraget detection * target parameter estimation Subject RIV: DG - Athmosphere Sciences, Meteorology OBOR OECD: Meteorology and atmospheric sciences http://science-gate.com/IJAAS/Articles/2017-4-11/18%202017-4-11-pp.116-120.pdf

  7. NMR signal analysis in the large COMPASS $^{14}$NH$_{3}$ target

    CERN Document Server

    Koivuniemi, J; Hess, C; Kisselev, Y U; Meyer, W; Radtke, E; Reicherz, G; Doshita, N; Iwata, T; Kondo, K; Michigami, T

    2009-01-01

    In the large COMPASS polarized proton target the 1508 cm$^{3}$ of irradiated granular ammonia is polarized with dynamic nuclear polarization method using 4 mm microwaves in 2.5 T eld. The nuclear polarization up to 90 - 93 % is determined with cw NMR. The properties of the observed ammonia proton signals are described and spin thermodynamics in high elds is presented. Also the second moment of the NMR line is estimated.

  8. Alteration of SHP-1/p-STAT3 Signaling: A Potential Target for Anticancer Therapy

    Directory of Open Access Journals (Sweden)

    Tzu-Ting Huang

    2017-06-01

    Full Text Available The Src homology 2 (SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1, a non-receptor protein tyrosine phosphatase, has been reported as a negative regulator of phosphorylated signal transducer and activator of transcription 3 (STAT3 and linked to tumor development. In this present review, we will discuss the importance and function of SHP-1/p-STAT3 signaling in nonmalignant conditions as well as malignancies, its cross-talk with other pathways, the current clinical development and the potential role of inhibitors of this pathway in anticancer therapy and clinical relevance of SHP-1/p-STAT3 in cancers. Lastly, we will summarize and highlight work involving novel drugs/compounds targeting SHP-1/p-STAT3 signaling and combined strategies that were/are discovered in our and our colleagues’ laboratories.

  9. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    2012-02-01

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  10. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-07-26

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  11. The antidepressant sertraline inhibits translation initiation by curtailing mammalian target of rapamycin signaling.

    Science.gov (United States)

    Lin, Chen-Ju; Robert, Francis; Sukarieh, Rami; Michnick, Stephen; Pelletier, Jerry

    2010-04-15

    Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner. (c) 2010 AACR.

  12. Monte Carlo Simulation of the Echo Signals from Low-Flying Targets for Airborne Radar

    Directory of Open Access Journals (Sweden)

    Mingyuan Man

    2014-01-01

    Full Text Available A demonstrated hybrid method based on the combination of half-space physical optics method (PO, graphical-electromagnetic computing (GRECO, and Monte Carlo method on echo signals from low-flying targets based on actual environment for airborne radar is presented in this paper. The half-space physical optics method , combined with the graphical-electromagnetic computing (GRECO method to eliminate the shadow regions quickly and rebuild the target automatically, is employed to calculate the radar cross section (RCS of the conductive targets in half space fast and accurately. The direct echo is computed based on the radar equation. The reflected paths from sea or ground surface cause multipath effects. In order to accurately obtain the echo signals, the phase factors are modified for fluctuations in multipath, and the statistical average value of the echo signals is obtained using the Monte Carlo method. A typical simulation is performed, and the numerical results show the accuracy of the proposed method.

  13. Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging.

    Science.gov (United States)

    Lankford, Miles; Behm, Carolyn Z; Yeh, James; Klibanov, Alexander L; Robinson, Peter; Lindner, Jonathan R

    2006-10-01

    Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

  14. Target of Rapamycin (TOR) Regulates Growth in Response to Nutritional Signals.

    Science.gov (United States)

    Weisman, Ronit

    2016-10-01

    All organisms can respond to the availability of nutrients by regulating their metabolism, growth, and cell division. Central to the regulation of growth in response to nutrient availability is the target of rapamycin (TOR) signaling that is composed of two structurally distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2). The TOR genes were first identified in yeast as target of rapamycin, a natural product of a soil bacterium, which proved beneficial as an immunosuppressive and anticancer drug and is currently being tested for a handful of other pathological conditions including diabetes, neurodegeneration, and age-related diseases. Studies of the TOR pathway unraveled a complex growth-regulating network. TOR regulates nutrient uptake, transcription, protein synthesis and degradation, as well as metabolic pathways, in a coordinated manner that ensures that cells grow or cease growth in response to nutrient availability. The identification of specific signals and mechanisms that stimulate TOR signaling is an active and exciting field of research that has already identified nitrogen and amino acids as key regulators of TORC1 activity. The signals, as well as the cellular functions of TORC2, are far less well understood. Additional open questions in the field concern the relationships between TORC1 and TORC2, as well as the links with other nutrient-responsive pathways. Here I review the main features of TORC1 and TORC2, with a particular focus on yeasts as model organisms.

  15. MicroRNA-99 family targets AKT/mTOR signaling pathway in dermal wound healing.

    Science.gov (United States)

    Jin, Yi; Tymen, Stéphanie D; Chen, Dan; Fang, Zong Juan; Zhao, Yan; Dragas, Dragan; Dai, Yang; Marucha, Phillip T; Zhou, Xiaofeng

    2013-01-01

    Recent studies suggest that microRNAs play important roles in dermal wound healing and microRNA deregulation has been linked with impaired wound repair. Here, using a mouse experimental wound healing model, we identified a panel of 63 differentially expressed microRNAs during dermal wound healing, including members of miR-99 family (miR-99a, miR-99b, miR-100). We further demonstrated that miR-99 family members regulate cell proliferation, cell migration, and AKT/mTOR signaling. Combined experimental and bioinformatics analyses revealed that miR-99 family members regulate AKT/mTOR signaling by targeting multiple genes, including known target genes (e.g., IGF1R, mTOR) and a new target (AKT1). The effects of miR-99 family members on the expression of IGF1R, mTOR and AKT1 were validated at both the mRNA and protein levels. Two adjacent miR-99 family targeting sites were identified in the 3'-UTR of the AKT1 mRNA. The direct interaction of miR-100 with these targeting sites was confirmed using luciferase reporter assays. The microRNA-100-directed recruitment of AKT1 mRNA to the RNAi-induced silencing complex (RISC) was confirmed by a ribonucleoprotein-IP assay. In summary, we identified a panel of differentially expressed microRNAs which may play important roles in wound healing. We provide evidence that miR-99 family members contribute to wound healing by regulating the AKT/mTOR signaling.

  16. MicroRNA expression, target genes, and signaling pathways in infants with a ventricular septal defect.

    Science.gov (United States)

    Chai, Hui; Yan, Zhaoyuan; Huang, Ke; Jiang, Yuanqing; Zhang, Lin

    2018-02-01

    This study aimed to systematically investigate the relationship between miRNA expression and the occurrence of ventricular septal defect (VSD), and characterize the miRNA target genes and pathways that can lead to VSD. The miRNAs that were differentially expressed in blood samples from VSD and normal infants were screened and validated by implementing miRNA microarrays and qRT-PCR. The target genes regulated by differentially expressed miRNAs were predicted using three target gene databases. The functions and signaling pathways of the target genes were enriched using the GO database and KEGG database, respectively. The transcription and protein expression of specific target genes in critical pathways were compared in the VSD and normal control groups using qRT-PCR and western blotting, respectively. Compared with the normal control group, the VSD group had 22 differentially expressed miRNAs; 19 were downregulated and three were upregulated. The 10,677 predicted target genes participated in many biological functions related to cardiac development and morphogenesis. Four target genes (mGLUR, Gq, PLC, and PKC) were involved in the PKC pathway and four (ECM, FAK, PI3 K, and PDK1) were involved in the PI3 K-Akt pathway. The transcription and protein expression of these eight target genes were significantly upregulated in the VSD group. The 22 miRNAs that were dysregulated in the VSD group were mainly downregulated, which may result in the dysregulation of several key genes and biological functions related to cardiac development. These effects could also be exerted via the upregulation of eight specific target genes, the subsequent over-activation of the PKC and PI3 K-Akt pathways, and the eventual abnormal cardiac development and VSD.

  17. MicroRNA-467g inhibits new bone regeneration by targeting Ihh/Runx-2 signaling.

    Science.gov (United States)

    Kureel, Jyoti; John, Aijaz A; Dixit, Manisha; Singh, Divya

    2017-04-01

    MicroRNAs are important post transcriptional regulators of gene expression and play critical role in osteoblast differentiation. In this study we report miR-467g, an uncharacterized novel miRNA, in regulation of osteoblast functions. Over-expression of miR-467g inhibited osteoblast differentiation. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay identified Runx-2 as a direct target of miR-467g. Over expression of miR-467g in osteoblasts down regulated Runx-2 and Ihh signaling components. Furthermore, silencing of miR-467g was done to see its role in Ihh and Runx-2 mediated bone healing and regeneration in a drill hole injury model in BALB/c mice. Silencing of miR-467g led to significant increase in new bone regeneration and Ihh and Runx-2 localization at injury site in a day dependent manner. In conclusion, miR-467g negatively regulates osteogenesis by targeting Ihh/Runx-2 signaling. We, thus, propose that therapeutic approaches targeting miR-467g could be useful in enhancing the new bone formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Cellular Signaling Pathway Alterations and Potential Targeted Therapies for Medullary Thyroid Carcinoma

    Directory of Open Access Journals (Sweden)

    Serena Giunti

    2013-01-01

    Full Text Available Parafollicular C-cell-derived medullary thyroid cancer (MTC comprises 3% to 4% of all thyroid cancers. While cytotoxic treatments have been shown to have limited efficacy, targeted molecular therapies that inhibit rearranged during transfection (RET and other tyrosine kinase receptors that are mainly involved in angiogenesis have shown great promise in the treatment of metastatic or locally advanced MTC. Multi-tyrosine kinase inhibitors such as vandetanib, which is already approved for the treatment of progressive MTC, and cabozantinib have shown distinct advantages with regard to rates of disease response and control. However, these types of tyrosine kinase inhibitor compounds are able to concurrently block several types of targets, which limits the understanding of RET as a specific target. Moreover, important resistances to tyrosine kinase inhibitors can occur, which limit the long-term efficacy of these treatments. Deregulated cellular signaling pathways and genetic alterations in MTC, particularly the activation of the RAS/mammalian target of rapamycin (mTOR cascades and RET crosstalk signaling, are now emerging as novel and potentially promising therapeutic treatments for aggressive MTC.

  19. Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

    Science.gov (United States)

    Putz, Eva M.; Guillerey, Camille; Kos, Kevin; Stannard, Kimberley; Miles, Kim; Delconte, Rebecca B.; Nicholson, Sandra E.; Huntington, Nicholas D.; Smyth, Mark J.

    2017-01-01

    ABSTRACT The cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy. PMID:28344878

  20. Targeting apoptosis signalling kinase-1 (ASK-1 does not prevent the development of neuropathy in streptozotocin-induced diabetic mice.

    Directory of Open Access Journals (Sweden)

    Victoria L Newton

    Full Text Available Apoptosis signal-regulating kinase-1 (ASK1 is a mitogen-activated protein 3 kinase (MAPKKK/MAP3K which lies upstream of the stress-activated MAPKs, JNK and p38. ASK1 may be activated by a variety of extracellular and intracellular stimuli. MAP kinase activation in the sensory nervous system as a result of diabetes has been shown in numerous preclinical and clinical studies. As a common upstream activator of both p38 and JNK, we hypothesised that activation of ASK1 contributes to nerve dysfunction in diabetic neuropathy. We therefore wanted to characterize the expression of ASK1 in sensory neurons, and determine whether the absence of functional ASK1 would protect against the development of neuropathy in a mouse model of experimental diabetes. ASK1 mRNA and protein is constitutively expressed by multiple populations of sensory neurons of the adult mouse lumbar DRG. Diabetes was induced in male C57BL/6 and transgenic ASK1 kinase-inactive (ASK1n mice using streptozotocin. Levels of ASK1 do not change in the DRG, spinal cord, or sciatic nerve following induction of diabetes. However, levels of ASK2 mRNA increase in the spinal cord at 4 weeks of diabetes, which could represent a future target for this field. Neither motor nerve conduction velocity deficits, nor thermal or mechanical hypoalgesia were prevented or ameliorated in diabetic ASK1n mice. These results suggest that activation of ASK1 is not responsible for the nerve deficits observed in this mouse model of diabetic neuropathy.

  1. The interaction of antimicrobial peptides with the membrane and intracellular targets of Staphylococcus aureus investigated by ATP leakage, DNA-binding analysis, and the expression of a LexA-controlled gene, recA

    DEFF Research Database (Denmark)

    Gottschalk, Sanne; Thomsen, Line Elnif

    2017-01-01

    The analysis of how antimicrobial peptides (AMPs) interact with bacterial membranes and intracellular targets is important for our understanding of how these molecules affect bacteria. Increased knowledge may aid the design of AMPs that work on their target bacterium without inducing bacterial...... resistance. Here, we describe different methods to investigate the mode of action of peptides against the Gram-positive bacterium Staphylococcus aureus. ATP leakage analysis can be used to evaluate the ability of AMPs to perturb bacteria. DNA-binding and SOS response induction can be analyzed to investigate...

  2. Identification of potential pathway mediation targets in Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Fan Li

    2009-02-01

    Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

  3. Sonic Hedgehog Signaling Drives Proliferation of Synoviocytes in Rheumatoid Arthritis: A Possible Novel Therapeutic Target

    Directory of Open Access Journals (Sweden)

    Mingxia Wang

    2014-01-01

    Full Text Available Sonic hedgehog (Shh signaling controls many aspects of human development, regulates cell growth and differentiation in adult tissues, and is activated in a number of malignancies. Rheumatoid arthritis (RA is characterized by chronic synovitis and pannus formation associated with activation of fibroblast-like synoviocytes (FLS. We investigated whether Shh signaling plays a role in the proliferation of FLS in RA. Expression of Shh signaling related components (Shh, Ptch1, Smo, and Gli1 in RA synovial tissues was examined by immunohistochemistry (IHC and in FLS by IHC, immunofluorescence (IF, quantitative RT-PCR, and western blotting. Expression of Shh, Smo, and Gli1 in RA synovial tissue was higher than that in control tissue (P<0.05. Cyclopamine (a specific inhibitor of Shh signaling decreased mRNA expression of Shh, Ptch1, Smo, and Gli1 in cultured RA FLS, Shh, and Smo protein expression, and significantly decreased FLS proliferation. Flow cytometry analysis suggested that cyclopamine treatment resulted in cell cycle arrest of FLS in G1 phase. Our data show that Shh signaling is activated in synovium of RA patients in vivo and in cultured FLS form RA patients in vitro, suggesting a role in the proliferation of FLS in RA. It may therefore be a novel therapeutic target in RA.

  4. MHC class I cross-talk with CD2 and CD28 induces specific intracellular signalling and leads to growth retardation and apoptosis via a p56(lck)-dependent mechanism

    DEFF Research Database (Denmark)

    Ruhwald, M; Pedersen, Anders Elm; Claesson, M H

    1999-01-01

    Ligation of the major histocompatibility complex class I molecules (MHC-I) on human T lymphoma cells (Jurkat) initiates p56(lck)-dependent intracellular signalling events (phosphotyrosine kinase activity; [Ca(2+)](i)) and leads to augmented growth inhibition and apoptosis. MHC-I ligation in concert...... of apoptosis. In parallel experiments with the p56(lck)-negative Jurkat mutant cell, JCaM1.6, cross-linking neither influenced cell signalling nor cellular growth functions, indicating a cardinal role of the src kinases in signal transduction via MHC-I, CD2 and CD28 molecules. The results presented here...... with ligation of CD2 or CD28 augments, changes or modifies the pattern of activation. Ligation of MHC-I and CD2 alone resulted in growth inhibition, whereas CD28 ligation alone had no effect on cell proliferation. Ligation of MHC-I together with CD2 augmented growth inhibition and enhanced the level...

  5. Targeting Sonic Hedgehog Signaling by Compounds and Derivatives from Natural Products

    Directory of Open Access Journals (Sweden)

    Yu-Chuen Huang

    2013-01-01

    Full Text Available Cancer stem cells (CSCs are a major cause of cancer treatment failure, relapse, and drug resistance and are known to be responsible for cancer cell invasion and metastasis. The Sonic hedgehog (Shh signaling pathway is crucial to embryonic development. Intriguingly, the aberrant activation of the Shh pathway plays critical roles in developing CSCs and leads to angiogenesis, migration, invasion, and metastasis. Natural compounds and chemical structure modified derivatives from complementary and alternative medicine have received increasing attention as cancer chemopreventives, and their antitumor effects have been demonstrated both in vitro and in vivo. However, reports for their bioactivity against CSCs and specifically targeting Shh signaling remain limited. In this review, we summarize investigations of the compounds cyclopamine, curcumin, epigallocatechin-3-gallate, genistein, resveratrol, zerumbone, norcantharidin, and arsenic trioxide, with a focus on Shh signaling blockade. Given that Shh signaling antagonism has been clinically proven as effective strategy against CSCs, this review may be exploitable for development of novel anticancer agents from complementary and alternative medicine.

  6. Targeting sTNF/TNFR1 Signaling as a New Therapeutic Strategy

    Directory of Open Access Journals (Sweden)

    Roman Fischer

    2015-03-01

    Full Text Available Deregulation of the tumor necrosis factor (TNF plays an important role in the initiation and perpetuation of chronic inflammation and has been implicated in the development of various autoimmune diseases. Accordingly, TNF-inhibitors are successfully used for the treatment of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, total inhibition of TNF can cause severe side effects such as an increased risk of inflammation and reactivation of tuberculosis. This is likely due to the different actions of the two TNF receptors. Whereas TNFR1 predominantly promotes inflammatory signaling pathways, TNFR2 mediates immune modulatory functions and promotes tissue homeostasis and regeneration. Therefore, the specific blockage of TNFR1 signaling, either by direct inhibition with TNFR1-selective antagonists or by targeting soluble TNF, which predominantly activates TNFR1, may prevent the detrimental effects associated with total TNF-inhibitors and constitute a next-generation approach to interfere with TNF.

  7. Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

    Directory of Open Access Journals (Sweden)

    William A. McEwan

    2016-11-01

    Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

  8. Magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway

    International Nuclear Information System (INIS)

    Lee, Cheol-Jung; Lee, Mee-Hyun; Yoo, Sun-Mi; Choi, Kyung-Il; Song, Ji-Hong; Jang, Jeong-Hoon; Oh, Sei-Ryang; Ryu, Hyung-Won; Lee, Hye-Suk; Surh, Young-Joon; Cho, Yong-Yeon

    2015-01-01

    Magnolin is a natural compound abundantly found in Magnolia flos, which has been traditionally used in oriental medicine to treat headaches, nasal congestion and anti-inflammatory reactions. Our recent results have demonstrated that magnolin targets the active pockets of ERK1 and ERK2, which are important signaling molecules in cancer cell metastasis. The aim of this study is to evaluate the effects of magnolin on cell migration and to further explore the molecular mechanisms involved. Magnolin-mediated signaling inhibition was confirmed by Western blotting using RSK2 +/+ and RSK2 −/− MEFs, A549 and NCI-H1975 lung cancer cells, and by NF-κB and Cox-2 promoter luciferase reporter assays. Inhibition of cell migration by magnolin was examined by wound healing and/or Boyden Chamber assays using JB6 Cl41 and A549 human lung cancer cells. The molecular mechanisms involved in cell migration and epithelial-to-mesenchymal transition were determined by zymography, Western blotting, real-time PCR and immunocytofluorescence. Magnolin inhibited NF-κB transactivation activity by suppressing the ERKs/RSK2 signaling pathway. Moreover, magnolin abrogated the increase in EGF-induced COX-2 protein levels and wound healing. In human lung cancer cells such as A549 and NCI-H1975, which harbor constitutive active Ras and EGFR mutants, respectively, magnolin suppressed wound healing and cell invasion as seen by a Boyden chamber assay. In addition, it was observed that magnolin inhibited MMP-2 and −9 gene expression and activity. The knockdown or knockout of RSK2 in A549 lung cancer cells or MEFs revealed that magnolin targeting ERKs/RSK2 signaling suppressed epithelial-to-mesenchymal transition by modulating EMT marker proteins such as N-cadherin, E-cadherin, Snail, Vimentin and MMPs. These results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway. The online version of this article (doi:10.1186/s12885-015-1580-7) contains

  9. Targeting dysfunctional beta-cell signaling for the potential treatment of type 1 diabetes mellitus.

    Science.gov (United States)

    Fenske, Rachel J; Kimple, Michelle E

    2018-03-01

    Since its discovery and purification by Frederick Banting in 1921, exogenous insulin has remained almost the sole therapy for type 1 diabetes mellitus. While insulin alleviates the primary dysfunction of the disease, many other aspects of the pathophysiology of type 1 diabetes mellitus are unaffected. Research aimed towards the discovery of novel type 1 diabetes mellitus therapeutics targeting different cell signaling pathways is gaining momentum. The focus of these efforts has been almost entirely on the impact of immunomodulatory drugs, particularly those that have already received FDA-approval for other autoimmune diseases. However, these drugs can often have severe side effects, while also putting already immunocompromised individuals at an increased risk for other infections. Potential therapeutic targets in the insulin-producing beta-cell have been largely ignored by the type 1 diabetes mellitus field, save the glucagon-like peptide 1 receptor. While there is preliminary evidence to support the clinical exploration of glucagon-like peptide 1 receptor-based drugs as type 1 diabetes mellitus adjuvant therapeutics, there is a vast space for other putative therapeutic targets to be explored. The alpha subunit of the heterotrimeric G z protein (Gα z ) has been shown to promote beta-cell inflammation, dysfunction, death, and failure to replicate in the context of diabetes in a number of mouse models. Genetic loss of Gα z or inhibition of the Gα z signaling pathway through dietary interventions is protective against the development of insulitis and hyperglycemia. The multifaceted effects of Gα z in regards to beta-cell health in the context of diabetes make it an ideal therapeutic target for further study. It is our belief that a low-risk, effective therapy for type 1 diabetes mellitus will involve a multidimensional approach targeting a number of regulatory systems, not the least of which is the insulin-producing beta-cell. Impact statement The expanding

  10. Pacific ciguatoxin-1b effect over Na+ and K+ currents, inositol 1,4,5-triphosphate content and intracellular Ca2+ signals in cultured rat myotubes

    Science.gov (United States)

    Hidalgo, Jorge; Liberona, José Luis; Molgó, Jordi; Jaimovich, Enrique

    2002-01-01

    The action of the main ciguatoxin involved in ciguatera fish poisoning in the Pacific region (P-CTX-1b) was studied in myotubes originated from rat skeletal muscle cells kept in primary culture. The effect of P-CTX-1b on sodium currents at short times of exposure (up to 1 min) showed a moderate increase in peak Na+ current. During prolonged exposures, P-CTX-1b decreased the peak Na+ current. This action was always accompanied by an increase of leakage currents, tail currents and outward Na+ currents, resulting in an intracellular Na+ accumulation. This effect is blocked by prior exposure to tetrodotoxin (TTX) and becomes evident only after washout of TTX. Low to moderate concentrations of P-CTX-1b (2–5 nM) partially blocked potassium currents in a manner that was dependent on the membrane potential. P-CTX-1b (2–12 nM) caused a small membrane depolarization (3–5 mV) and an increase in the frequency of spontaneous action potential discharges that reached in general low frequencies (0.1–0.5 Hz). P-CTX-1b (10 nM) caused a transient increase of intracellular inositol 1,4,5-trisphosphate (IP3) mass levels, which was blocked by TTX. In the presence of P-CTX-1b (10 nM) and in the absence of external Ca2+, the intracellular Ca2+ levels show a transient increase in the cytoplasm as well as in the nuclei. The time course of this effect may reflect the action of IP3 over internal stores activated by P-CTX-1b-induced membrane depolarization. PMID:12429578

  11. Rapamycin and Glucose-Target of Rapamycin (TOR) Protein Signaling in Plants*

    Science.gov (United States)

    Xiong, Yan; Sheen, Jen

    2012-01-01

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs. PMID:22134914

  12. Target-assistant Zn2+-dependent DNAzyme for signal-on electrochemiluminescent biosensing

    International Nuclear Information System (INIS)

    Huang, Yin; Lei, Jianping; Cheng, Yan; Ju, Huangxian

    2015-01-01

    Highlights: • The sensing strategy is based on cleavage reaction of target-assistant Zn 2+ -dependent DNAzyme. • A dual quenching mechanism of ECL is identified. • A sensitive and selective ECL sensor is constructed for detection of ATP. • The biosensor can detect ATP in serum samples with good accuracy. - Abstract: A signal-on electrochemiluminescent (ECL) approach for ultrasensitive ATP detection was developed using target-assistant Zn 2+ -dependent DNAzyme via a dual quenching pathway between quantum dots (QDs) and Au nanoclusters (Au NCs). The facile ECL biosensor was constructed by covalent assembly of Au NCs-labeled hairpin DNA on QDs modified glassy carbon electrode. A dual quenching ECL mechanism was identified to be via resonance energy transfer between QDs and Au NCs and electrocatalytic reduction of coreactant oxygen by Au NCs. With the assistance of two help DNAs, the G-quadruplex structure of ATP aptamer was formed, and thus narrowed the two fragments of Zn 2+ -dependent DNAzyme. In the presence of Zn 2+ , Zn 2+ -dependent DNAzyme can be generated in situ on the biosensor's surface. The as-prepared DNAzyme can cleave the substrate strand, and release the Au NCs from the electrode, resulting in the signal-on ECL state. This biosensor showed good analytical performance with 4 orders magnitude linear range, excellent specificity, and acceptable stability. The biosensor had been applied in detection of ATP in real serum sample and provided significant potential application in clinical analysis

  13. Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers

    Science.gov (United States)

    Ricketts, Christopher J.; Wei, Darmood; Yang, Youfeng; Baranes, Sarah M.; Gibbs, Benjamin K.; Ohanjanian, Lernik; Spencer Krane, L.; Scroggins, Bradley T.; Keith Killian, J.; Wei, Ming-Hui; Kijima, Toshiki; Meltzer, Paul S.; Citrin, Deborah E.; Neckers, Len; Vocke, Cathy D.; Marston Linehan, W.

    2018-01-01

    Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15–20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC. PMID:29535838

  14. New Challenges in Targeting Signaling Pathways in Acute Lymphoblastic Leukemia by NGS Approaches: An Update

    Science.gov (United States)

    Hernández-Rivas, Jesús María

    2018-01-01

    The identification and study of genetic alterations involved in various signaling pathways associated with the pathogenesis of acute lymphoblastic leukemia (ALL) and the application of recent next-generation sequencing (NGS) in the identification of these lesions not only broaden our understanding of the involvement of various genetic alterations in the pathogenesis of the disease but also identify new therapeutic targets for future clinical trials. The present review describes the main deletions, amplifications, sequence mutations, epigenetic lesions, and new structural DNA rearrangements detected by NGS in B-ALL and T-ALL and their clinical importance for therapeutic procedures. We reviewed the molecular basis of pathways including transcriptional regulation, lymphoid differentiation and development, TP53 and the cell cycle, RAS signaling, JAK/STAT, NOTCH, PI3K/AKT/mTOR, Wnt/β-catenin signaling, chromatin structure modifiers, and epigenetic regulators. The implementation of NGS strategies has enabled important mutated genes in each pathway, their associations with the genetic subtypes of ALL, and their outcomes, which will be described further. We also discuss classic and new cryptic DNA rearrangements in ALL identified by mRNA-seq strategies. Novel cooperative abnormalities in ALL could be key prognostic and/or predictive biomarkers for selecting the best frontline treatment and for developing therapies after the first relapse or refractory disease. PMID:29642462

  15. Monoglyceride lipase as a drug target: At the crossroads of arachidonic acid metabolism and endocannabinoid signaling.

    Science.gov (United States)

    Grabner, Gernot F; Zimmermann, Robert; Schicho, Rudolf; Taschler, Ulrike

    2017-07-01

    Monoglyerides (MGs) are short-lived, intermediary lipids deriving from the degradation of phospho- and neutral lipids, and monoglyceride lipase (MGL), also designated as monoacylglycerol lipase (MAGL), is the major enzyme catalyzing the hydrolysis of MGs into glycerol and fatty acids. This distinct function enables MGL to regulate a number of physiological and pathophysiological processes since both MGs and fatty acids can act as signaling lipids or precursors thereof. The most prominent MG species acting as signaling lipid is 2-arachidonoyl glycerol (2-AG) which is the most abundant endogenous agonist of cannabinoid receptors in the body. Importantly, recent observations demonstrate that 2-AG represents a quantitatively important source for arachidonic acid, the precursor of prostaglandins and other inflammatory mediators. Accordingly, MGL-mediated 2-AG degradation affects lipid signaling by cannabinoid receptor-dependent and independent mechanisms. Recent genetic and pharmacological studies gave important insights into MGL's role in (patho-)physiological processes, and the enzyme is now considered as a promising drug target for a number of disorders including cancer, neurodegenerative and inflammatory diseases. This review summarizes the basics of MG (2-AG) metabolism and provides an overview on the therapeutic potential of MGL. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity

    KAUST Repository

    Othman, Basmah A.

    2016-04-01

    ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn2+ concentration prior to cell death. The response of the cells within the population to intracellular Zn2+ is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.

  17. Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity

    KAUST Repository

    Othman, Basmah A.; Greenwood, Christina; AbuElela, Ayman; Bharath, Anil A.; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen; Porter, Alexandra E.

    2016-01-01

    ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn2+ concentration prior to cell death. The response of the cells within the population to intracellular Zn2+ is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.

  18. Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy.

    Science.gov (United States)

    Pasquier, Florence; Cabagnols, Xenia; Secardin, Lise; Plo, Isabelle; Vainchenker, William

    2014-09-01

    The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review. Copyright © 2014

  19. Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.

    Science.gov (United States)

    Zhang, Xu; Belkina, Natalya; Jacob, Harrys Kishore Charles; Maity, Tapan; Biswas, Romi; Venugopalan, Abhilash; Shaw, Patrick G; Kim, Min-Sik; Chaerkady, Raghothama; Pandey, Akhilesh; Guha, Udayan

    2015-01-01

    Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http

  20. Arcing and rf signal generation during target irradiation by a high-energy, pulsed neutral particle beam

    International Nuclear Information System (INIS)

    Robiscoe, R.T.

    1988-02-01

    We present a theory describing the dynamics of arc discharges in bulk dielectric materials on board space-based vehicles. Such ''punch-through'' arcs can occur in target satellites irradiated by high-energy (250 MeV), pulsed (100 mA x 10 ms) neutral particle beams. We treat the arc as a capacitively limited avalanche current in the target dielectric material, and we find expressions for the arc duration, charge transport, currents, and discharge energy. These quantities are adjusted to be consistent with known scaling laws for the area of charge depleted by the arc. After a brief account of the statistical distribution of voltages at which the arc starts and stops, we calculate the signal strength and frequency spectrum of the electromagnetic radiation broadcast by the arc. We find that arcs from thick (/similar to/1 cm) targets can generate rf signals detectable up to 1000 km from the target, bu a radio receiver operating at frequency 80 MHz, bandwidth 100 kHz, and detection threshold -105 dBm. These thick-target arc signals are 10 to 20 dB above ambient noise at the receiver, and they provide target hit assessment if the signal spectrum can be sampled at several frequencies in the nominal range 30-200 MHz. Thin-target (/similar to/1 mm) arc signals are much weaker, but when they are detecable in conjunction with thick-target signals, target discrimination is possible by comparing the signal frequency spectra. 24 refs., 12 figs

  1. Target of rapamycin signalling mediates the lifespan-extending effects of dietary restriction by essential amino acid alteration

    NARCIS (Netherlands)

    Emran, S.; Yang, M.Y.; He, X.L.; Zandveld, J.; Piper, M.D.W.

    2014-01-01

    Dietary restriction (DR), defined as a moderate reduction in food intake short of malnutrition, has been shown to extend healthy lifespan in a diverse range of organisms, from yeast to primates. Reduced signalling through the insulin/IGF-like (IIS) and Target of Rapamycin (TOR) signalling pathways

  2. An intracellular study on low-frequency acoustic signal processing in locust——Structure and function of the cercus-to-giant interneuron system

    Institute of Scientific and Technical Information of China (English)

    沈钧贤; 徐智敏

    1995-01-01

    The structure and function of the cercus-to-giant interneuron system,relevant to the receptionof low-frequency sound,within the terminal abdominal ganglion of the locust Locusta migratoria were revealedby using intracellular electrophysiological recording and dye labeling technique.This system consists of 4 bilater-al pairs of the giant interneurons(GIs 1—4).Each GI has distinct dendritic branching fields,position of thesoma,and location and orientation of its major axon.The characteristics of the system in responseto low-frequency sound,such as discharge patterns,the relationships between response threshold-frequency,in-tensity curves,and encoding of stimulus frequency,were also studied.The role of the system in low-frequencysound communication was discussed.

  3. Signal and data processing of small targets 1992; Proceedings of the Meeting, Orlando, FL, Apr. 20-22, 1992

    Science.gov (United States)

    Drummond, Oliver E.

    This volume on signal and data processing of small targets contains chapters devoted to signal processing, low observable detection, systems and simulations, association and filtering in tracking, multiple sensor processing and fusion, and data processing. Papers included are on multisensor predetection fusion, adaptive whitening filters for small target detection, unified framework for IR target detection and tracking, and target detection from image sequences using pixel-based decision criterion. Attention is also given to automatic acquisition and tracking of rounds and targets for electrooptic fire control, advanced surveillance testbed and background modeling, an interacting-multiple-model algorithm for tracking targets that maneuver through coordinated turns, and angular momentum and ballistic tracking. Other papers are on a data integration (fusion) tree paradigm, single-scan tracking using N IR sensors, and track monitoring with single and multiple 2D passive sensors. (No individual items are abstracted in this volume)

  4. Intracellular calcium is a target of modulation of apoptosis in MCF-7 cells in the presence of IgA adsorbed to polyethylene glycol

    Science.gov (United States)

    Honorio-França, Adenilda Cristina; Nunes, Gabriel Triches; Fagundes, Danny Laura Gomes; de Marchi, Patrícia Gelli Feres; Fernandes, Rubian Trindade da Silva; França, Juliana Luzia; França-Botelho, Aline do Carmo; Moraes, Lucélia Campelo Albuquerque; Varotti, Fernando de Pilla; França, Eduardo Luzía

    2016-01-01

    Purpose Clinical and epidemiological studies have indicated that breastfeeding has a protective effect on breast cancer risk. Protein-based drugs, including antibodies, are being developed to attain better forms of cancer therapy. Secretory IgA (SIgA) is the antibody class in human breast milk, and its activity can be linked to the protective effect of breastfeeding. The aim of this study was to investigate the effect of polyethylene glycol (PEG) microspheres with adsorbed SIgA on MCF-7 human breast cancer cells. Methods The PEG microspheres were characterized by flow cytometry and fluorescence microscopy. The MCF-7 cells were obtained from American Type Culture Collection. MCF-7 cells were pre-incubated for 24 hours with or without SIgA (100 ng/mL), PEG microspheres or SIgA adsorbed in PEG microspheres (100 ng/mL). Viability, intracellular calcium release, and apoptosis in MCF-7 cells were determined by flow cytometry. Results Fluorescence microscopy and flow cytometry analyses revealed that SIgA was able to adsorb to the PEG microspheres. The MCF-7 cells that were incubated with PEG microspheres with adsorbed SIgA showed decreased viability. MCF-7 cells that were incubated with SIgA or PEG microspheres with adsorbed SIgA had increased intracellular Ca2+ levels. In the presence of SIgA, an increase in the percentage of apoptotic cells was observed. The highest apoptosis index was observed when the cells were treated with PEG microspheres with adsorbed SIgA. Conclusion These data suggest that colostral SIgA adsorbed to PEG microspheres has antitumor effects on human MCF-7 breast cancer cells and that the presence of large amounts of this protein in secreted breast milk may provide protection against breast tumors in women who breastfed. PMID:26893571

  5. Molecular Pathways: Cachexia Signaling-A Targeted Approach to Cancer Treatment.

    Science.gov (United States)

    Miyamoto, Yuji; Hanna, Diana L; Zhang, Wu; Baba, Hideo; Lenz, Heinz-Josef

    2016-08-15

    Cancer cachexia is a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass, which negatively affects quality of life and portends a poor prognosis. Numerous molecular substrates and mechanisms underlie the dysregulation of skeletal muscle synthesis and degradation observed in cancer cachexia, including proinflammatory cytokines (TNFα, IL1, and IL6), and the NF-κB, IGF1/AKT/mTOR, and myostatin/activin-SMAD pathways. Recent preclinical and clinical studies have demonstrated that anti-cachexia drugs (such as MABp1 and soluble receptor antagonist of myostatin/activin) not only prevent muscle wasting but also may prolong overall survival. In this review, we focus on the significance of cachexia signaling in patients with cancer and highlight promising drugs targeting tumor cachexia in clinical development. Clin Cancer Res; 22(16); 3999-4004. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Bmi-1-targeting suppresses osteosarcoma aggressiveness through the NF-κB signaling pathway

    Science.gov (United States)

    Liu, Jiaguo; Luo, Bin; Zhao, Meng

    2017-01-01

    Bone cancer is one of the most lethal malignancies and the specific causes of tumor initiation are not well understood. B-cell-specific Moloney murine leukemia virus integration site 1 protein (Bmi-1) has been reported to be associated with the initiation and progression of osteosarcoma, and as a prognostic indicator in the clinic. In the current study, a full-length antibody targeting Bmi-1 (AbBmi-1) was produced and the preclinical value of Bmi-1-targeted therapy was evaluated in bone carcinoma cells and tumor xenograft mice. The results indicated that the Bmi-1 expression level was markedly upregulated in bone cancer cell lines, and inhibition of Bmi-1 by AbBmi-1 reduced the invasiveness and migration of osteosarcoma cells. Overexpression of Bmi-1 promoted proliferation and angiogenesis, and increased apoptosis resistance induced by cisplatin via the nuclear factor-κB (NF-κB) signal pathway. In addition, AbBmi-1 treatment inhibited the tumorigenicity of osteosarcoma cells in vivo. Furthermore, AbBmi-1 blocked NF-κB signaling and reduced MMP-9 expression. Furthermore, Bmi-1 promoted osteosarcoma tumor growth, whereas AbBmi-1 significantly inhibited osteosarcoma tumor growth in vitro and in vivo. Notably, AbBmi-1 decreased the percentages of Ki67-positive cells and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells in tumors compared with Bmi-1-treated and PBS controls. Notably, MMP-9 and NF-κB expression were downregulated by treatment with AbBmi-1 in MG-63 osteosarcoma cells. In conclusion, the data provides evidence that AbBmi-1 inhibited the progression of osteosarcoma, suggesting that AbBmi-1 may be a novel anti-cancer agent through the inhibition of Bmi-1 via activating the NF-κB pathway in osteosarcoma. PMID:28983587

  7. Andrographolide suppresses TRIF-dependent signaling of toll-like receptors by targeting TBK1.

    Science.gov (United States)

    Kim, Ah-Yeon; Shim, Hyun-Jin; Shin, Hyeon-Myeong; Lee, Yoo Jung; Nam, Hyeonjeong; Kim, Su Yeon; Youn, Hyung-Sun

    2018-04-01

    Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro. These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Quorum sensing communication between bacteria and human cells: signals, targets and functions

    Directory of Open Access Journals (Sweden)

    Angelika eHolm

    2014-06-01

    Full Text Available Both direct and long-range interactions between pathogenic Pseudomonas aeruginosa bacteria and their eukaryotic hosts are important in the outcome of infections. For cell-to-cell communication, these bacteria employ the quorum sensing (QS system to pass on information of the density of the bacterial population and collectively switch on virulence factor production, biofilm formation and resistance development. Thus, QS allows bacteria to behave as a community to perform tasks which would be impossible for individual cells, e.g. to overcome defense and immune systems and establish infections in higher organisms. This review highlights these aspects of QS and our own recent research on how P.aeruginosa communicates with human cells using the small QS signal molecules N-acyl homoserine lactones (AHL. We focus on how this conversation changes the behavior and function of neutrophils, macrophages and epithelial cells and on how the signaling machinery in human cells responsible for the recognition of AHL. Understanding the bacteria-host relationships at both cellular and molecular levels is essential for the identification of new targets and for the development of novel strategies to fight bacterial infections in the future.

  9. ATP as a Multi-target Danger Signal in the Brain

    Directory of Open Access Journals (Sweden)

    Ricardo J Rodrigues

    2015-04-01

    Full Text Available ATP is released in an activity-dependent manner from different cell types in the brain, fulfilling different roles as a neurotransmitter, neuromodulator, astrocyte-to-neuron communication, propagating astrocytic responses and formatting microglia responses. This involves the activation of different ATP P2 receptors (P2R as well as adenosine receptors upon extracellular ATP catabolism by ecto-nucleotidases. Notably, brain noxious stimuli trigger a sustained increase of extracellular ATP, which plays a key role as danger signal in the brain. This involves a combined action of extracellular ATP in different cell types, namely increasing the susceptibility of neurons to damage, promoting astrogliosis and recruiting and formatting microglia to mount neuroinflammatory responses. Such actions involve the activation of different receptors, as heralded by neuroprotective effects resulting from blockade mainly of P2X7R, P2Y1R and adenosine A2A receptors (A2AR, which hierarchy, cooperation and/or redundancy is still not resolved. These pleiotropic functions of ATP as a danger signal in brain damage prompt a therapeutic interest to multi-target different purinergic receptors to provide maximal opportunities for neuroprotection.

  10. Therapeutic Targeting of Redox Signaling in Myofibroblast Differentiation and Age-Related Fibrotic Disease

    Directory of Open Access Journals (Sweden)

    Natalie Sampson

    2012-01-01

    Full Text Available Myofibroblast activation plays a central role during normal wound healing. Whereas insufficient myofibroblast activation impairs wound healing, excessive myofibroblast activation promotes fibrosis in diverse tissues (including benign prostatic hyperplasia, BPH leading to organ dysfunction and also promotes a stromal response that supports tumor progression. The incidence of impaired wound healing, tissue fibrosis, BPH, and certain cancers strongly increases with age. This paper summarizes findings from in vitro fibroblast-to-myofibroblast differentiation systems that serve as cellular models to study fibrogenesis of diverse tissues. Supported by substantial in vivo data, a large body of evidence indicates that myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor beta is driven by a prooxidant shift in redox homeostasis due to elevated production of NADPH oxidase 4 (NOX4-derived hydrogen peroxide and supported by concomitant decreases in nitric oxide/cGMP signaling and reactive oxygen species (ROS scavenging enzymes. Fibroblast-to-myofibroblast differentiation can be inhibited and reversed by restoring redox homeostasis using antioxidants or NOX4 inactivation as well as enhancing nitric oxide/cGMP signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases. Current evidence indicates the therapeutic potential of targeting the prooxidant shift in redox homeostasis for the treatment of age-related diseases associated with myofibroblast dysregulation.

  11. Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression

    Directory of Open Access Journals (Sweden)

    Stephen A. Rosenberg

    2015-02-01

    Full Text Available Our group has previously reported that the majority of human melanomas (>60% express the metabotropic glutamate receptor 1 (GRM1 and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy.

  12. Targeting the NFκB signaling pathways for breast cancer prevention and therapy.

    Science.gov (United States)

    Wang, Wei; Nag, Subhasree A; Zhang, Ruiwen

    2015-01-01

    The activation of nuclear factor-kappaB (NFκB), a proinflammatory transcription factor, is a commonly observed phenomenon in breast cancer. It facilitates the development of a hormone-independent, invasive, high-grade, and late-stage tumor phenotype. Moreover, the commonly used cancer chemotherapy and radiotherapy approaches activate NFκB, leading to the development of invasive breast cancers that show resistance to chemotherapy, radiotherapy, and endocrine therapy. Inhibition of NFκB results in an increase in the sensitivity of cancer cells to the apoptotic effects of chemotherapeutic agents and radiation and restoring hormone sensitivity, which is correlated with increased disease-free survival in patients with breast cancer. In this review article, we focus on the role of the NFκB signaling pathways in the development and progression of breast cancer and the validity of NFκB as a potential target for breast cancer prevention and therapy. We also discuss the recent findings that NFκB may have tumor suppressing activity in certain cancer types. Finally, this review also covers the state-of-the-art development of NFκB inhibitors for cancer therapy and prevention, the challenges in targeting validation, and pharmacology and toxicology evaluations of these agents from the bench to the bedside.

  13. Estrogen enhanced cell-cell signalling in breast cancer cells exposed to targeted irradiation

    International Nuclear Information System (INIS)

    Shao, Chunlin; Folkard, Melvyn; Held, Kathryn D; Prise, Kevin M

    2008-01-01

    Radiation-induced bystander responses, where cells respond to their neighbours being irradiated are being extensively studied. Although evidence shows that bystander responses can be induced in many types of cells, it is not known whether there is a radiation-induced bystander effect in breast cancer cells, where the radiosensitivity may be dependent on the role of the cellular estrogen receptor (ER). This study investigated radiation-induced bystander responses in estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells. The influence of estrogen and anti-estrogen treatments on the bystander response was determined by individually irradiating a fraction of cells within the population with a precise number of helium-3 using a charged particle microbeam. Damage was scored as chromosomal damage measured as micronucleus formation. A bystander response measured as increased yield of micronucleated cells was triggered in both MCF-7 and MDA-MB-231 cells. The contribution of the bystander response to total cell damage in MCF-7 cells was higher than that in MDA-MB-231 cells although the radiosensitivity of MDA-MB-231 was higher than MCF-7. Treatment of cells with 17β-estradiol (E2) increased the radiosensitivity and the bystander response in MCF-7 cells, and the effect was diminished by anti-estrogen tamoxifen (TAM). E2 also increased the level of intracellular reactive oxygen species (ROS) in MCF-7 cells in the absence of radiation. In contrast, E2 and TAM had no influence on the bystander response and ROS levels in MDA-MB-231 cells. Moreover, the treatment of MCF-7 cells with antioxidants eliminated both the E2-induced ROS increase and E2-enhanced bystander response triggered by the microbeam irradiation, which indicates that ROS are involved in the E2-enhanced bystander micronuclei formation after microbeam irradiation. The observation of bystander responses in breast tumour cells may offer new potential targets for radiation

  14. Cyclic AMP-dependent signaling system is a primary metabolic target for non-thermal effect of microwaves on heart muscle hydration.

    Science.gov (United States)

    Narinyan, Lilia; Ayrapetyan, Sinerik

    2017-01-01

    Previously, we have suggested that cell hydration is a universal and extra-sensitive sensor for the structural changes of cell aqua medium caused by the impact of weak chemical and physical factors. The aim of present work is to elucidate the nature of the metabolic messenger through which physiological solution (PS) treated by non-thermal (NT) microwaves (MW) could modulate heart muscle hydration of rats. For this purpose, the effects of NT MW-treated PS on heart muscle hydration, [ 3 H]-ouabain binding with cell membrane, 45 Ca 2+ uptake and intracellular cyclic nucleotides contents in vivo and in vitro experiments were studied. It is shown that intraperitoneal injections of both Sham-treated PS and NT MW-treated PS elevate heart muscle hydration. However, the effect of NT MW-treated PS on muscle hydration is more pronounced than the effect of Sham-treated PS. In vitro experiments NT MW-treated PS has dehydration effect on muscle, which is not changed by decreasing Na + gradients on membrane. Intraperitoneal injection of Sham- and NT MW-treated PS containing 45 Ca 2+ have similar dehydration effect on muscle, while NT MW-treated PS has activation effect on Na + /Ca 2+ exchange in reverse mode. The intraperitoneal injection of NT MW-treated PS depresses [ 3 H]-ouabain binding with its high-affinity membrane receptors, elevates intracellular cAMP and decreases cGMP contents. Based on the obtained data, it is suggested that cAMP-dependent signaling system serves as a primary metabolic target for NT MW effect on heart muscle hydration.

  15. Targeting Wnt signaling in colorectal cancer. A Review in the Theme: Cell Signaling: Proteins, Pathways and Mechanisms

    Science.gov (United States)

    Novellasdemunt, Laura; Antas, Pedro

    2015-01-01

    The evolutionarily conserved Wnt signaling pathway plays essential roles during embryonic development and tissue homeostasis. Notably, comprehensive genetic studies in Drosophila and mice in the past decades have demonstrated the crucial role of Wnt signaling in intestinal stem cell maintenance by regulating proliferation, differentiation, and cell-fate decisions. Wnt signaling has also been implicated in a variety of cancers and other diseases. Loss of the Wnt pathway negative regulator adenomatous polyposis coli (APC) is the hallmark of human colorectal cancers (CRC). Recent advances in high-throughput sequencing further reveal many novel recurrent Wnt pathway mutations in addition to the well-characterized APC and β-catenin mutations in CRC. Despite attractive strategies to develop drugs for Wnt signaling, major hurdles in therapeutic intervention of the pathway persist. Here we discuss the Wnt-activating mechanisms in CRC and review the current advances and challenges in drug discovery. PMID:26289750

  16. Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals

    International Nuclear Information System (INIS)

    Popovici, Cornel; Fallet, Mathieu; Marguet, Didier; Birnbaum, Daniel; Roubin, Regine

    2006-01-01

    The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals (i) synergy of several NLS and (ii) attenuated secretion signal

  17. Poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticles with polyethylenimine coat as simple, safe, and versatile vehicles for cell targeting: population characteristics, cell uptake, and intracellular trafficking.

    Science.gov (United States)

    Wu, Lin-Ping; Wang, Danyang; Parhamifar, Ladan; Hall, Arnaldur; Chen, Guo-Qiang; Moghimi, Seyed M

    2014-06-01

    A simple and highly safe poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticulate delivery system that targets different cell types is developed. A sub-cytotoxic level of polyethylenimine coat mediates universal cell targeting. Internalized nanoparticles traffic along endolysosomal compartments, endoplasmic reticulum and the Golgi complex. Nanoparticles have no detrimental effects on cell morphology and respiration. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling

    Science.gov (United States)

    Corral-Jara, Karla F.; Gómez-Leyva, Juan F.; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  19. Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

    Science.gov (United States)

    Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

    2014-01-01

    The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

  20. Discovery of a Novel Inhibitor of the Hedgehog Signaling Pathway through Cell-based Compound Discovery and Target Prediction.

    Science.gov (United States)

    Kremer, Lea; Schultz-Fademrecht, Carsten; Baumann, Matthias; Habenberger, Peter; Choidas, Axel; Klebl, Bert; Kordes, Susanne; Schöler, Hans R; Sterneckert, Jared; Ziegler, Slava; Schneider, Gisbert; Waldmann, Herbert

    2017-10-09

    Cell-based assays enable monitoring of small-molecule bioactivity in a target-agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small-molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell-based bioactive-compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell-based assay, we identified the pyrazolo-imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch +/- medulloblastoma cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy.

    Science.gov (United States)

    Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F; Tang, Jen-Yang; Chang, Hsueh-Wei

    2017-07-14

    Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer.

  2. TRAIL, Wnt, Sonic Hedgehog, TGFβ, and miRNA Signalings Are Potential Targets for Oral Cancer Therapy

    Science.gov (United States)

    Farooqi, Ammad Ahmad; Shu, Chih-Wen; Huang, Hurng-Wern; Wang, Hui-Ru; Chang, Yung-Ting; Fayyaz, Sundas; Yuan, Shyng-Shiou F.; Tang, Jen-Yang

    2017-01-01

    Clinical studies and cancer cell models emphasize the importance of targeting therapies for oral cancer. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is highly expressed in cancer, and is a selective killing ligand for oral cancer. Signaling proteins in the wingless-type mouse mammary tumor virus (MMTV) integration site family (Wnt), Sonic hedgehog (SHH), and transforming growth factor β (TGFβ) pathways may regulate cell proliferation, migration, and apoptosis. Accordingly, the genes encoding these signaling proteins are potential targets for oral cancer therapy. In this review, we focus on recent advances in targeting therapies for oral cancer and discuss the gene targets within TRAIL, Wnt, SHH, and TGFβ signaling for oral cancer therapies. Oncogenic microRNAs (miRNAs) and tumor suppressor miRNAs targeting the genes encoding these signaling proteins are summarized, and the interactions between Wnt, SHH, TGFβ, and miRNAs are interpreted. With suitable combination treatments, synergistic effects are expected to improve targeting therapies for oral cancer. PMID:28708091

  3. Individual differences in ethanol locomotor sensitization are associated with dopamine D1 receptor intra-cellular signaling of DARPP-32 in the nucleus accumbens.

    Directory of Open Access Journals (Sweden)

    Karina Possa Abrahao

    Full Text Available In mice there are clear individual differences in the development of behavioral sensitization to ethanol, a progressive potentiation of its psychomotor stimulant effect. Variability in the behavioral responses to ethanol has been associated with alcohol preference. Here we investigated if the functional hyperresponsiveness of D1 receptors observed in ethanol sensitized mice leads to an increased activation of DARPP-32, a central regulatory protein in medium spiny neurons, in the nucleus accumbens - a brain region known to play a role in drug reinforcement. Swiss Webster mice received ethanol (2.2 g/kg/day or saline i.p. administrations for 21 days and were weekly evaluated regarding their locomotor activity. From those treated with ethanol, the 33% with the highest levels of locomotor activity were classified as "sensitized" and the 33% with the lowest levels as "non-sensitized". The latter presented similar locomotor levels to those of saline-treated mice. Different subgroups of mice received intra-accumbens administrations of saline and, 48 h later, SKF-38393, D1 receptor agonist 0.1 or 1 µg/side. Indeed, sensitized mice presented functional hyperresponsiveness of D1 receptors in the accumbens. Two weeks following the ethanol treatment, other subgroups received systemic saline or SKF 10 mg/kg, 20 min before the euthanasia. The nucleus accumbens were dissected for the Western Blot analyses of total DARPP-32 and phospho-Thr34-DARPP-32 expression. D1 receptor activation induced higher phospho-Thr34-DARPP-32 expression in sensitized mice than in non-sensitized or saline. The functionally hyperresponsiveness of D1 receptors in the nucleus accumbens is associated with an increased phospho-Thr34-DARPP-32 expression after D1 receptor activation. These data suggest that an enduring increase in the sensitivity of the dopamine D1 receptor intracellular pathway sensitivity represents a neurobiological correlate associated with the development of

  4. 6-OHDA-induced apoptosis and mitochondrial dysfunction are mediated by early modulation of intracellular signals and interaction of Nrf2 and NF-κB factors

    International Nuclear Information System (INIS)

    Tobón-Velasco, Julio C.; Limón-Pacheco, Jorge H.; Orozco-Ibarra, Marisol; Macías-Silva, Marina; Vázquez-Victorio, Genaro; Cuevas, Elvis; Ali, Syed F.

    2013-01-01

    6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson's disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage

  5. ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex.

    Science.gov (United States)

    Park, Kyungho; Ikushiro, Hiroko; Seo, Ho Seong; Shin, Kyong-Oh; Kim, Young Il; Kim, Jong Youl; Lee, Yong-Moon; Yano, Takato; Holleran, Walter M; Elias, Peter; Uchida, Yoshikazu

    2016-03-08

    We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

  6. Signal and data processing of small targets 1991; Proceedings of the Meeting, Orlando, FL, Apr. 1-3, 1991

    Science.gov (United States)

    Drummond, Oliver E.

    Attention is given to signal processing; track-before-detect; systems and simulations; association and filtering in tracking; and data processing. Particular attention is given to a linear modeling algorithm for tracking time-varying signals, an optoelectric Gabor detector for transient signals, small-target acquisition and typing by AASAP, model-based analysis of 3D spatial-temporal IR clutter suppression filtering, algorithms and architectures for implementing large-velocity filter banks, an end-to-end scenario-generating model for IRST performance analysis, detection and tracking of small targets in persistence, an incremental model for target maneuver estimation, implementation of an angle-only tracking filter, a global modeling approach for multisensor problems, passive-sensor data fusion, midcourse multitarget racking using continuous representation, neural data association, and statistical initial orbit determination. (For individual items see A93-26797 to A93-26799)

  7. Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chao-Yuan [Department of Oncology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Radiological Technology, Yuanpei University, Hsinchu, Taiwan (China); Tai, Wei-Tien [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan (China); Wu, Szu-Yuan [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Radiation Oncology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Department of Biotechnology, Hungkuang University, Taichung, Taiwan (China); Shih, Chih-Ting; Chen, Min-Hsuan; Tsai, Ming-Hsien [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); Kuo, Chiung-Wen [Department of Medical Imaging and Radiological Technology, Yuanpei University of Medical Technology, Hsinchu, Taiwan (China); Shiau, Chung-Wai [Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan (China); Hung, Man-Hsin, E-mail: cindybeaty@gmail.com [Division of Medical Oncology, Department of Oncology, Taipei Veterans General Hospital, Taipei, Taiwan (China); Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan (China); Program in Molecular Medicine, School of Life Science, National Yang-Ming University, Taipei, Taiwan (China); School of Medicine, National Yang-Ming University, Taipei, Taiwan (China); Chen, Kuen-Feng, E-mail: kfchen1970@ntu.edu.tw [Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan (China); National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan (China)

    2016-06-01

    Purpose: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Methods and Materials: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Results: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. Conclusions: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

  8. Targeting Cannabinoid Signaling in the Immune System: “High”-ly Exciting Questions, Possibilities, and Challenges

    Directory of Open Access Journals (Sweden)

    Attila Oláh

    2017-11-01

    Full Text Available It is well known that certain active ingredients of the plants of Cannabis genus, i.e., the “phytocannabinoids” [pCBs; e.g., (−-trans-Δ9-tetrahydrocannabinol (THC, (−-cannabidiol, etc.] can influence a wide array of biological processes, and the human body is able to produce endogenous analogs of these substances [“endocannabinoids” (eCB, e.g., arachidonoylethanolamine (anandamide, AEA, 2-arachidonoylglycerol (2-AG, etc.]. These ligands, together with multiple receptors (e.g., CB1 and CB2 cannabinoid receptors, etc., and a complex enzyme and transporter apparatus involved in the synthesis and degradation of the ligands constitute the endocannabinoid system (ECS, a recently emerging regulator of several physiological processes. The ECS is widely expressed in the human body, including several members of the innate and adaptive immune system, where eCBs, as well as several pCBs were shown to deeply influence immune functions thereby regulating inflammation, autoimmunity, antitumor, as well as antipathogen immune responses, etc. Based on this knowledge, many in vitro and in vivo studies aimed at exploiting the putative therapeutic potential of cannabinoid signaling in inflammation-accompanied diseases (e.g., multiple sclerosis or in organ transplantation, and to dissect the complex immunological effects of medical and “recreational” marijuana consumption. Thus, the objective of the current article is (i to summarize the most recent findings of the field; (ii to highlight the putative therapeutic potential of targeting cannabinoid signaling; (iii to identify open questions and key challenges; and (iv to suggest promising future directions for cannabinoid-based drug development.

  9. Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

    International Nuclear Information System (INIS)

    Huang, Chao-Yuan; Tai, Wei-Tien; Wu, Szu-Yuan; Shih, Chih-Ting; Chen, Min-Hsuan; Tsai, Ming-Hsien; Kuo, Chiung-Wen; Shiau, Chung-Wai; Hung, Man-Hsin; Chen, Kuen-Feng

    2016-01-01

    Purpose: Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Methods and Materials: HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Results: Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC in vitro and in vivo. Conclusions: SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

  10. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

    DEFF Research Database (Denmark)

    Deshmukh, Atul S.; Treebak, Jonas Thue; Long, Yun Chau

    2008-01-01

    AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK...... activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from...... (Thr37/46) (P mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued...

  11. Elimination of head and neck cancer initiating cells through targeting glucose regulated protein78 signaling

    Directory of Open Access Journals (Sweden)

    Huang Chih-Yang

    2010-10-01

    Full Text Available Abstract Background Head and neck squamous cell carcinoma (HNSCC is a highly lethal cancer that contains cellular and functional heterogeneity. Previously, we enriched a subpopulation of highly tumorigenic head and neck cancer initiating cells (HN-CICs from HNSCC. However, the molecular mechanisms by which to govern the characteristics of HN-CICs remain unclear. GRP78, a stress-inducible endoplasmic reticulum chaperone, has been reported to play a crucial role in the maintenance of embryonic stem cells, but the role of GRP78 in CICs has not been elucidated. Results Initially, we recognized GRP78 as a putative candidate on mediating the stemness and tumorigenic properties of HN-CICs by differential systemic analyses. Subsequently, cells with GRP78 anchored at the plasma membrane (memGRP78+ exerted cancer stemness properties of self-renewal, differentiation and radioresistance. Of note, xenotransplantation assay indicated merely 100 memGRP78+ HNSCCs resulted in tumor growth. Moreover, knockdown of GRP78 significantly reduced the self-renewal ability, side population cells and expression of stemness genes, but inversely promoted cell differentiation and apoptosis in HN-CICs. Targeting GRP78 also lessened tumorigenicity of HN-CICs both in vitro and in vivo. Clinically, co-expression of GRP78 and Nanog predicted the worse survival prognosis of HNSCC patients by immunohistochemical analyses. Finally, depletion of GRP78 in HN-CICs induced the expression of Bax, Caspase 3, and PTEN. Conclusions In summary, memGRP78 should be a novel surface marker for isolation of HN-CICs, and targeting GRP78 signaling might be a potential therapeutic strategy for HNSCC through eliminating HN-CICs.

  12. Targeted Gene-Silencing Reveals the Functional Significance of Myocardin Signaling in the Failing Heart

    Science.gov (United States)

    Torrado, Mario; Iglesias, Raquel; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T.

    2011-01-01

    Background Myocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear. Methodology/Principal Findings The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. Conclusions/Significance These data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view. PMID:22028870

  13. Targeted gene-silencing reveals the functional significance of myocardin signaling in the failing heart.

    Directory of Open Access Journals (Sweden)

    Mario Torrado

    Full Text Available BACKGROUND: Myocardin (MYOCD, a potent transcriptional coactivator of smooth muscle (SM and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF. However, the molecular and functional consequences of myocd upregulation in HF are still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox-induced diastolic HF (DHF model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1 a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2 amelioration of impaired diastolic dysfunction, and (3 higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that a moderate inhibition (e.g., normalization of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

  14. Investigating Internalization and Intracellular Trafficking of GPCRs

    DEFF Research Database (Denmark)

    Foster, Simon R; Bräuner-Osborne, Hans

    2017-01-01

    for signal transduction. One of the major mechanisms for GPCR regulation involves their endocytic trafficking, which serves to internalize the receptors from the plasma membrane and thereby attenuate G protein-dependent signaling. However, there is accumulating evidence to suggest that GPCRs can signal...... independently of G proteins, as well as from intracellular compartments including endosomes. It is in this context that receptor internalization and intracellular trafficking have attracted renewed interest within the GPCR field. In this chapter, we will review the current understanding and methodologies...

  15. Signal and data processing of small targets 1989; Proceedings of the Meeting, Orlando, FL, Mar. 27-29, 1989

    Science.gov (United States)

    Drummond, Oliver E. (Editor)

    1989-01-01

    The present conference on digital signal processing, association and filtering techniques, and multiple-sensor/multiple-tracking techniques, discusses single-frame velocity estimation, efficient target extraction for laser radar imagery, precision target tracking for small extended objects, IR clutter partitioning for matched filter design, the maximum-likelihood approach to gamma circumvention, position estimation for optical point targets using staring detector arrays, and a multiple-scan signal processing technique for area-moving target indication. Also discussed are a proportional integral estimator, the prediction of track purity in tracking performance evaluations, synchronization and fault-tolerance in a distributed tracker, the benefits of soft sensors and probabilistic fusion, and testing track initiation algorithms fusing two-dimensional tracks.

  16. Cell fate reprogramming by control of intracellular network dynamics

    Science.gov (United States)

    Zanudo, Jorge G. T.; Albert, Reka

    Identifying control strategies for biological networks is paramount for practical applications that involve reprogramming a cell's fate, such as disease therapeutics and stem cell reprogramming. Although the topic of controlling the dynamics of a system has a long history in control theory, most of this work is not directly applicable to intracellular networks. Here we present a network control method that integrates the structural and functional information available for intracellular networks to predict control targets. Formulated in a logical dynamic scheme, our control method takes advantage of certain function-dependent network components and their relation to steady states in order to identify control targets, which are guaranteed to drive any initial state to the target state with 100% effectiveness and need to be applied only transiently for the system to reach and stay in the desired state. We illustrate our method's potential to find intervention targets for cancer treatment and cell differentiation by applying it to a leukemia signaling network and to the network controlling the differentiation of T cells. We find that the predicted control targets are effective in a broad dynamic framework. Moreover, several of the predicted interventions are supported by experiments. This work was supported by NSF Grant PHY 1205840.

  17. Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling.

    Science.gov (United States)

    Queval, Christophe J; Song, Ok-Ryul; Carralot, Jean-Philippe; Saliou, Jean-Michel; Bongiovanni, Antonino; Deloison, Gaspard; Deboosère, Nathalie; Jouny, Samuel; Iantomasi, Raffaella; Delorme, Vincent; Debrie, Anne-Sophie; Park, Sei-Jin; Gouveia, Joana Costa; Tomavo, Stanislas; Brosch, Roland; Yoshimura, Akihiko; Yeramian, Edouard; Brodin, Priscille

    2017-09-26

    Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Mycobacterium tuberculosis Controls Phagosomal Acidification by Targeting CISH-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Christophe J. Queval

    2017-09-01

    Full Text Available Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH, which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive.

  19. Regional reliability of quantitative signal targeting with alternating radiofrequency (STAR) labeling of arterial regions (QUASAR).

    Science.gov (United States)

    Tatewaki, Yasuko; Higano, Shuichi; Taki, Yasuyuki; Thyreau, Benjamin; Murata, Takaki; Mugikura, Shunji; Ito, Daisuke; Takase, Kei; Takahashi, Shoki

    2014-01-01

    Quantitative signal targeting with alternating radiofrequency labeling of arterial regions (QUASAR) is a recent spin labeling technique that could improve the reliability of brain perfusion measurements. Although it is considered reliable for measuring gray matter as a whole, it has never been evaluated regionally. Here we assessed this regional reliability. Using a 3-Tesla Philips Achieva whole-body system, we scanned four times 10 healthy volunteers, in two sessions 2 weeks apart, to obtain QUASAR images. We computed perfusion images and ran a voxel-based analysis within all brain structures. We also calculated mean regional cerebral blood flow (rCBF) within regions of interest configured for each arterial territory distribution. The mean CBF over whole gray matter was 37.74 with intraclass correlation coefficient (ICC) of .70. In white matter, it was 13.94 with an ICC of .30. Voxel-wise ICC and coefficient-of-variation maps showed relatively lower reliability in watershed areas and white matter especially in deeper white matter. The absolute mean rCBF values were consistent with the ones reported from PET, as was the relatively low variability in different feeding arteries. Thus, QUASAR reliability for regional perfusion is high within gray matter, but uncertain within white matter. © 2014 The Authors. Journal of Neuroimaging published by the American Society of Neuroimaging.

  20. Nuclear localization signal targeting to macronucleus and micronucleus in binucleated ciliate Tetrahymena thermophila.

    Science.gov (United States)

    Iwamoto, Masaaki; Mori, Chie; Osakada, Hiroko; Koujin, Takako; Hiraoka, Yasushi; Haraguchi, Tokuko

    2018-06-08

    Ciliated protozoa possess two morphologically and functionally distinct nuclei: a macronucleus (MAC) and a micronucleus (MIC). The MAC is transcriptionally active and functions in all cellular events. The MIC is transcriptionally inactive during cell growth, but functions in meiotic events to produce progeny nuclei. Thus, these two nuclei must be distinguished by the nuclear proteins required for their distinct functions during cellular events such as cell proliferation and meiosis. To understand the mechanism of the nuclear transport specific to either MAC or MIC, we identified specific nuclear localization signals (NLSs) in two MAC- and MIC-specific nuclear proteins, macronuclear histone H1 and micronuclear linker histone-like protein (Mlh1), respectively. By expressing GFP-fused fragments of these proteins in Tetrahymena thermophila cells, two distinct regions in macronuclear histone H1 protein were assigned as independent MAC-specific NLSs and two distinct regions in Mlh1 protein were assigned as independent MIC-specific NLSs. These NLSs contain several essential lysine residues responsible for the MAC- and MIC-specific nuclear transport, but neither contains any consensus sequence with known monopartite or bipartite NLSs in other model organisms. Our findings contribute to understanding how specific nuclear targeting is achieved to perform distinct nuclear functions in binucleated ciliates. © 2018 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  1. Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

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    Eun-Mi Noh

    2016-01-01

    Full Text Available Reactive oxygen species (ROS play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3 metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

  2. Resistance exercise-induced increases in putative anabolic hormones do not enhance muscle protein synthesis or intracellular signalling in young men.

    Science.gov (United States)

    West, Daniel W D; Kujbida, Gregory W; Moore, Daniel R; Atherton, Philip; Burd, Nicholas A; Padzik, Jan P; De Lisio, Michael; Tang, Jason E; Parise, Gianni; Rennie, Michael J; Baker, Steven K; Phillips, Stuart M

    2009-11-01

    We aimed to determine whether exercise-induced elevations in systemic concentration of testosterone, growth hormone (GH) and insulin-like growth factor-1 (IGF-1) enhanced post-exercise myofibrillar protein synthesis (MPS) and phosphorylation of signalling proteins important in regulating mRNA translation. Eight young men (20 +/- 1.1 years, BMI = 26 +/- 3.5 kg m(-2)) completed two exercise protocols designed to maintain basal hormone concentrations (low hormone, LH) or elicit increases in endogenous hormones (high hormone, HH). In the LH protocol, participants performed a bout of unilateral resistance exercise with the elbow flexors. The HH protocol consisted of the same elbow flexor exercise with the contralateral arm followed immediately by high-volume leg resistance exercise. Participants consumed 25 g of protein after arm exercise to maximize MPS. Muscle biopsies and blood samples were taken as appropriate. There were no changes in serum testosterone, GH or IGF-1 after the LH protocol, whereas there were marked elevations after HH (testosterone, P anabolic hormones do not enhance fed-state anabolic signalling or MPS following resistance exercise. Local mechanisms are likely to be of predominant importance for the post-exercise increase in MPS.

  3. Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

    Science.gov (United States)

    Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

    2010-12-01

    l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

  4. The Conserved Arginine Cluster in the Insert of the Third Cytoplasmic Loop of the Long Form of the D2 Dopamine Receptor (D2L-R Acts as an Intracellular Retention Signal

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    Valentina Kubale

    2016-07-01

    Full Text Available This study examined whether the conserved arginine cluster present within the 29-amino acid insert of the long form of the D2 dopamine receptor (D2L-R confers its predominant intracellular localization. We hypothesized that the conserved arginine cluster (RRR located within the insert could act as an RXR-type endoplasmic reticulum (ER retention signal. Arginine residues (R within the cluster at positions 267, 268, and 269 were charge-reserved to glutamic acids (E, either individually or in clusters, thus generating single, double, and triple D2L-R mutants. Through analyses of cellular localization by confocal microscopy and enzyme-linked immunosorbent assay (ELISA, radioligand binding assay, bioluminescence resonance energy transfer (BRET2 β-arrestin 2 (βarr2 recruitment assay, and cAMP signaling, it was revealed that charge reversal of the R residues at all three positions within the motif impaired their colocalization with ER marker calnexin and led to significantly improved cell surface expression. Additionally, these data demonstrate that an R to glutamic acid (E substitution at position 2 within the RXR motif is not functionally permissible. Furthermore, all generated D2L-R mutants preserved their functional integrity regarding ligand binding, agonist-induced βarr2 recruitment and Gαi-mediated signaling. In summary, our results show that the conserved arginine cluster within the 29-amino acid insert of third cytoplasmic loop (IC3 of the D2L-R appears to be the ER retention signal.

  5. TPL-2-ERK1/2 signaling promotes host resistance against intracellular bacterial infection by negative regulation of type I IFN production.

    Science.gov (United States)

    McNab, Finlay W; Ewbank, John; Rajsbaum, Ricardo; Stavropoulos, Evangelos; Martirosyan, Anna; Redford, Paul S; Wu, Xuemei; Graham, Christine M; Saraiva, Margarida; Tsichlis, Philip; Chaussabel, Damien; Ley, Steven C; O'Garra, Anne

    2013-08-15

    Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading cause of mortality and morbidity worldwide, causing ≈ 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL-12, IL-1, and TNF-α, as well as IFN-γ and CD4(+) Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I IFN have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL-10 is known to inhibit the immune response to M. tuberculosis in murine models through the negative regulation of key proinflammatory cytokines and the subsequent Th1 response. We show in this study, using a combination of transcriptomic analysis, genetics, and pharmacological inhibitors, that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I IFN production. The TPL-2-ERK1/2 signaling pathway regulated production by macrophages of several cytokines important in the immune response to M. tuberculosis as well as regulating induction of a large number of additional genes, many in a type I IFN-dependent manner. In the absence of TPL-2 in vivo, excess type I IFN promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I IFN may promote susceptibility to this important disease.

  6. fundTPL-2 – ERK1/2 Signaling Promotes Host Resistance against Intracellular Bacterial Infection by Negative Regulation of Type I Interferon Production3

    Science.gov (United States)

    McNab, Finlay W.; Ewbank, John; Rajsbaum, Ricardo; Stavropoulos, Evangelos; Martirosyan, Anna; Redford, Paul S.; Wu, Xuemei; Graham, Christine M.; Saraiva, Margarida; Tsichlis, Philip; Chaussabel, Damien; Ley, Steven C.; O’Garra, Anne

    2013-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of mortality and morbidity worldwide, causing approximately 1.4 million deaths per year. Key immune components for host protection during tuberculosis include the cytokines IL-12, IL-1 and TNF-α, as well as IFN-γ and CD4+ Th1 cells. However, immune factors determining whether individuals control infection or progress to active tuberculosis are incompletely understood. Excess amounts of type I interferon have been linked to exacerbated disease during tuberculosis in mouse models and to active disease in patients, suggesting tight regulation of this family of cytokines is critical to host resistance. In addition, the immunosuppressive cytokine IL-10 is known to inhibit the immune response to Mtb in murine models through the negative regulation of key pro-inflammatory cytokines and the subsequent Th1 response. We show here, using a combination of transcriptomic analysis, genetics and pharmacological inhibitors that the TPL-2-ERK1/2 signaling pathway is important in mediating host resistance to tuberculosis through negative regulation of type I interferon production. The TPL-2-ERK1/2 signalling pathway regulated production by macrophages of several cytokines important in the immune response to Mtb as well as regulating induction of a large number of additional genes, many in a type I IFN dependent manner. In the absence of TPL-2 in vivo, excess type I interferon promoted IL-10 production and exacerbated disease. These findings describe an important regulatory mechanism for controlling tuberculosis and reveal mechanisms by which type I interferon may promote susceptibility to this important disease. PMID:23842752

  7. Adaptive Traffic Signal Control: Deep Reinforcement Learning Algorithm with Experience Replay and Target Network

    OpenAIRE

    Gao, Juntao; Shen, Yulong; Liu, Jia; Ito, Minoru; Shiratori, Norio

    2017-01-01

    Adaptive traffic signal control, which adjusts traffic signal timing according to real-time traffic, has been shown to be an effective method to reduce traffic congestion. Available works on adaptive traffic signal control make responsive traffic signal control decisions based on human-crafted features (e.g. vehicle queue length). However, human-crafted features are abstractions of raw traffic data (e.g., position and speed of vehicles), which ignore some useful traffic information and lead t...

  8. Genomic Targets and Features of BarA-UvrY (-SirA Signal Transduction Systems.

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    Tesfalem R Zere

    Full Text Available The two-component signal transduction system BarA-UvrY of Escherichia coli and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA binding in E. coli and Salmonella enterica. The csrB and csrC genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY in vitro only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including cspA, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating csrB and csrC transcription. We also used in vivo crosslinking and other experimental approaches to reveal new features of csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with fliW, which encodes a protein that binds to CsrA and antagonizes its activity in Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species.

  9. The Macrophage Galactose-Type Lectin-1 (MGL1 Recognizes Taenia crassiceps Antigens, Triggers Intracellular Signaling, and Is Critical for Resistance to This Infection

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    Daniel Montero-Barrera

    2015-01-01

    Full Text Available C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1 recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1−/− mice showed less binding ability toward parasite antigens than their wild-type (WT counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1−/− macrophages. Following T. crassiceps infection, MGL1−/− mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1−/− mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1−/− macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance.

  10. Signal processing, sensor fusion, and target recognition; Proceedings of the Meeting, Orlando, FL, Apr. 20-22, 1992

    Science.gov (United States)

    Libby, Vibeke; Kadar, Ivan

    Consideration is given to a multiordered mapping technique for target prioritization, a neural network approach to multiple-target-tracking problems, a multisensor fusion algorithm for multitarget multibackground classification, deconvolutiom of multiple images of the same object, neural networks and genetic algorithms for combinatorial optimization of sensor data fusion, classification of atmospheric acoustic signals from fixed-wing aircraft, and an optics approach to sensor fusion for target recognition. Also treated are a zoom lens for automatic target recognition, a hybrid model for the analysis of radar sensors, an innovative test bed for developing and assessing air-to-air noncooperative target identification algorithms, SAR imagery scene segmentation using fractal processing, sonar feature-based bandwidth compression, laboratory experiments for a new sonar system, computational algorithms for discrete transform using fixed-size filter matrices, and pattern recognition for power systems.

  11. Polyplex micelle installing intracellular self-processing functionalities without free catiomers for safe and efficient systemic gene therapy through tumor vasculature targeting.

    Science.gov (United States)

    Chen, Qixian; Osada, Kensuke; Ge, Zhishen; Uchida, Satoshi; Tockary, Theofilus A; Dirisala, Anjaneyulu; Matsui, Akitsugu; Toh, Kazuko; Takeda, Kaori M; Liu, Xueying; Nomoto, Takahiro; Ishii, Tekihiko; Oba, Makoto; Matsumoto, Yu; Kataoka, Kazunori

    2017-01-01

    Both efficiency and safety profiles are crucial for promotion of gene delivery systems towards practical applications. A promising template system was previously developed based on block catiomer of poly(ethylene glycol) (PEG)-b-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide}-cholesteryl [PEG-PAsp(DET)-cholesteryl] with strategies of ligand conjugation at the α-terminus for specific affinity to the targeted cells and cholesteryl conjugation at the ω-terminus for structural stabilization to obtain systemic retention. Aiming for advocating this formulation towards practical applications, in the current study, the binding profile of this polymer to plasmid DNA (pDNA) was carefully studied to address an issue of toxicity origin. Quantification of free polymer composition confirmed that the toxicity mainly results from unbound polymer and polyplex micelle itself has negligible toxicity. This evaluation allowed for identifying an optimal condition to prepare safe polyplex micelles for systemic application that possess maximal polymer-binding but exclude free polymers. The identified polyplex micelles then faced a drawback of limited transfection efficiency due to the absence of free polymer, which is an acknowledged tendency found in various synthetic gene carriers. Thus, series of functional components was strategically compiled to improve the transfection efficiency such as attachment of cyclic (Arg-Gly-Asp) (cRGD) peptide as a ligand onto the polyplex micelles to facilitate cellular uptake, use of endosome membrane disruptive catiomer of PAsp(DET) for facilitating endosome escape along with use of the conjugated cholesteryl group to amplify the effect of PAsp(DET) on membrane disruption, so as to obtain efficient transfection. The mechanistic investigation respecting the appreciated pH dependent protonation behavior of PAsp(DET) permitted to depict an intriguing scenario how the block catiomers manage to escape from the endosome entrapment in response to the p

  12. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

    Science.gov (United States)

    Singh, M B; Hough, T; Theerakulpisut, P; Avjioglu, A; Davies, S; Smith, P M; Taylor, P; Simpson, R J; Ward, L D; McCluskey, J

    1991-01-01

    We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain. Images PMID:1671715

  13. DOA Estimation of Low Altitude Target Based on Adaptive Step Glowworm Swarm Optimization-multiple Signal Classification Algorithm

    Directory of Open Access Journals (Sweden)

    Zhou Hao

    2015-06-01

    Full Text Available The traditional MUltiple SIgnal Classification (MUSIC algorithm requires significant computational effort and can not be employed for the Direction Of Arrival (DOA estimation of targets in a low-altitude multipath environment. As such, a novel MUSIC approach is proposed on the basis of the algorithm of Adaptive Step Glowworm Swarm Optimization (ASGSO. The virtual spatial smoothing of the matrix formed by each snapshot is used to realize the decorrelation of the multipath signal and the establishment of a fullorder correlation matrix. ASGSO optimizes the function and estimates the elevation of the target. The simulation results suggest that the proposed method can overcome the low altitude multipath effect and estimate the DOA of target readily and precisely without radar effective aperture loss.

  14. The role of the class A scavenger receptors, SR-A and MARCO, in the immune system. Part 1. The structure of receptors, their ligand binding repertoires and ability to initiate intracellular signaling

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    Szczepan Józefowski

    2012-02-01

    Full Text Available  Recognition of pathogens by innate immune cells is mediated by pattern recognition receptors (PRR, which include scavenger receptors (SR. The class A SR, SR-A/CD204 and MARCO, are characterized by the presence of collagenous and SR cysteine-rich domains in their extracellular portions. Both receptors are expressed mainly on macrophages and dendritic cells. Thanks to their ability to bind to a wide range of polyanionic ligands, the class A SR may participate in numerous functions of these cells, such as endocytosis, and adhesion to extracellular matrix and to other cells. Among SR-A ligands are oxidized lipoproteins and β-amyloid fibrils, which link SR-A to the pathogenesis of arteriosclerosis and Alzheimer’s disease. Despite the demonstration of class A SR involvement in so many processes, the lack of selective ligands precluded reaching definite conclusions concerning their signaling abilities. Using specific receptor ligation with antibodies, we showed that SR-A and MARCO trigger intracellular signaling, modulating pro-inflammatory and microbicidal activities of macrophages. Surprisingly, despite similarities in structure and ligand binding repertoires, SR-A and MARCO exert opposite effects on interleukin-12 (IL-12 production in macrophages. SR-A ligation also stimulated H2O2 and IL-10 production, but had no effect on the release of several other cytokines. These limited effects of specific SR-A ligation contrast with generalized enhancement of immune responses observed in SR-A-deficient mice. Recent studies have revealed that many of these effects of SR-A deficiency may be caused by compensatory changes in the expression of other receptors and/or disinhibition of signal transduction from receptors belonging to the Toll/IL-1R family, rather than by the loss of the receptor function of SR-A.

  15. A model system for targeted drug release triggered by biomolecular signals logically processed through enzyme logic networks.

    Science.gov (United States)

    Mailloux, Shay; Halámek, Jan; Katz, Evgeny

    2014-03-07

    A new Sense-and-Act system was realized by the integration of a biocomputing system, performing analytical processes, with a signal-responsive electrode. A drug-mimicking release process was triggered by biomolecular signals processed by different logic networks, including three concatenated AND logic gates or a 3-input OR logic gate. Biocatalytically produced NADH, controlled by various combinations of input signals, was used to activate the electrochemical system. A biocatalytic electrode associated with signal-processing "biocomputing" systems was electrically connected to another electrode coated with a polymer film, which was dissolved upon the formation of negative potential releasing entrapped drug-mimicking species, an enzyme-antibody conjugate, operating as a model for targeted immune-delivery and consequent "prodrug" activation. The system offers great versatility for future applications in controlled drug release and personalized medicine.

  16. Getting “Inside” Type I IFNs: Type I IFNs in Intracellular Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Deann T. Snyder

    2017-01-01

    Full Text Available Type I interferons represent a unique and complex group of cytokines, serving many purposes during innate and adaptive immunity. Discovered in the context of viral infections, type I IFNs are now known to have myriad effects in infectious and autoimmune disease settings. Type I IFN signaling during bacterial infections is dependent on many factors including whether the infecting bacterium is intracellular or extracellular, as different signaling pathways are activated. As such, the repercussions of type I IFN induction can positively or negatively impact the disease outcome. This review focuses on type I IFN induction and downstream consequences during infection with the following intracellular bacteria: Chlamydia trachomatis, Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium, Francisella tularensis, Brucella abortus, Legionella pneumophila, and Coxiella burnetii. Intracellular bacterial infections are unique because the bacteria must avoid, circumvent, and even co-opt microbial “sensing” mechanisms in order to reside and replicate within a host cell. Furthermore, life inside a host cell makes intracellular bacteria more difficult to target with antibiotics. Because type I IFNs are important immune effectors, modulating this pathway may improve disease outcomes. But first, it is critical to understand the context-dependent effects of the type I IFN pathway in intracellular bacterial infections.

  17. Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately.

    Science.gov (United States)

    Shi, Yan; Shu, Bin; Yang, Ronghua; Xu, Yingbin; Xing, Bangrong; Liu, Jian; Chen, Lei; Qi, Shaohai; Liu, Xusheng; Wang, Peng; Tang, Jinming; Xie, Julin

    2015-06-16

    Wnt and Notch signaling pathways are critically involved in relative cell fate decisions within the development of cutaneous tissues. Moreover, several studies identified the above two pathways as having a significant role during wound healing. However, their biological effects during cutaneous tissues repair are unclear. We employed a self-controlled model (Sprague-Dawley rats with full-thickness skin wounds) to observe the action and effect of Wnt/β-catenin and Notch signalings in vivo. The quality of wound repair relevant to the gain/loss-of-function Wnt/β-catenin and Notch activation was estimated by hematoxylin-and-eosin and Masson staining. Immunofluorescence analysis and Western blot analysis were used to elucidate the underlying mechanism of the regulation of Wnt and Notch signaling pathways in wound healing. Meanwhile, epidermal stem cells (ESCs) were cultured in keratinocyte serum-free medium with Jaggedl or in DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl) to investigate whether the interruption of Notch signaling contributes to the expression of Wnt/β-catenin signaling. The results showed that in vivo the gain-of-function Wnt/β-catenin and Notch activation extended the ability to promote wound closure. We further determined that activation or inhibition of Wnt signaling and Notch signaling can affect the proliferation of ESCs, the differentiation and migration of keratinocytes, and follicle regeneration by targeting c-Myc and Hes1, which ultimately lead to enhanced or delayed wound healing. Furthermore, Western blot analysis suggested that the two pathways might interact in vivo and in vitro. These results suggest that Wnt and Notch signalings play important roles in cutaneous repair by targeting c-Myc and Hes1 separately. What's more, interaction between the above two pathways might act as a vital role in regulation of wound healing.

  18. Signal Processing of Ground Penetrating Radar Using Spectral Estimation Techniques to Estimate the Position of Buried Targets

    Directory of Open Access Journals (Sweden)

    Shanker Man Shrestha

    2003-11-01

    Full Text Available Super-resolution is very important for the signal processing of GPR (ground penetration radar to resolve closely buried targets. However, it is not easy to get high resolution as GPR signals are very weak and enveloped by the noise. The MUSIC (multiple signal classification algorithm, which is well known for its super-resolution capacity, has been implemented for signal and image processing of GPR. In addition, conventional spectral estimation technique, FFT (fast Fourier transform, has also been implemented for high-precision receiving signal level. In this paper, we propose CPM (combined processing method, which combines time domain response of MUSIC algorithm and conventional IFFT (inverse fast Fourier transform to obtain a super-resolution and high-precision signal level. In order to support the proposal, detailed simulation was performed analyzing SNR (signal-to-noise ratio. Moreover, a field experiment at a research field and a laboratory experiment at the University of Electro-Communications, Tokyo, were also performed for thorough investigation and supported the proposed method. All the simulation and experimental results are presented.

  19. Cell signaling heterogeneity is modulated by both cell-intrinsic and -extrinsic mechanisms: An integrated approach to understanding targeted therapy.

    Science.gov (United States)

    Kim, Eunjung; Kim, Jae-Young; Smith, Matthew A; Haura, Eric B; Anderson, Alexander R A

    2018-03-01

    During the last decade, our understanding of cancer cell signaling networks has significantly improved, leading to the development of various targeted therapies that have elicited profound but, unfortunately, short-lived responses. This is, in part, due to the fact that these targeted therapies ignore context and average out heterogeneity. Here, we present a mathematical framework that addresses the impact of signaling heterogeneity on targeted therapy outcomes. We employ a simplified oncogenic rat sarcoma (RAS)-driven mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase-protein kinase B (PI3K-AKT) signaling pathway in lung cancer as an experimental model system and develop a network model of the pathway. We measure how inhibition of the pathway modulates protein phosphorylation as well as cell viability under different microenvironmental conditions. Training the model on this data using Monte Carlo simulation results in a suite of in silico cells whose relative protein activities and cell viability match experimental observation. The calibrated model predicts distributional responses to kinase inhibitors and suggests drug resistance mechanisms that can be exploited in drug combination strategies. The suggested combination strategies are validated using in vitro experimental data. The validated in silico cells are further interrogated through an unsupervised clustering analysis and then integrated into a mathematical model of tumor growth in a homogeneous and resource-limited microenvironment. We assess posttreatment heterogeneity and predict vast differences across treatments with similar efficacy, further emphasizing that heterogeneity should modulate treatment strategies. The signaling model is also integrated into a hybrid cellular automata (HCA) model of tumor growth in a spatially heterogeneous microenvironment. As a proof of concept, we simulate tumor responses to targeted therapies in a spatially segregated tissue structure containing tumor

  20. Signal and data processing of small targets 1990; Proceedings of the Meeting, Orlando, FL, Apr. 16-18, 1990

    Science.gov (United States)

    Drummond, Oliver E.

    Various papers on signal and data processing of small targets are presented. Individual topics addressed include: clutter rejection using multispectral processing, new sensor for automatic guidance, Doppler domain localized generalized-likelihood-ratio detector, linear filter for resolution of point sources, analysis of order-statistic filters for robust detection, distribution functions for additive Gaussian and gamma noise, temperature discrimination of closely space objects, knowledge-based tracking algorithm, detecting and tracking low-observable targets using IR, weak target detection using the entropy concept, measurement-based neural-net multitarget tracker, object-track closed-form solution in angle space, passive-sensor data association for tracking. Also discussed are: application of MHT to dim moving targets, automatic static covariance analysis with mathematica, neural network implementation of plot/track association, application of Bayesian networks to multitarget tracking, tracking clusters and extended objects with multiple sensors, target tracking by human operator, finite impulse response estimator, multitarget tracking using an extended Kalman filter, maneuvering target tracking using a two-state model algorithm, mixture reduction algorithms for target tracking in clutter, scene interpretation approach to high-level target tracking.

  1. Hsa-miR-11181 regulates Wnt signaling pathway through targeting of APC2 transcripts in SW480 cell line.

    Science.gov (United States)

    Dokanehiifard, Sadat; Soltani, Bahram M

    2018-01-30

    Wnt signaling plays important roles in differentiation, morphogenesis and development. This signaling pathway is highly regulated at all levels and microRNAs are small noncoding RNAs regulating Wnt signaling. Here, we intended to investigate hsa-miR-11181 (a novel miRNA located in TrkC gene) effect on Wnt signaling pathway in SW480 cell line. TOP/FOP flash assay indicated up-regulation of Wnt signaling, following the overexpression of hsa-miR-11181, verified through RT-qPCR. Bioinformatics analysis predicted APC1, APC2 and Axin1 might be targeted by hsa-miR-11181. Then, RT-qPCR analysis indicated that APC2 and Axin1 have been significantly down-regulated following the hsa-miR-11181 overexpression. However dual luciferase assay analysis supported only APC2 3'-UTR is directly targeted by this miRNA. Then, treatment of SW480 cells with Wnt-inhibitory small molecules supported the effect of hsa-miR-11181 at the inhibitory complex level containing APC2 protein. Consistently, viability of SW480 cells overexpressing hsa-miR-11181 was significantly elevated, measured through MTT assay. Overall, these results suggest that hsa-miR-11181 may play a crucial role in Wnt signaling regulation and confirmed that APC2 3'-UTR is targeted by hsa-miR-11181 and propose the presence of its recognition sites in the promoter or coding regions of Axin1 gene. Copyright © 2017. Published by Elsevier B.V.

  2. Characterization of Leptin Intracellular Trafficking

    Directory of Open Access Journals (Sweden)

    E Walum

    2009-12-01

    Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

  3. Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

    Directory of Open Access Journals (Sweden)

    Bin Tang

    Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

  4. Activin signaling targeted by insulin/dFOXO regulates aging and muscle proteostasis in Drosophila.

    Directory of Open Access Journals (Sweden)

    Hua Bai

    2013-11-01

    Full Text Available Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.

  5. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  6. miR-958 inhibits Toll signaling and Drosomycin expression via direct targeting of Toll and Dif in Drosophila melanogaster.

    Science.gov (United States)

    Li, Shengjie; Li, Yao; Shen, Li; Jin, Ping; Chen, Liming; Ma, Fei

    2017-02-01

    Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well studied, the precise mechanisms of posttranscriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. In this study, we used an in silico strategy in combination with the Gal80 ts -Gal4 driver system to identify microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila We report that overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. We further demonstrate in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958 sponge rescued the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results, not only revealed a novel function and modulation pattern of miR-958, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity. Copyright © 2017 the American Physiological Society.

  7. Protein kinase C α is a central signaling node and therapeutic target for breast cancer stem cells.

    Science.gov (United States)

    Tam, Wai Leong; Lu, Haihui; Buikhuisen, Joyce; Soh, Boon Seng; Lim, Elgene; Reinhardt, Ferenc; Wu, Zhenhua Jeremy; Krall, Jordan A; Bierie, Brian; Guo, Wenjun; Chen, Xi; Liu, Xiaole Shirley; Brown, Myles; Lim, Bing; Weinberg, Robert A

    2013-09-09

    The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. New intracellular activities of matrix metalloproteinases shine in the moonlight.

    Science.gov (United States)

    Jobin, Parker G; Butler, Georgina S; Overall, Christopher M

    2017-11-01

    Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal

    NARCIS (Netherlands)

    Faber, Klaas Nico; Keizer-Gunnink, Ineke; Pluim, Dick; Harder, Willem; AB, Geert; Veenhuis, Marten

    1995-01-01

    Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the

  10. Series-nonuniform rational B-spline signal feedback: From chaos to any embedded periodic orbit or target point

    Energy Technology Data Exchange (ETDEWEB)

    Shao, Chenxi, E-mail: cxshao@ustc.edu.cn; Xue, Yong; Fang, Fang; Bai, Fangzhou [Department of Computer Science and Technology, University of Science and Technology of China, Hefei 230027 (China); Yin, Peifeng [Department of Computer Science and Engineering, Pennsylvania State University, State College, Pennsylvania 16801 (United States); Wang, Binghong [Department of Modern Physics, University of Science and Technology of China, Hefei 230026 (China)

    2015-07-15

    The self-controlling feedback control method requires an external periodic oscillator with special design, which is technically challenging. This paper proposes a chaos control method based on time series non-uniform rational B-splines (SNURBS for short) signal feedback. It first builds the chaos phase diagram or chaotic attractor with the sampled chaotic time series and any target orbit can then be explicitly chosen according to the actual demand. Second, we use the discrete timing sequence selected from the specific target orbit to build the corresponding external SNURBS chaos periodic signal, whose difference from the system current output is used as the feedback control signal. Finally, by properly adjusting the feedback weight, we can quickly lead the system to an expected status. We demonstrate both the effectiveness and efficiency of our method by applying it to two classic chaotic systems, i.e., the Van der Pol oscillator and the Lorenz chaotic system. Further, our experimental results show that compared with delayed feedback control, our method takes less time to obtain the target point or periodic orbit (from the starting point) and that its parameters can be fine-tuned more easily.

  11. Series-nonuniform rational B-spline signal feedback: From chaos to any embedded periodic orbit or target point.

    Science.gov (United States)

    Shao, Chenxi; Xue, Yong; Fang, Fang; Bai, Fangzhou; Yin, Peifeng; Wang, Binghong

    2015-07-01

    The self-controlling feedback control method requires an external periodic oscillator with special design, which is technically challenging. This paper proposes a chaos control method based on time series non-uniform rational B-splines (SNURBS for short) signal feedback. It first builds the chaos phase diagram or chaotic attractor with the sampled chaotic time series and any target orbit can then be explicitly chosen according to the actual demand. Second, we use the discrete timing sequence selected from the specific target orbit to build the corresponding external SNURBS chaos periodic signal, whose difference from the system current output is used as the feedback control signal. Finally, by properly adjusting the feedback weight, we can quickly lead the system to an expected status. We demonstrate both the effectiveness and efficiency of our method by applying it to two classic chaotic systems, i.e., the Van der Pol oscillator and the Lorenz chaotic system. Further, our experimental results show that compared with delayed feedback control, our method takes less time to obtain the target point or periodic orbit (from the starting point) and that its parameters can be fine-tuned more easily.

  12. Pleiotrophin Signaling Through PTNR in Breast Cancer

    National Research Council Canada - National Science Library

    Powers, Ciaron

    2001-01-01

    ... of intracellular signaling cascades. The pleiotrophin signaling pathway is known to be important in angiogenesis and breast cancer growth, but the exact mechanisms of pleiotrophin signaling remain undefined...

  13. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1.

    Directory of Open Access Journals (Sweden)

    Gokhan Demirkan

    Full Text Available Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR complex 1 (mTORC1, which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood.We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2 affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates.In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

  14. Targeting cFMS signaling to restore immune function and eradicate HIV reservoirs

    Science.gov (United States)

    Gerngross, Lindsey

    While combination anti-retroviral therapy (cART) has improved the length and quality of life of individuals living with HIV-1 infection, the prevalence of HIV-associated neurocognitive disorders (HAND) has increased and remains a significant clinical concern. The neuropathogenesis of HAND is not completely understood, however, latent HIV infection in the central nervous system (CNS) and chronic neuroinflammation are believed to play a prominent role. CNS-associated macrophages and resident microglia are significant contributors to CNS inflammation and constitute the chief reservoir of HIV-1 infection in the CNS. Previous studies from our lab suggest monocyte/macrophage invasion of the CNS in HIV may be driven by altered monocyte/macrophage homeostasis. We have reported expansion of a monocyte subset (CD14+CD16 +CD163+) in peripheral blood of HIV+ patients that is phenotypically similar to macrophages/microglia that accumulate in the CNS as seen in post-mortem tissue. The factors driving the expansion of this monocyte subset are unknown, however, signaling through cFMS, a type III receptor tyrosine kinase (RTK), may play a role. Macrophage-colony stimulating factor (M-CSF), a ligand of cFMS, has been shown to be elevated in the cerebral spinal fluid (CSF) of individuals with the most severe form of HAND, HIV-associated dementia (HAD). M-CSF promotes a Macrophage-2-like phenotype and increases CD16 and CD163 expression in cultured monocytes. M-CSF has also been shown to increase the susceptibility of macrophages to HIV infection and enhance virus production. These findings, in addition to the known function of M-CSF in promoting macrophage survival, supports a role for M-CSF in the development and maintenance of macrophage viral reservoirs in tissues where these cells accumulate, including the CNS. Interestingly, a second ligand for cFMS, IL-34, was recently identified and reported to share some functions with M-CSF, suggesting that both ligands may contribute to HIV

  15. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  16. [Neurotensin: reception and intracellular mechanisms of signaling].

    Science.gov (United States)

    Osadchiĭ, O E

    2006-01-01

    The review coveres the features of neurotensin receptor, functional role ot its structural elements, nature of conjugation with effectoral cell systems, and mechanisms of receptor decensitization developing as results of prolonged effect of agonist. The author provides pharmacological description of neurotensin antagonists and special features of three subtypes of its receptors. The author reviews the research results establishing a correlation between structural modification of various section of neurotensin molecula and manifestations of its physiological activity. Special focus is mage on discussion of neurotensin's physiological effects developing as results of its modulating impact on discharge of other biologically active substances.

  17. Detection and localization of multiple short range targets using FMCW radar signal

    KAUST Repository

    Jardak, Seifallah

    2016-07-26

    In this paper, a 24 GHz frequency-modulated continuous wave radar is used to detect and localize both stationary and moving targets. Depending on the application, the implemented software offers different modes of operation. For example, it can simply output raw data samples for advanced offline processing or directly carry out a two dimensional fast Fourier transform to estimate the location and velocity of multiple targets. To suppress clutter and detect only moving targets, two methods based on the background reduction and the slow time processing techniques are implemented. A trade-off between the two methods is presented based on their performance and the required processing time. © 2016 IEEE.

  18. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    International Nuclear Information System (INIS)

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-01-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence 916 NFNHIHKRIRRVADKYLSG 934 comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence 1414 SKKTNRGSQLHKYYMKRRTL 1433 , a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS

  19. ROS and ROS-Mediated Cellular Signaling

    Directory of Open Access Journals (Sweden)

    Jixiang Zhang

    2016-01-01

    Full Text Available It has long been recognized that an increase of reactive oxygen species (ROS can modify the cell-signaling proteins and have functional consequences, which successively mediate pathological processes such as atherosclerosis, diabetes, unchecked growth, neurodegeneration, inflammation, and aging. While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF-κB, MAPKs, Keap1-Nrf2-ARE, and PI3K-Akt, ion channels and transporters (Ca2+ and mPTP, and modifying protein kinase and Ubiquitination/Proteasome System.

  20. Detection and localization of multiple short range targets using FMCW radar signal

    KAUST Repository

    Jardak, Seifallah; Kiuru, Tero; Metso, Mikko; Pursula, Pekka; Hakli, Janne; Hirvonen, Mervi; Ahmed, Sajid; Alouini, Mohamed-Slim

    2016-01-01

    In this paper, a 24 GHz frequency-modulated continuous wave radar is used to detect and localize both stationary and moving targets. Depending on the application, the implemented software offers different modes of operation. For example, it can

  1. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    Directory of Open Access Journals (Sweden)

    Neil Dani

    Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

  2. Tetherin Suppresses Type I Interferon Signaling by Targeting MAVS for NDP52-Mediated Selective Autophagic Degradation in Human Cells.

    Science.gov (United States)

    Jin, Shouheng; Tian, Shuo; Luo, Man; Xie, Weihong; Liu, Tao; Duan, Tianhao; Wu, Yaoxing; Cui, Jun

    2017-10-19

    Tetherin (BST2/CD317) is an interferon-inducible antiviral factor known for its ability to block the release of enveloped viruses from infected cells. Yet its role in type I interferon (IFN) signaling remains poorly defined. Here, we demonstrate that Tetherin is a negative regulator of RIG-I like receptor (RLR)-mediated type I IFN signaling by targeting MAVS. The induction of Tetherin by type I IFN accelerates MAVS degradation via ubiquitin-dependent selective autophagy in human cells. Moreover, Tetherin recruits E3 ubiquitin ligase MARCH8 to catalyze K27-linked ubiquitin chains on MAVS at lysine 7, which serves as a recognition signal for NDP52-dependent autophagic degradation. Taken together, our findings reveal a negative feedback loop of RLR signaling generated by Tetherin-MARCH8-MAVS-NDP52 axis and provide insights into a better understanding of the crosstalk between selective autophagy and optimal deactivation of type I IFN signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Analysis of Maneuvering Targets with Complex Motions by Two-Dimensional Product Modified Lv's Distribution for Quadratic Frequency Modulation Signals.

    Science.gov (United States)

    Jing, Fulong; Jiao, Shuhong; Hou, Changbo; Si, Weijian; Wang, Yu

    2017-06-21

    For targets with complex motion, such as ships fluctuating with oceanic waves and high maneuvering airplanes, azimuth echo signals can be modeled as multicomponent quadratic frequency modulation (QFM) signals after migration compensation and phase adjustment. For the QFM signal model, the chirp rate (CR) and the quadratic chirp rate (QCR) are two important physical quantities, which need to be estimated. For multicomponent QFM signals, the cross terms create a challenge for detection, which needs to be addressed. In this paper, by employing a novel multi-scale parametric symmetric self-correlation function (PSSF) and modified scaled Fourier transform (mSFT), an effective parameter estimation algorithm is proposed-referred to as the Two-Dimensional product modified Lv's distribution (2D-PMLVD)-for QFM signals. The 2D-PMLVD is simple and can be easily implemented by using fast Fourier transform (FFT) and complex multiplication. These measures are analyzed in the paper, including the principle, the cross term, anti-noise performance, and computational complexity. Compared to the other three representative methods, the 2D-PMLVD can achieve better anti-noise performance. The 2D-PMLVD, which is free of searching and has no identifiability problems, is more suitable for multicomponent situations. Through several simulations and analyses, the effectiveness of the proposed estimation algorithm is verified.

  4. Functions of the APC tumor suppressor protein dependent and independent of canonical WNT signaling: implications for therapeutic targeting.

    Science.gov (United States)

    Hankey, William; Frankel, Wendy L; Groden, Joanna

    2018-03-01

    The acquisition of biallelic mutations in the APC gene is a rate-limiting step in the development of most colorectal cancers and occurs in the earliest lesions. APC encodes a 312-kDa protein that localizes to multiple subcellular compartments and performs diverse functions. APC participates in a cytoplasmic complex that promotes the destruction of the transcriptional licensing factor β-catenin; APC mutations that abolish this function trigger constitutive activation of the canonical WNT signaling pathway, a characteristic found in almost all colorectal cancers. By negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis, and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus. APC also suppresses tumor initiation and progression in the colorectal epithelium through functions that are independent of canonical WNT signaling. APC regulates the mitotic spindle to facilitate proper chromosome segregation, localizes to the cell periphery and cell protrusions to establish cell polarity and appropriate directional migration, and inhibits DNA replication by interacting directly with DNA. Mutations in APC are often frameshifts, insertions, or deletions that introduce premature stop codons and lead to the production of truncated APC proteins that lack its normal functions and possess tumorigenic properties. Therapeutic approaches in development for the treatment of APC-deficient tumors are focused on the inhibition of canonical WNT signaling, especially through targets downstream of APC in the pathway, or on the restoration of wild-type APC expression.

  5. c-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals.

    Science.gov (United States)

    Nishimura, Christopher D; Brenner, Daniel A; Mukherjee, Malini; Hirsch, Rachel A; Ott, Leah; Wu, Meng-Fen; Liu, Hao; Dakhova, Olga; Orange, Jordan S; Brenner, Malcolm K; Lin, Charles Y; Arber, Caroline

    2017-12-21

    Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL + polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents. © 2017 by The American Society of Hematology.

  6. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

    Science.gov (United States)

    Pan, Yuan; Laird, Joseph G; Yamaguchi, David M; Baker, Sheila A

    2015-06-01

    Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.

  7. Pathogenic mechanisms of intracellular bacteria.

    Science.gov (United States)

    Niller, Hans Helmut; Masa, Roland; Venkei, Annamária; Mészáros, Sándor; Minarovits, Janos

    2017-06-01

    We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.

  8. The Rac1 hypervariable region in targeting and signaling: a tail of many stories

    NARCIS (Netherlands)

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control

  9. Locating proteins in the cell using TargetP, SignalP and related tools

    DEFF Research Database (Denmark)

    Emanuelsson, O.; Brunak, Søren; von Heijne, G.

    2007-01-01

    of methods to predict subcellular localization based on these sorting signals and other sequence properties. We then outline how to use a number of internet-accessible tools to arrive at a reliable subcellular localization prediction for eukaryotic and prokaryotic proteins. In particular, we provide detailed...

  10. Sphingosine-1-Phosphate (S1P) and S1P Signaling Pathway: Therapeutic Targets in Autoimmunity and Inflammation.

    Science.gov (United States)

    Tsai, Hsing-Chuan; Han, May H

    2016-07-01

    Sphingosine-1-phosphate (S1P) and S1P receptors (S1PR) are ubiquitously expressed. S1P-S1PR signaling has been well characterized in immune trafficking and activation in innate and adaptive immune systems. However, the full extent of its involvement in the pathogenesis of autoimmune diseases is not well understood. FTY720 (fingolimod), a non-selective S1PR modulator, significantly decreased annualized relapse rates in relapsing-remitting multiple sclerosis (MS). FTY720, which primarily targets S1P receptor 1 as a functional antagonist, arrests lymphocyte egress from secondary lymphoid tissues and reduces neuroinflammation in the central nervous system (CNS). Recent studies suggest that FTY720 also decreases astrogliosis and promotes oligodendrocyte differentiation within the CNS and may have therapeutic benefit to prevent brain atrophy. Since S1P signaling is involved in multiple immune functions, therapies targeting S1P axis may be applicable to treat autoimmune diseases other than MS. Currently, over a dozen selective S1PR and S1P pathway modulators with potentially superior therapeutic efficacy and better side-effect profiles are in the pipeline of drug development. Furthermore, newly characterized molecules such as apolipoprotein M (ApoM) (S1P chaperon) and SPNS2 (S1P transporter) are also potential targets for treatment of autoimmune diseases. Finally, the application of therapies targeting S1P and S1P signaling pathways may be expanded to treat several other immune-mediated disorders (such as post-infectious diseases, post-stroke and post-stroke dementia) and inflammatory conditions beyond their application in primary autoimmune diseases.

  11. Targeting the phosphatidylinositol 3-kinase/Akt/mechanistic target of rapamycin signaling pathway in B-lineage acute lymphoblastic leukemia: An update.

    Science.gov (United States)

    Simioni, Carolina; Martelli, Alberto M; Zauli, Giorgio; Vitale, Marco; McCubrey, James A; Capitani, Silvano; Neri, Luca M

    2018-04-18

    Despite considerable progress in treatment protocols, B-lineage acute lymphoblastic leukemia (B-ALL) displays a poor prognosis in about 15-20% of pediatric cases and about 60% of adult patients. In addition, life-long irreversible late effects from chemo- and radiation therapy, including secondary malignancies, are a growing problem for leukemia survivors. Targeted therapy holds promising perspectives for cancer treatment as it may be more effective and have fewer side effects than conventional therapies. The phosphatidylinositol 3-phosphate kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway is a key regulatory cascade which controls proliferation, survival and drug-resistance of cancer cells, and it is frequently upregulated in the different subtypes of B-ALL, where it plays important roles in the pathophysiology, maintenance and progression of the disease. Moreover, activation of this signaling cascade portends a poorer prognosis in both pediatric and adult B-ALL patients. Promising preclinical data on PI3K/Akt/mTOR inhibitors have documented their anticancer activity in B-ALL and some of these novel drugs have entered clinical trials as they could lead to a longer event-free survival and reduce therapy-associated toxicity for patients with B-ALL. This review highlights the current status of PI3K/Akt/mTOR inhibitors in B-ALL, with an emphasis on emerging evidence of the superior efficacy of synergistic combinations involving the use of traditional chemotherapeutics or other novel, targeted agents. © 2018 Wiley Periodicals, Inc.

  12. Targeting Epidermal Growth Factor Receptor-Related Signaling Pathways in Pancreatic Cancer.

    Science.gov (United States)

    Philip, Philip A; Lutz, Manfred P

    2015-10-01

    Pancreatic cancer is aggressive, chemoresistant, and characterized by complex and poorly understood molecular biology. The epidermal growth factor receptor (EGFR) pathway is frequently activated in pancreatic cancer; therefore, it is a rational target for new treatments. However, the EGFR tyrosine kinase inhibitor erlotinib is currently the only targeted therapy to demonstrate a very modest survival benefit when added to gemcitabine in the treatment of patients with advanced pancreatic cancer. There is no molecular biomarker to predict the outcome of erlotinib treatment, although rash may be predictive of improved survival; EGFR expression does not predict the biologic activity of anti-EGFR drugs in pancreatic cancer, and no EGFR mutations are identified as enabling the selection of patients likely to benefit from treatment. Here, we review clinical studies of EGFR-targeted therapies in combination with conventional cytotoxic regimens or multitargeted strategies in advanced pancreatic cancer, as well as research directed at molecules downstream of EGFR as alternatives or adjuncts to receptor targeting. Limitations of preclinical models, patient selection, and trial design, as well as the complex mechanisms underlying resistance to EGFR-targeted agents, are discussed. Future clinical trials must incorporate translational research end points to aid patient selection and circumvent resistance to EGFR inhibitors.

  13. Multimodal Nanomedicine Strategies for Targeting Cancer Cells as well as Cancer Stem Cell Signalling Mechanisms.

    Science.gov (United States)

    Kanwar, Jagat R; Samarasinghe, Rasika M; Kamalapuram, Sishir K; Kanwar, Rupinder K

    2017-01-01

    Increasing evidence suggests that stem cells, a small population of cells with unique selfrenewable and tumour regenerative capacity, are aiding tumour re-growth and multidrug resistance. Conventional therapies are highly ineffective at eliminating these cells leading to relapse of disease and formation of chemoresistance tumours. Cancer and stem cells targeted therapies that utilizes nanotherapeutics to delivery anti-cancer drugs to specific sites are continuously investigated. This review focuses on recent research using nanomedicine and targeting entities to eliminate cancer cells and cancer stem cells. Current nanotherapeutics in clinical trials along with more recent publications on targeted therapies are addressed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. Purinergic signalling and diabetes

    DEFF Research Database (Denmark)

    Burnstock, Geoffrey; Novak, Ivana

    2013-01-01

    , and common and divergent roles of receptors for nucleotides and nucleosides in different organ systems will be given. This integrated picture will aid our understanding of the challenges of the potential and currently used drugs targeted to specific organ/cells or disorders associated with diabetes.......The pancreas is an organ with a central role in nutrient breakdown, nutrient sensing and release of hormones regulating whole body nutrient homeostasis. In diabetes mellitus, the balance is broken-cells can be starving in the midst of plenty. There are indications that the incidence of diabetes...... type 1 and 2, and possibly pancreatogenic diabetes, is rising globally. Events leading to insulin secretion and action are complex, but there is emerging evidence that intracellular nucleotides and nucleotides are not only important as intracellular energy molecules but also as extracellular signalling...

  15. Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Li-Ching Fan

    2015-09-01

    Full Text Available STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC. Our previous data demonstrated that regorafenib (Stivarga is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1 that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3Tyr705 level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43–induced apoptosis and decreased p-STAT3Tyr705 level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3Tyr705 level. These data suggest that SC-43–induced apoptosis mediated through the loss of p-STAT3Tyr705 was dependent on SHP-1 function. Importantly, SC-43–enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3Tyr705 signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3Tyr705expression in CRC patients (P = .038. Patients with strong SHP-1 and weak p-STAT3Tyr705 expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3Tyr705 expression (P = .029. In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC.

  16. Immunohistochemical analysis of the mechanistic target of rapamycin and hypoxia signalling pathways in basal cell carcinoma and trichoepithelioma.

    Directory of Open Access Journals (Sweden)

    Tjinta Brinkhuizen

    Full Text Available BACKGROUND: Basal cell carcinoma (BCC is the most common cancer in Caucasians. Trichoepithelioma (TE is a benign neoplasm that strongly resembles BCC. Both are hair follicle (HF tumours. HFs are hypoxic microenvironments, therefore we hypothesized that hypoxia-induced signalling pathways could be involved in BCC and TE as they are in other human malignancies. Hypoxia-inducible factor 1 (HIF1 and mechanistic/mammalian target of rapamycin (mTOR are key players in these pathways. OBJECTIVES: To determine whether HIF1/mTOR signalling is involved in BCC and TE. METHODS: We used immunohistochemical staining of formalin-fixed paraffin-embedded BCC (n = 45 and TE (n = 35 samples to assess activity of HIF1, mTORC1 and their most important target genes. The percentage positive tumour cells was assessed manually in a semi-quantitative manner and categorized (0%, 80%. RESULTS: Among 45 BCC and 35 TE examined, expression levels were respectively 81% and 57% (BNIP3, 73% and 75% (CAIX, 79% and 86% (GLUT1, 50% and 19% (HIF1α, 89% and 88% (pAKT, 55% and 61% (pS6, 15% and 25% (pMTOR, 44% and 63% (PHD2 and 44% and 49% (VEGF-A. CAIX, Glut1 and PHD2 expression levels were significantly higher in TE when only samples with at least 80% expression were included. CONCLUSIONS: HIF and mTORC1 signalling seems active in both BCC and TE. There are no appreciable differences between the two with respect to pathway activity. At this moment immunohistochemical analyses of HIF, mTORC1 and their target genes does not provide a reliable diagnostic tool for the discrimination of BCC and TE.

  17. Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation

    DEFF Research Database (Denmark)

    Napoletano, Chiara; Zizzari, Ilaria G; Rughetti, Aurelia

    2012-01-01

    NAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL......Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner....... Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for a-N-acetylgalactosamine (Gal...

  18. Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

    Science.gov (United States)

    2013-06-01

    metabolic changes and results in muscle dystrophy . Cell Metab 8: 411–424 72. Schieke SM, Phillips D, McCoy JP, Aponte AM, Shen RF, Balaban RS, Finkel T...alterations in melting temperature following TALEN treatment and reductions in mRNA levels, indicating that mutations are produced (see example of the...peak was detected following CRISPR treatment indicating the production of mutations (see example of the targeted yellow gene in Figure 2D

  19. Targeting activator protein 1 signaling pathway by bioactive natural agents: Possible therapeutic strategy for cancer prevention and intervention.

    Science.gov (United States)

    Tewari, Devesh; Nabavi, Seyed Fazel; Nabavi, Seyed Mohammad; Sureda, Antoni; Farooqi, Ammad Ahmad; Atanasov, Atanas G; Vacca, Rosa Anna; Sethi, Gautam; Bishayee, Anupam

    2018-02-01

    Activator protein 1 (AP-1) is a key transcription factor in the control of several cellular processes responsible for cell survival proliferation and differentiation. Dysfunctional AP-1 expression and activity are involved in several severe diseases, especially inflammatory disorders and cancer. Therefore, targeting AP-1 has recently emerged as an attractive therapeutic strategy for cancer prevention and therapy. This review summarizes our current understanding of AP-1 biology and function as well as explores and discusses several natural bioactive compounds modulating AP-1-associated signaling pathways for cancer prevention and intervention. Current limitations, challenges, and future directions of research are also critically discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Chloroquine targets pancreatic cancer stem cells via inhibition of CXCR4 and hedgehog signaling

    DEFF Research Database (Denmark)

    Balic, Anamaria; Sørensen, Morten Dræby; Trabulo, Sara Maria

    2014-01-01

    effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent...... is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer...

  1. The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Eskildsen, Tilde V; Schneider, Mikael; Sandberg, Maria B

    2015-01-01

    INTRODUCTION: MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular...... in silico and in vitro experiments to identify miR-132/212 molecular targets in primary rat cardiac fibroblasts. RESULTS: MiR-132/212 overexpression increased fibroblast cell size and mRNA arrays detected several hundred genes that were differentially expressed, including a wide panel of receptors...... pathways that fine-tuned by miR-132/212, suggesting a role for this miRNA family as master signalling switches in cardiac fibroblasts. Our data underscore the potential for miRNA tools to manipulate a large array of molecules and thereby control biological function....

  2. HLH-29 regulates ovulation in C. elegans by targeting genes in the inositol triphosphate signaling pathway

    Directory of Open Access Journals (Sweden)

    Ana White

    2012-02-01

    The reproductive cycle in the nematode Caenorhabditis elegans depends in part on the ability of the mature oocyte to ovulate into the spermatheca, fuse with the sperm during fertilization, and then exit the spermatheca as a fertilized egg. This cycle requires the integration of signals between the germ cells and the somatic gonad and relies heavily on the precise control of inositol 1,4,5 triphosphate (IP3levels. The HLH-29 protein, one of five Hairy/Enhancer of Split (HES homologs in C. elegans, was previously shown to affect development of the somatic gonad. Here we show that HLH-29 expression in the adult spermatheca is strongly localized to the distal spermatheca valve and to the spermatheca-uterine valve, and that loss of hlh-29 activity interferes with oocyte entry into and egg exit from the spermatheca. We show that HLH-29 can regulate the transcriptional activity of the IP3 signaling pathway genes ppk-1, ipp-5, and plc-1 and provide evidence that hlh-29 acts in a genetic pathway with each of these genes. We propose that the HES-like protein HLH-29 acts in the spermatheca of larval and adult animals to effectively increase IP3 levels during the reproductive cycle.

  3. Ultrasound-targeted microbubble destruction enhances delayed BMC delivery and attenuates post-infarction cardiac remodelling by inducing engraftment signals.

    Science.gov (United States)

    Chen, Yanmei; Zhang, Chuanxi; Shen, Shuxin; Guo, Shengcun; Zhong, Lintao; Li, Xinzhong; Chen, Guojun; Chen, Gangbin; He, Xiang; Huang, Chixiong; He, Nvqin; Liao, Wangjun; Liao, Yulin; Bin, Jianping

    2016-12-01

    Delayed administration of bone marrow cells (BMCs) at 2-4 weeks after successful reperfusion in patients with acute myocardial infarction (MI) does not improve cardiac function. The reduction in engraftment signals observed following this time interval might impair the effects of delayed BMC treatment. In the present study, we aimed to determine whether ultrasound-targeted microbubble destruction (UTMD) treatment could increase engraftment signals, enhance the delivery of delayed BMCs and subsequently attenuate post-infarction cardiac remodelling. A myocardial ischaemia/reperfusion (I/R) model was induced in Wistar rats via left coronary ligation for 45 min followed by reperfusion. Western blotting revealed that engraftment signals peaked at 7 days post-I/R and were dramatically lower at 14 days post-I/R. The lower engraftment signals at 14 days post-I/R could be triggered by UTMD treatment at a mechanical index of 1.0-1.9. The troponin I levels in the 1.9 mechanical index group were higher than in the other groups. Simultaneous haematoxylin and eosin staining and fluorescence revealed that the number of engrafted BMCs in the ischaemic zone was greater in the group treated with both UTMD and delayed BMC transplantation than in the control groups (PBMC transplantation improved cardiac function and decreased cardiac fibrosis at 4 weeks after treatment, as compared with control groups (both PBMC transplantation increased capillary density, myocardial cell proliferation and c-kit + cell proliferation. These findings indicated that UTMD treatment could induce engraftment signals and enhance homing of delayed BMCs to ischaemic myocardium, attenuating post-infarction cardiac remodelling by promoting neovascularization, cardiomyogenesis and expansion of cardiac c-kit + cells. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  4. Crosstalk between bone niche and immune system: osteoimmunology signaling as a potential target for cancer treatment.

    Science.gov (United States)

    Criscitiello, Carmen; Viale, Giulia; Gelao, Lucia; Esposito, Angela; De Laurentiis, Michele; De Placido, Sabino; Santangelo, Michele; Goldhirsch, Aron; Curigliano, Giuseppe

    2015-02-01

    There is a well recognized link between the bone and the immune system and in recent years there has been a major effort to elucidate the multiple functions of the molecules expressed in both bone and immune cells. Several molecules that were initially identified and studied in the immune system have been shown to have essential functions also in the bone. An interdisciplinary field embracing immune and bone biology has been brought together and called "osteoimmunology". The co-regulation of the skeletal and immune systems strikingly exemplifies the extreme complexity of such an interaction. Their interdependency must be considered in designing therapeutic approaches for either of the two systems. In other words, it is necessary to think of the osteoimmune system as a complex physiological unit. Denosumab was originally introduced to specifically target bone resorption, but it is now under evaluation for its effect on the long term immune response. Similarly, our current and still growing knowledge of the intimate link between the immune system and bone will be beneficial for the safety of drugs targeting either of these integrated systems. Given the large number of molecules exerting functions on both the skeletal and immune systems, osteoimmunological understanding is becoming increasingly important. Both bone and immune systems are frequently disrupted in cancer; and they may be crucial in regulating tumor growth and progression. Some therapies - such as bisphosphonates and receptor activator of NF-κB ligand (RANKL) targeted drugs - that aim at reducing pathologic osteolysis in cancer may interact with the immune system, thus providing potential favorable effects on survival. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids

    DEFF Research Database (Denmark)

    Valls, L A; Winther, Jakob R.; Stevens, T H

    1990-01-01

    The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations...... sort and deliver only the wild-type molecule to the vacuole. These results indicate that the PRC1 missorting mutations are cis-dominant, implying that the mutant forms of proCPY are secreted as a consequence of failing to interact with the sorting apparatus, rather than a general poisoning...

  6. Targeting Ligand Dependent and Ligand Independent Androgen Receptor Signaling in Prostate Cancer

    Science.gov (United States)

    2014-10-01

    flanking residues around the LXXLL motif to our lead D2 compound. * To facilitate the synthesis of tris-benzamide analogues designed to target AR...we used only alkyl groups such as n- propyl , isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl and isopentyl groups for side chain diversity of the...by reacting 3-alkoxy-4-nitrobenzoyl chlorides 2 with methyl 3-alkoxy-4-aminobenzoates 3 (Scheme 1).21 NO2 O NHO R2 O R1 O O NH2 O R1 O Oa N H NO2 O

  7. Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

    Science.gov (United States)

    Tkatch, Tatiana; Greotti, Elisa; Baranauskas, Gytis; Pendin, Diana; Roy, Soumitra; Nita, Luliaoana I; Wettmarshausen, Jennifer; Prigge, Matthias; Yizhar, Ofer; Shirihai, Orian S; Fishman, Daniel; Hershfinkel, Michal; Fleidervish, Ilya A; Perocchi, Fabiana; Pozzan, Tullio; Sekler, Israel

    2017-06-27

    Key mitochondrial functions such as ATP production, Ca 2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH + ) across the inner membrane. Although several drugs can modulate ΔμH + , their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψ m ) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca 2+ dynamics, and respiratory metabolism. By directly modulating Δψ m , the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

  8. Targeting Adenosine Signaling in Parkinson's Disease: From Pharmacological to Non-pharmacological Approaches

    Directory of Open Access Journals (Sweden)

    Luiza R. Nazario

    2017-11-01

    Full Text Available Parkinson's disease (PD is one of the most prevalent neurodegenerative disease displaying negative impacts on both the health and social ability of patients and considerable economical costs. The classical anti-parkinsonian drugs based in dopaminergic replacement are the standard treatment, but several motor side effects emerge during long-term use. This mini-review presents the rationale to several efforts from pre-clinical and clinical studies using adenosine receptor antagonists as a non-dopaminergic therapy. As several studies have indicated that the monotherapy with adenosine receptor antagonists reaches limited efficacy, the usage as a co-adjuvant appeared to be a promising strategy. The formulation of multi-targeted drugs, using adenosine receptor antagonists and other neurotransmitter systems than the dopaminergic one as targets, have been receiving attention since Parkinson's disease presents a complex biological impact. While pharmacological approaches to cure or ameliorate the conditions of PD are the leading strategy in this area, emerging positive aspects have arisen from non-pharmacological approaches and adenosine function inhibition appears to improve both strategies.

  9. Targeting the PI3K signaling pathway in KRAS mutant colon cancer

    International Nuclear Information System (INIS)

    Hong, Suntaek; Kim, SoYoung; Kim, Hye Youn; Kang, Myunghee; Jang, Ho Hee; Lee, Won-Suk

    2015-01-01

    Metastatic colorectal cancer (CRC) patients with v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are resistant to monoclonal antibody that targets the epidermal growth factor receptor such as cetuximab. BKM120 targets phosphatidylinositide-3-kinase (PIK3CA), but it is unknown whether BKM120 can reverse cetuximab resistance in KRAS mutant CRC. Human CRC cell lines with KRAS mutations (DLD-1, HCT116, and LoVo) were used to test the effect of cetuximab, BKM120, and cetuximab plus BKM120 on cell proliferation in vitro and in vivo. BKM120 reduced cell proliferation in a concentration-dependent manner in the LoVo (PI3KCA wild type) as well as the HCT116 and DLD1 cells (that carry a PI3KCA mutation). BKM120 only inhibited ERK phosphorylation in LoVo cells (PIK3CA wild type), but not in DLD1 or HCT116 cells at a concentration of 1 μmol/L. Treatment with cetuximab and BKM120 significantly reduced the growth of xenograft tumors originating from KRAS mutant cells compared with cetuximab alone (P = 0.034). BKM120 may overcome cetuximab resistance in colon cancer cells with KRAS mutation

  10. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

    Science.gov (United States)

    Rivera, Mariela; Ramos, Yanilda; Rodríguez-Valentín, Madeline; López-Acevedo, Sheila; Cubano, Luis A.; Zou, Jin; Zhang, Qiang; Wang, Guangdi

    2017-01-01

    Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses. PMID:28628644

  11. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Mariela Rivera

    Full Text Available Curcumin, an extract from the turmeric rhizome (Curcuma longa, is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12 and Poly (ADP-ribose polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

  12. Targeting TGF-β Signaling by Antisense Oligonucleotide-mediated Knockdown of TGF-β Type I Receptor

    Directory of Open Access Journals (Sweden)

    Dwi U Kemaladewi

    2014-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5. Antisense oligonucleotides (AONs were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD.

  13. The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

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    Bond Charles S

    2011-03-01

    Full Text Available Abstract Background The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. Results To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. Conclusions Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.

  14. Peroxisome Proliferator-Activated Receptor-γ Ligands: Potential Pharmacological Agents for Targeting the Angiogenesis Signaling Cascade in Cancer

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    Costas Giaginis

    2008-01-01

    Full Text Available Peroxisome proliferator-activated receptor-γ (PPAR-γ has currently been considered as molecular target for the treatment of human metabolic disorders. Experimental data from in vitro cultures, animal models, and clinical trials have shown that PPAR-γ ligand activation regulates differentiation and induces cell growth arrest and apoptosis in a variety of cancer types. Tumor angiogenesis constitutes a multifaceted process implicated in complex downstream signaling pathways that triggers tumor growth, invasion, and metastasis. In this aspect, accumulating in vitro and in vivo studies have provided extensive evidence that PPAR-γ ligands can function as modulators of the angiogenic signaling cascade. In the current review, the crucial role of PPAR-γ ligands and the underlying mechanisms participating in tumor angiogenesis are summarized. Targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments with conventional chemotherapy; however, special attention should be taken as there is also substantial evidence to support that PPAR-γ ligands can enhance angiogenic phenotype in tumoral cells.

  15. THE HYDROGENOSOMAL ENZYME HYDROGENASE FROM THE ANAEROBIC FUNGUS NEOCALLIMASTIX SP L2 IS RECOGNIZED BY ANTIBODIES, DIRECTED AGAINST THE C-TERMINAL MICROBODY PROTEIN TARGETING SIGNAL SKL

    NARCIS (Netherlands)

    MARVINSIKKEMA, FD; KRAAK, MN; VEENHUIS, M; GOTTSCHAL, JC; PRINS, RA

    The question was addressed whether antibodies directed against the general microbody C-terminal protein targeting signal SKL recognized hydrogenosomal proteins from Neocallimastix sp. L2. Immunofluorescence, immunocytochemistry and Western blotting experiments using these antibodies indicated the

  16. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

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    Binod Kumar

    2016-11-01

    Full Text Available Kaposi’s sarcoma associated herpesvirus (KSHV is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS, integrins (α3β1, αVβ3 and αVβ5, and EphA2 receptor tyrosine kinase (EphA2R. This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR, inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of

  17. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma.

    Science.gov (United States)

    Saha, Manujendra N; Jiang, Hua; Yang, Yijun; Zhu, Xiaoyun; Wang, Xiaoming; Schimmer, Aaron D; Qiu, Lugui; Chang, Hong

    2012-01-01

    The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK

  18. Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma.

    Directory of Open Access Journals (Sweden)

    Manujendra N Saha

    Full Text Available The low frequency of p53 alterations e.g., mutations/deletions (∼10% in multiple myeloma (MM makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP analysis showed that activated c-Jun binds to the activator protein-1 (AP-1 binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with

  19. Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo

    DEFF Research Database (Denmark)

    Hönes, G. Sebastian; Rakov, Helena; Logan, John

    2017-01-01

    Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger s...

  20. MicroRNA-1297 inhibits prostate cancer cell proliferation and invasion by targeting the AEG-1/Wnt signaling pathway

    International Nuclear Information System (INIS)

    Liang, Xuan; Li, Hecheng; Fu, Delai; Chong, Tie; Wang, Ziming; Li, Zhaolun

    2016-01-01

    MicroRNAs (miRNAs) have been known to be implicated in tumorigenic programs. miR-1297 has been reported to be dysregulated and involved in cancer progression in many types of human cancers. However, the expression level and the role of miR-1297 in prostate cancer remain unclear. Herein, we aimed to investigate the potential role and molecular mechanism of miR-1297 in prostate cancer progression. We found that miR-1297 was significantly downregulated in human prostate cancer specimens as well as in several prostate cancer cell lines. In addition, functional experiments demonstrated that overexpression of miR-1297 remarkably inhibited prostate cancer cell proliferation and invasion whereas miR-1297 suppression significantly promoted prostate cancer cell proliferation and invasion. Bioinformatics analysis showed that the Astrocyte elevated gene-1 (AEG-1), a well-known oncogene, is a predicted target of miR-1297. Dual-luciferase reporter assay showed that miR-1297 was able to directly target the 3’-untranslated region of AEG-1. In addition, RT-qPCR and Western blot analysis showed that miR-1297 regulated the mRNA and protein expression levels of AEG-1. We also showed that miR-1297 was able to regulate the Wnt signaling pathway. Moreover, rescue assays indicated that AEG-1 contributed to miR-1297-endowed effects on cell proliferation and invasion as well as Wnt signaling pathway. Taken together, these findings suggest that miR-1297 inhibits prostate cancer proliferation and invasion by targeting AEG-1, thereby providing novel insight into understanding the pathogenesis of prostate cancer. Thus, miR-1297 may be a novel potential therapeutic candidate to treat prostate cancer. - Highlights: • miR-1297 is decreased in prostate cancer. • miR-1297 inhibits prostate cancer cell proliferation and invasion. • miR-1297 targets and inhibits AEG-1. • miR-1297 regulates AEG-1/Wnt signaling pathway.

  1. The crosstalk between Target of Rapamycin (TOR) and Jasmonic Acid (JA) signaling existing in Arabidopsis and cotton.

    Science.gov (United States)

    Song, Yun; Zhao, Ge; Zhang, Xueyan; Li, Linxuan; Xiong, Fangjie; Zhuo, Fengping; Zhang, Chaojun; Yang, Zuoren; Datla, Raju; Ren, Maozhi; Li, Fuguang

    2017-04-04

    Target of rapamycin (TOR) acts as an important regulator of cell growth, development and stress responses in most examined diploid eukaryotes. However, little is known about TOR in tetraploid species such as cotton. Here, we show that TORC1-S6K-RPS6, the major signaling components, are conserved and further expanded in cotton genome. Though the cotton seedlings are insensitive to rapamycin, AZD8055, the second-generation inhibitor of TOR, can significantly suppress the growth in cotton. Global transcriptome analysis revealed that genes associated with jasmonic acid (JA) biosynthesis and transduction were significantly altered in AZD8055 treated cotton seedlings, suggesting the potential crosstalk between TOR and JA signaling. Pharmacological and genetic approaches have been employed to get further insights into the molecular mechanism of the crosstalk between TOR and JA. Combination of AZD8055 with methyl jasmonate can synergistically inhibit cotton growth, and additionally JA levels were significantly increased when cotton seedlings were subjected to AZD8055. JA biosynthetic and signaling mutants including jar1, coi1-2 and myc2-2 displayed TOR inhibitor-resistant phenotypes, whereas COI1 overexpression transgenic lines and jaz10 exhibited sensitivity to AZD8055. Consistently, cotton JAZ can partially rescue TOR-suppressed phenotypes in Arabidopsis. These evidences revealed that the crosstalk between TOR and JA pathway operates in cotton and Arabidopsis.

  2. R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m6A/MYC/CEBPA Signaling.

    Science.gov (United States)

    Su, Rui; Dong, Lei; Li, Chenying; Nachtergaele, Sigrid; Wunderlich, Mark; Qing, Ying; Deng, Xiaolan; Wang, Yungui; Weng, Xiaocheng; Hu, Chao; Yu, Mengxia; Skibbe, Jennifer; Dai, Qing; Zou, Dongling; Wu, Tong; Yu, Kangkang; Weng, Hengyou; Huang, Huilin; Ferchen, Kyle; Qin, Xi; Zhang, Bin; Qi, Jun; Sasaki, Atsuo T; Plas, David R; Bradner, James E; Wei, Minjie; Marcucci, Guido; Jiang, Xi; Mulloy, James C; Jin, Jie; He, Chuan; Chen, Jianjun

    2018-01-11

    R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N 6 -methyladenosine (m 6 A) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m 6 A/MYC/CEBPA signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma.

    Science.gov (United States)

    Peponi, Evangelia; Drakos, Elias; Reyes, Guadalupe; Leventaki, Vasiliki; Rassidakis, George Z; Medeiros, L Jeffrey

    2006-12-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

  4. Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.

    Directory of Open Access Journals (Sweden)

    Mariana Rodova

    Full Text Available Dysregulation of the sonic hedgehog (Shh signaling pathway has been associated with cancer stem cells (CSC and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN, an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4 as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway

  5. Synergistic effects of targeted PI3K signaling inhibition and chemotherapy in liposarcoma.

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    Shang Guo

    Full Text Available While liposarcoma is the second most common soft tissue malignant tumor, the molecular pathogenesis in this malignancy is poorly understood. Our goal was therefore to expand the understanding of molecular mechanisms that drive liposarcoma and identify therapeutically-susceptible genetic alterations. We studied a cohort of high-grade liposarcomas and benign lipomas across multiple disease sites, as well as two liposarcoma cell lines, using multiplexed mutational analysis. Nucleic acids extracted from diagnostic patient tissue were simultaneously interrogated for 150 common mutations across 15 essential cancer genes using a clinically-validated platform for cancer genotyping. Western blot analysis was implemented to detect activation of downstream pathways. Liposarcoma cell lines were used to determine the effects of PI3K targeted drug treatment with or without chemotherapy. We identified mutations in the PIK3CA gene in 4 of 18 human liposarcoma patients (22%. No PIK3CA mutations were identified in benign lipomas. Western blot analysis confirmed downstream activation of AKT in both PIK3CA mutant and non-mutant liposarcoma samples. PI-103, a dual PI3K/mTOR inhibitor, effectively inhibited the activation of the PI3K/AKT in liposarcoma cell lines and induced apoptosis. Importantly, combination with PI-103 treatment strongly synergized the growth-inhibitory effects of the chemotherapy drugs doxorubicin and cisplatin in liposarcoma cells. Taken together, these findings suggest that activation of the PI3K/AKT pathway is an important cancer mechanism in liposarcoma. Targeting the PI3K/AKT/pathway with small molecule inhibitors in combination with chemotherapy could be exploited as a novel strategy in the treatment of liposarcoma.

  6. Induction of biogenic magnetization and redox control by a component of the target of rapamycin complex 1 signaling pathway.

    Directory of Open Access Journals (Sweden)

    Keiji Nishida

    Full Text Available Most organisms are simply diamagnetic, while magnetotactic bacteria and migratory animals are among organisms that exploit magnetism. Biogenic magnetization not only is of fundamental interest, but also has industrial potential. However, the key factor(s that enable biogenic magnetization in coordination with other cellular functions and metabolism remain unknown. To address the requirements for induction and the application of synthetic bio-magnetism, we explored the creation of magnetism in a simple model organism. Cell magnetization was first observed by attraction towards a magnet when normally diamagnetic yeast Saccharomyces cerevisiae were grown with ferric citrate. The magnetization was further enhanced by genetic modification of iron homeostasis and introduction of ferritin. The acquired magnetizable properties enabled the cells to be attracted to a magnet, and be trapped by a magnetic column. Superconducting quantum interference device (SQUID magnetometry confirmed and quantitatively characterized the acquired paramagnetism. Electron microscopy and energy-dispersive X-ray spectroscopy showed electron-dense iron-containing aggregates within the magnetized cells. Magnetization-based screening of gene knockouts identified Tco89p, a component of TORC1 (Target of rapamycin complex 1, as important for magnetization; loss of TCO89 and treatment with rapamycin reduced magnetization in a TCO89-dependent manner. The TCO89 expression level positively correlated with magnetization, enabling inducible magnetization. Several carbon metabolism genes were also shown to affect magnetization. Redox mediators indicated that TCO89 alters the intracellular redox to an oxidized state in a dose-dependent manner. Taken together, we demonstrated that synthetic induction of magnetization is possible and that the key factors are local redox control through carbon metabolism and iron supply.

  7. Targeting Fibroblast Growth Factor 23 Signaling with Antibodies and Inhibitors, Is There a Rationale?

    Directory of Open Access Journals (Sweden)

    Seiji Fukumoto

    2018-02-01

    Full Text Available Fibroblast growth factor 23 (FGF23 is a phosphotropic hormone mainly produced by bone. FGF23 reduces serum phosphate by suppressing intestinal phosphate absorption through reducing 1,25-dihydroxyvitamin D and proximal tubular phosphate reabsorption. Excessive actions of FG23 result in several kinds of hypophosphatemic rickets/osteomalacia including X-linked hypophosphatemic rickets (XLH and tumor-induced osteomalacia. While neutral phosphate and active vitamin D are standard therapies for child patients with XLH, these medications have several limitations both in their effects and adverse events. Several approaches that inhibit FGF23 actions including anti-FGF23 antibodies and inhibitors of FGF signaling have been shown to improve phenotypes of model mice for FG23-related hypophosphatemic diseases. In addition, clinical trials indicated that a humanized anti-FGF23 antibody increased serum phosphate and improved quality of life in patients with XLH. Furthermore, circulatory FGF23 is high in patients with chronic kidney disease (CKD. Many epidemiological studies indicated the association between high FGF23 levels and various adverse events especially in patients with CKD. However, it is not known whether the inhibition of FGF23 activities in patients with CKD is beneficial for these patients. In this review, recent findings concerning the modulation of FGF23 activities are discussed.

  8. Therapeutic Potential of Thymoquinone in Glioblastoma Treatment: Targeting Major Gliomagenesis Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Fabliha Ahmed Chowdhury

    2018-01-01

    Full Text Available Glioblastoma multiforme (GBM is one of the most devastating brain tumors with median survival of one year and presents unique challenges to therapy because of its aggressive behavior. Current treatment strategy involves surgery, radiotherapy, immunotherapy, and adjuvant chemotherapy even though optimal management requires a multidisciplinary approach and knowledge of potential complications from both the disease and its treatment. Thymoquinone (TQ, the main bioactive component of Nigella sativa L., has exhibited anticancer effects in numerous preclinical studies. Due to its multitargeting nature, TQ interferes in a wide range of tumorigenic processes and counteract carcinogenesis, malignant growth, invasion, migration, and angiogenesis. TQ can specifically sensitize tumor cells towards conventional cancer treatments and minimize therapy-associated toxic effects in normal cells. Its potential to enter brain via nasal pathway due to volatile nature of TQ adds another advantage in overcoming blood-brain barrier. In this review, we summarized the potential role of TQ in different signaling pathways in GBM that have undergone treatment with standard therapeutic modalities or with TQ. Altogether, we suggest further comprehensive evaluation of TQ in preclinical and clinical level to delineate its implied utility as novel therapeutics to combat the challenges for the treatment of GBM.

  9. Reverting doxorubicin resistance in colon cancer by targeting a key signaling protein, steroid receptor coactivator.

    Science.gov (United States)

    Xiong, Sang; Xiao, Gong-Wei

    2018-04-01

    Although there have been notable improvements in treatments against cancer, further research is required. In colon cancer, nearly all patients eventually experience drug resistance and stop responding to the approved drugs, making treatment difficult. Steroid receptor coactivator (SRC) is an oncogenic nuclear receptor coactivator that serves an important role in drug resistance. The present study generated a doxorubicin-resistant colon cancer cell line, in which the upregulation/activation of SRC was responsible for drug resistance, which in turn activated AKT. Overexpression of receptor tyrosine kinase-like epidermal growth factor receptor and insulin-like growth factor 1 receptor also induced SRC expression. It was observed that doxorubicin resistance in colon cancer also induced epithelial to mesenchymal transition, a decrease in expression of epithelial marker E-cadherin and an increase in the expression of mesenchymal markers, including N-cadherin and vimentin. Additionally, the present study indicated that SRC acts as a common signaling node, and inhibiting SRC in combination with doxorubicin treatment in doxorubicin-resistant cells aids in reversing the resistance. Thus, the present study suggests that activation of SRC is responsible for doxorubicin resistance in colon cancer. However, further research is required to understand the complete mechanism of how drug resistance occurs and how it may be tackled to treat patients.

  10. Kaempferol inhibits cell proliferation and glycolysis in esophagus squamous cell carcinoma via targeting EGFR signaling pathway.

    Science.gov (United States)

    Yao, Shihua; Wang, Xiaowei; Li, Chunguang; Zhao, Tiejun; Jin, Hai; Fang, Wentao

    2016-08-01

    Antitumor activity of kaempferol has been studied in various tumor types, but its potency in esophagus squamous cell carcinoma is rarely known. Here, we reported the activity of kaempferol against esophagus squamous cell carcinoma as well as its antitumor mechanisms. Results of cell proliferation and colony formation assay showed that kaempferol substantially inhibited tumor cell proliferation and clone formation in vitro. Flow cytometric analysis demonstrated that tumor cells were induced G0/G1 phase arrest after kaempferol treatment, and the expression of protein involved in cell cycle regulation was dramatically changed. Except the potency on cell proliferation, we also discovered that kaempferol had a significant inhibitory effect against tumor glycolysis. With the downregulation of hexokinase-2, glucose uptake and lactate production in tumor cells were dramatically declined. Mechanism studies revealed kaempferol had a direct effect on epidermal growth factor receptor (EGFR) activity, and along with the inhibition of EGFR, its downstream signaling pathways were also markedly suppressed. Further investigations found that exogenous overexpression of EGFR in tumor cells substantially attenuated glycolysis suppression induced by kaempferol, which implied that EGFR also played an important role in kaempferol-mediated glycolysis inhibition. Finally, the antitumor activity of kaempferol was validated in xenograft model and kaempferol prominently restrained tumor growth in vivo. Meanwhile, dramatic decrease of EGFR activity and hexokinase-2 expression were observed in kaempferol-treated tumor tissue, which confirmed these findings in vitro. Briefly, these studies suggested that kaempferol, or its analogues, may serve as effective candidates for esophagus squamous cell carcinoma management.

  11. Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways and novel therapeutic targets.

    Science.gov (United States)

    Tham, Yow Keat; Bernardo, Bianca C; Ooi, Jenny Y Y; Weeks, Kate L; McMullen, Julie R

    2015-09-01

    The onset of heart failure is typically preceded by cardiac hypertrophy, a response of the heart to increased workload, a cardiac insult such as a heart attack or genetic mutation. Cardiac hypertrophy is usually characterized by an increase in cardiomyocyte size and thickening of ventricular walls. Initially, such growth is an adaptive response to maintain cardiac function; however, in settings of sustained stress and as time progresses, these changes become maladaptive and the heart ultimately fails. In this review, we discuss the key features of pathological cardiac hypertrophy and the numerous mediators that have been found to be involved in the pathogenesis of cardiac hypertrophy affecting gene transcription, calcium handling, protein synthesis, metabolism, autophagy, oxidative stress and inflammation. We also discuss new mediators including signaling proteins, microRNAs, long noncoding RNAs and new findings related to the role of calcineurin and calcium-/calmodulin-dependent protein kinases. We also highlight mediators and processes which contribute to the transition from adaptive cardiac remodeling to maladaptive remodeling and heart failure. Treatment strategies for heart failure commonly include diuretics, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers and β-blockers; however, mortality rates remain high. Here, we discuss new therapeutic approaches (e.g., RNA-based therapies, dietary supplementation, small molecules) either entering clinical trials or in preclinical development. Finally, we address the challenges that remain in translating these discoveries to new and approved therapies for heart failure.

  12. A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.

    Science.gov (United States)

    Min, Junseon; Song, Eun Kyung; Kim, Hansol; Kim, Kyoung Taek; Park, Tae Joo; Kang, Sebyung

    2016-04-11

    We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.

  13. Oligomerization of Paramagnetic Substrates Result in Signal Amplification and Can be Used for MR Imaging of Molecular Targets

    Directory of Open Access Journals (Sweden)

    Alexei Bogdanov

    2002-01-01

    Full Text Available Magnetic resonance imaging (MRI has evolved into a sophisticated, noninvasive imaging modality capable of high-resolution anatomical and functional characterization of transgenic animals. To expand the capabilities MRI, we have developed a novel MR signal amplification (MRamp strategy based on enzyme-mediated polymerization of paramagnetic substrates into oligomers of higher magnetic relaxivity. The substrates consist of chelated gadolinium covalently bound to phenols, which then serve as electron donors during enzymatic hydrogen peroxide reduction by peroxidase. The converted monomers undergo rapid condensation into paramagnetic oligomers leading to a threefold increase in atomic relaxivity (R1/Gd. The observed relaxivity changes are largely due to an increase in the rotational correlation time τr of the lanthanide. Three applications of the developed system are demonstrated: (1 imaging of nanomolar amounts of an oxidoreductase (peroxidase; (2 detection of a model ligand using an enzyme-linked immunoadsorbent assay format; and (3 imaging of E-selectin on the surface of endothelial cells probed for with an anti-E-selectin – peroxidase conjugate. The development of “enzyme sensing” probes is expected to have utility for a number of applications including in vivo detection of specific molecular targets. One particular advantage of the MRamp technique is that the same paramagnetic substrate can be potentially used to identify different molecular targets by attaching enzymes to various antibodies or other target-seeking molecules.

  14. Adrenergic Signaling: A Targetable Checkpoint Limiting Development of the Antitumor Immune Response

    Science.gov (United States)

    Qiao, Guanxi; Chen, Minhui; Bucsek, Mark J.; Repasky, Elizabeth A.; Hylander, Bonnie L.

    2018-01-01

    An immune response must be tightly controlled so that it will be commensurate with the level of response needed to protect the organism without damaging normal tissue. The roles of cytokines and chemokines in orchestrating these processes are well known, but although stress has long been thought to also affect immune responses, the underlying mechanisms were not as well understood. Recently, the role of nerves and, specifically, the sympathetic nervous system, in regulating immune responses is being revealed. Generally, an acute stress response is beneficial but chronic stress is detrimental because it suppresses the activities of effector immune cells while increasing the activities of immunosuppressive cells. In this review, we first discuss the underlying biology of adrenergic signaling in cells of both the innate and adaptive immune system. We then focus on the effects of chronic adrenergic stress in promoting tumor growth, giving examples of effects on tumor cells and immune cells, explaining the methods commonly used to induce stress in preclinical mouse models. We highlight how this relates to our observations that mandated housing conditions impose baseline chronic stress on mouse models, which is sufficient to cause chronic immunosuppression. This problem is not commonly recognized, but it has been shown to impact conclusions of several studies of mouse physiology and mouse models of disease. Moreover, the fact that preclinical mouse models are chronically immunosuppressed has critical ramifications for analysis of any experiments with an immune component. Our group has found that reducing adrenergic stress by housing mice at thermoneutrality or treating mice housed at cooler temperatures with β-blockers reverses immunosuppression and significantly improves responses to checkpoint inhibitor immunotherapy. These observations are clinically relevant because there are numerous retrospective epidemiological studies concluding that cancer patients who were

  15. A concerted kinase interplay identifies PPARgamma as a molecular target of ghrelin signaling in macrophages.

    Directory of Open Access Journals (Sweden)

    Annie Demers

    2009-11-01

    Full Text Available The peroxisome proliferator-activator receptor PPARgamma plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARgamma. Although the interplay between CD36 and PPARgamma in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARgamma remains unknown. Here, we demonstrate that ghrelin triggers PPARgamma activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRalpha and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARgamma phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARgamma Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARgamma activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARgamma response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Galphaq-dependent manner, resulting in Akt recruitment to PPARgamma, enhanced PPARgamma phosphorylation and activation independently of Ser-84, and increased expression of LXRalpha and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Galphaq/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARgamma to ghrelin in macrophages.

  16. MAVS dimer is a crucial signaling component of innate immunity and the target of hepatitis C virus NS3/4A protease.

    Science.gov (United States)

    Baril, Martin; Racine, Marie-Eve; Penin, François; Lamarre, Daniel

    2009-02-01

    The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-kappaB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-kappaB in antiviral innate immunity.

  17. A pH and solvent optimized reverse-phase ion-paring-LC–MS/MS method that leverages multiple scan-types for targeted absolute quantification of intracellular metabolites

    DEFF Research Database (Denmark)

    McCloskey, Douglas; Gangoiti, Jon A.; Palsson, Bernhard O.

    2015-01-01

    Comprehensive knowledge of intracellular biochemistry is needed to accurately understand, model, and manipulate metabolism for industrial and therapeutic applications. Quantitative metabolomics has been driven by advances in analytical instrumentation and can add valuable knowledge to the underst......Comprehensive knowledge of intracellular biochemistry is needed to accurately understand, model, and manipulate metabolism for industrial and therapeutic applications. Quantitative metabolomics has been driven by advances in analytical instrumentation and can add valuable knowledge...... existing reverse phase ion-paring liquid chromatography methods for separation and detection of polar and anionic compounds that comprise key nodes of intracellular metabolism by optimizing pH and solvent composition. In addition, the presented method utilizes multiple scan types provided by hybrid...

  18. An Epstein-Barr Virus MicroRNA Blocks Interleukin-1 (IL-1) Signaling by Targeting IL-1 Receptor 1.

    Science.gov (United States)

    Skinner, Camille M; Ivanov, Nikita S; Barr, Sarah A; Chen, Yan; Skalsky, Rebecca L

    2017-11-01

    Epstein-Barr virus (EBV) encodes >44 viral microRNAs (miRNAs) that are differentially expressed throughout infection, can be detected in Epstein-Barr virus (EBV)-positive tumors, and manipulate several biological processes, including cell proliferation, apoptosis, and immune responses. Here, we show that EBV BHRF1-2 miRNAs block NF-κB activation following treatment with proinflammatory cytokines, specifically interleukin-1β (IL-1β). Analysis of EBV PAR-CLIP miRNA targetome data sets combined with pathway analysis revealed multiple BHRF1-2 miRNA targets involved in interleukin signaling pathways. By further analyzing changes in cellular gene expression patterns, we identified the IL-1 receptor 1 (IL1R1) as a direct target of miR-BHRF1-2-5p. Targeting the IL1R1 3' untranslated region (UTR) by EBV miR-BHRF1-2-5p was confirmed using 3'-UTR luciferase reporter assays and Western blot assays. Manipulation of EBV BHRF1-2 miRNA activity in latently infected B cells altered steady-state cytokine levels and disrupted IL-1β responsiveness. These studies demonstrate functionally relevant BHRF1-2 miRNA interactions during EBV infection, which is an important step in understanding their roles in pathogenesis. IMPORTANCE IL-1 signaling plays an important role in inflammation and early activation of host innate immune responses following virus infection. Here, we demonstrate that a viral miRNA downregulates the IL-1 receptor 1 during EBV infection, which consequently alters the responsiveness of cells to IL-1 stimuli and changes the cytokine expression levels within infected cell populations. We postulate that this viral miRNA activity not only disrupts IL-1 autocrine and paracrine signaling loops that can alert effector cells to sites of infection but also provides a survival advantage by dampening excessive inflammation that may be detrimental to the infected cell. Copyright © 2017 American Society for Microbiology.

  19. Theobromine, the primary methylxanthine found in Theobroma cacao, prevents malignant glioblastoma proliferation by negatively regulating phosphodiesterase-4, extracellular signal-regulated kinase, Akt/mammalian target of rapamycin kinase, and nuclear factor-kappa B.

    Science.gov (United States)

    Sugimoto, Naotoshi; Miwa, Shinji; Hitomi, Yoshiaki; Nakamura, Hiroyuki; Tsuchiya, Hiroyuki; Yachie, Akihiro

    2014-01-01

    Theobromine, a caffeine derivative, is the primary methylxanthine produced by Theobroma cacao. We previously showed that methylxanthines, including caffeine and theophylline, have antitumor and antiinflammatory effects, which are in part mediated by their inhibition of phosphodiesterase (PDE). A member of the PDE family, PDE4, is widely expressed in and promotes the growth of glioblastoma, the most common type of brain tumor. The purpose of this study was to determine whether theobromine could exert growth inhibitory effects on U87-MG, a cell line derived from human malignant glioma. We show that theobromine treatment elevates intracellular cAMP levels and increases the activity of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, whereas it attenuates p44/42 extracellular signal-regulated kinase activity and the Akt/mammalian target of rapamycin kinase and nuclear factor-kappa B signal pathways. It also inhibits cell proliferation. These results suggest that foods and beverages containing cocoa bean extracts, including theobromine, might be extremely effective in preventing human glioblastoma.

  20. Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus

    DEFF Research Database (Denmark)

    Launholt, Dorte; Merkle, Thomas; Houben, Andreas

    2006-01-01

    In plants, the chromatin-associated high mobility group (HMG) proteins occur in twosubfamilies termedHMGAandHMGB.The HMGAproteins are characterized by the presence of four AT-hookDNAbinding motifs, and theHMGBproteins contain anHMG boxDNAbinding domain. As architectural factors, theHMGproteins ap......In plants, the chromatin-associated high mobility group (HMG) proteins occur in twosubfamilies termedHMGAandHMGB.The HMGAproteins are characterized by the presence of four AT-hookDNAbinding motifs, and theHMGBproteins contain anHMG boxDNAbinding domain. As architectural factors, the...... of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C...

  1. HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors.

    Science.gov (United States)

    Dailey, Deanna D; Anfinsen, Kristin P; Pfaff, Liza E; Ehrhart, E J; Charles, J Brad; Bønsdorff, Tina B; Thamm, Douglas H; Powers, Barbara E; Jonasdottir, Thora J; Duval, Dawn L

    2013-07-01

    Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression. Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify HES1, HEY1, NOTCH1 and NOTCH2 gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of  300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes. Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. HES1 mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI 300 days. NOTCH2 and HEY1 mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between

  2. TGF-β signaling controls FSHR signaling-reduced ovarian granulosa cell apoptosis through the SMAD4/miR-143 axis.

    Science.gov (United States)

    Du, Xing; Zhang, Lifan; Li, Xinyu; Pan, Zengxiang; Liu, Honglin; Li, Qifa

    2016-11-24

    Follicle-stimulating hormone receptor (FSHR) and its intracellular signaling control mammalian follicular development and female infertility. Our previous study showed that FSHR is downregulated during follicular atresia of porcine ovaries. However, its role and regulation in follicular atresia remain unclear. Here, we showed that FSHR knockdown induced porcine granulosa cell (pGC) apoptosis and follicular atresia, and attenuated the levels of intracellular signaling molecules such as PKA, AKT and p-AKT. FSHR was identified as a target of miR-143, a microRNA that was upregulated during porcine follicular atresia. miR-143 enhanced pGC apoptosis by targeting FSHR, and reduced the levels of intracellular signaling molecules. SMAD4, the final molecule in transforming growth factor (TGF)-β signaling, bound to the promoter and induced significant downregulation of miR-143 in vitro and in vivo. Activated TGF-β signaling rescued miR-143-reduced FSHR and intracellular signaling molecules, and miR-143-induced pGC apoptosis. Overall, our findings offer evidence to explain how TGF-β signaling influences and FSHR signaling for regulation of pGC apoptosis and follicular atresia by a specific microRNA, miR-143.

  3. Chimeric peptides as modulators of CK2-dependent signaling: Mechanism of action and off-target effects.

    Science.gov (United States)

    Zanin, Sofia; Sandre, Michele; Cozza, Giorgio; Ottaviani, Daniele; Marin, Oriano; Pinna, Lorenzo A; Ruzzene, Maria

    2015-10-01

    Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127-7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Fluorescent nanosensors for intracellular measurements: synthesis, characterisation, calibration and measurement

    Directory of Open Access Journals (Sweden)

    Arpan Shailesh Desai

    2014-01-01

    Full Text Available Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer and a pH-insensitive reference fluorophore (internal standard immobilised in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesised using standard laboratory equipment and are detectable by non-invasive widely accessibly imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: 1 synthesis and characterisation of polyacrylamide and silica based nanosensors 2 nanosensor calibration and 3 performing measurements using fluorescence microscopy.

  5. Signaling Network Assessment of Mutations and Copy Number Variations Predict Breast Cancer Subtype-Specific Drug Targets

    Directory of Open Access Journals (Sweden)

    Naif Zaman

    2013-10-01

    Full Text Available Individual cancer cells carry a bewildering number of distinct genomic alterations (e.g., copy number variations and mutations, making it a challenge to uncover genomic-driven mechanisms governing tumorigenesis. Here, we performed exome sequencing on several breast cancer cell lines that represent two subtypes, luminal and basal. We integrated these sequencing data and functional RNAi screening data (for the identification of genes that are essential for cell proliferation and survival onto a human signaling network. Two subtype-specific networks that potentially represent core-signaling mechanisms underlying tumorigenesis were identified. Within both networks, we found that genes were differentially affected in different cell lines; i.e., in some cell lines a gene was identified through RNAi screening, whereas in others it was genomically altered. Interestingly, we found that highly connected network genes could be used to correctly classify breast tumors into subtypes on the basis of genomic alterations. Further, the networks effectively predicted subtype-specific drug targets, which were experimentally validated.

  6. Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease.

    Science.gov (United States)

    Mercado, Gabriela; Castillo, Valentina; Soto, Paulina; López, Nélida; Axten, Jeffrey M; Sardi, Sergio P; Hoozemans, Jeroen J M; Hetz, Claudio

    2018-04-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. miRNA targeted signaling pathway in the early stage of denervated fast and slow muscle atrophy

    Directory of Open Access Journals (Sweden)

    Gang Li

    2016-01-01

    Full Text Available Denervation often results in skeletal muscle atrophy. Different mechanisms seem to be involved in the determination between denervated slow and fast skeletal muscle atrophy. At the epigenetic level, miRNAs are thought to be highly involved in the pathophysiological progress of denervated muscles. We used miRNA microarrays to determine miRNA expression profiles from a typical slow muscle (soleus muscle and a typical fast muscle (tibialis anterior muscle at an early denervation stage in a rat model. Results showed that miR-206, miR-195, miR-23a, and miR-30e might be key factors in the transformation process from slow to fast muscle in denervated slow muscles. Additionally, certain miRNA molecules (miR-214, miR-221, miR-222, miR-152, miR-320, and Let-7e could be key regulatory factors in the denervated atrophy process involved in fast muscle. Analysis of signaling pathway networks revealed the miRNA molecules that were responsible for regulating certain signaling pathways, which were the final targets (e.g., p38 MAPK pathway; Pax3/Pax7 regulates Utrophin and follistatin by HDAC4; IGF1/PI3K/Akt/mTOR pathway regulates atrogin-1 and MuRF1 expression via FoxO phosphorylation. Our results provide a better understanding of the mechanisms of denervated skeletal muscle pathophysiology.

  8. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  9. miR-150 suppresses the proliferation and tumorigenicity of leukemia stem cells by targeting the Nanog signaling pathway

    Directory of Open Access Journals (Sweden)

    Dan-dan Xu

    2016-11-01

    Full Text Available Proliferation, a key feature of cancer cells, accounts for the majority of cancer-related diseases resulting in mortality. MicroRNAs (miRNAs plays important post-transcriptional modulation roles by acting on multiple signaling pathways, but the underlying mechanism in proliferation and tumorigenicity is unclear. Here, we identified the role of miR-150 in proliferation and tumorigenicity in leukemia stem cells (LSCs (CD34+CD38- cells. miR-150 expression was significantly down-regulated in LSCs from leukemia cell lines and clinical samples. Functional assays demonstrated that increased miR-150 expression inhibited proliferation and clonal and clonogenic growth, enhanced chemosensitivity, and attenuated tumorigenic activity of LSCs in vitro. Transplantation animal studies revealed that miR-150 overexpression progressively abrogates tumour growth. Immunohistochemistry assays demonstrated that miR-150 overexpression enhanced caspase-3 level and reduced Ki-67 level. Moreover, luciferase reporter assays indicated Nanog is a direct and functional target of miR-150. Nanog silencing using small interfering RNA recapitulated anti-proliferation and tumorigenicity inhibition effects. Furthermore, miR-150 directly down-regulated the expression of other cancer stem cell factors including Notch2 and CTNNB1. These results provide insights into the specific biological behaviour of miR-150 in regulating LSC proliferation and tumorigenicity. Targeting this miR-150/Nanog axis would be a helpful therapeutic strategy to treat acute myeloid leukemia.

  10. ONC201 Targets AR and AR-V7 Signaling, Reduces PSA, and Synergizes with Everolimus in Prostate Cancer.

    Science.gov (United States)

    Lev, Avital; Lulla, Amriti R; Ross, Brian C; Ralff, Marie D; Makhov, Petr B; Dicker, David T; El-Deiry, Wafik S

    2018-05-01

    Androgen receptor (AR) signaling plays a key role in prostate cancer progression, and androgen deprivation therapy (ADT) is a mainstay clinical treatment regimen for patients with advanced disease. Unfortunately, most prostate cancers eventually become androgen-independent and resistant to ADT with patients progressing to metastatic castration-resistant prostate cancer (mCRPC). Constitutively activated AR variants (AR-V) have emerged as mediators of resistance to AR-targeted therapy and the progression of mCRPC, and they represent an important therapeutic target. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant, and its expression correlates with ADT resistance. ONC201/TIC10 is the founding member of the imipridone class of small molecules and has shown anticancer activity in a broad range of tumor types. ONC201 is currently being tested in phase I/II clinical trials for advanced solid tumors, including mCRPC, and hematologic malignancies. There has been promising activity observed in patients in early clinical testing. This study demonstrates preclinical single-agent efficacy of ONC201 using in vitro and in vivo models of prostate cancer. ONC201 has potent antiproliferative and proapoptotic effects in both castration-resistant and -sensitive prostate cancer cells. Furthermore, the data demonstrate that ONC201 downregulates the expression of key drivers of prostate cancer such as AR-V7 and downstream target genes including the clinically used biomarker PSA (KLK3). Finally, the data also provide a preclinical rationale for combination of ONC201 with approved therapeutics for prostate cancer such as enzalutamide, everolimus (mTOR inhibitor), or docetaxel. Implications: The preclinical efficacy of ONC201 as a single agent or in combination, in hormone-sensitive or castration-resistant prostate cancer, suggests the potential for immediate clinical translation. Mol Cancer Res; 16(5); 754-66. ©2018 AACR . ©2018 American Association for Cancer

  11. Silibinin inhibits fibronectin induced motility, invasiveness and survival in human prostate carcinoma PC3 cells via targeting integrin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Deep, Gagan [Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO (United States); University of Colorado Cancer Center, University of Colorado Denver, Aurora, CO (United States); Kumar, Rahul; Jain, Anil K. [Department of Pharmaceutical Sciences, Skaggs School of