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Sample records for activates cellular signaling

  1. Sleep Loss Activates Cellular Inflammatory Signaling

    PubMed Central

    Irwin, Michael R.; Wang, Minge; Ribeiro, Denise; Cho, Hyong Jin; Olmstead, Richard; Breen, Elizabeth Crabb; Martinez-Maza, Otoniel; Cole, Steve

    2008-01-01

    Background Accumulating evidence suggests that sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. This study was undertaken to test the effects of sleep loss on activation of nuclear factor (NF) -κB, a transcription factor that serves a critical role in the inflammatory signaling cascade. Methods In 14 healthy adults (7 females; 7 males), peripheral blood mononuclear cell NF-κB was repeatedly assessed, along with enumeration of lymphocyte subpopulations, in the morning after baseline sleep, partial sleep deprivation (awake from 23:00 h to 03:00 h), and recovery sleep. Results In the morning after a night of sleep loss, mononuclear cell NF-κB activation was significantly greater compared with morning levels following uninterrupted baseline or recovery sleep, in which the response was found in females but not in males. Conclusions These results identify NF-κB activation as a molecular pathway by which sleep disturbance may influence leukocyte inflammatory gene expression and the risk of inflammation-related disease. PMID:18561896

  2. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling

    SciTech Connect

    Huang, Pengxiang; Nedelcu, Daniel; Watanabe, Miyako; Jao, Cindy; Kim, Youngchang; Liu, Jing; Salic, Adrian

    2016-08-01

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.

  3. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function.

    PubMed

    Indo, Hiroko P; Hawkins, Clare L; Nakanishi, Ikuo; Matsumoto, Ken-Ichiro; Matsui, Hirofumi; Suenaga, Shigeaki; Davies, Michael J; St Clair, Daret K; Ozawa, Toshihiko; Majima, Hideyuki J

    2017-02-08

    Mitochondria are a major source of intracellular energy and reactive oxygen species in cells, but are also increasingly being recognized as a controller of cell death. Here, we review evidence of signal transduction control by mitochondrial superoxide generation via the nuclear factor-κB (NF-κB) and GATA signaling pathways. We have also reviewed the effects of ROS on the activation of MMP and HIF. There is significant evidence to support the hypothesis that mitochondrial superoxide can initiate signaling pathways following transport into the cytosol. In this study, we provide evidence of TATA signal transductions by mitochondrial superoxide. Oxidative phosphorylation via the electron transfer chain, glycolysis, and generation of superoxide from mitochondria could be important factors in regulating signal transduction, cellular homeostasis, and cell death.

  4. Morbilliviruses Use Signaling Lymphocyte Activation Molecules (CD150) as Cellular Receptors

    PubMed Central

    Tatsuo, Hironobu; Ono, Nobuyuki; Yanagi, Yusuke

    2001-01-01

    Morbilliviruses comprise measles virus, canine distemper virus, rinderpest virus, and several other viruses that cause devastating human and animal diseases accompanied by severe immunosuppression and lymphopenia. Recently, we have shown that human signaling lymphocyte activation molecule (SLAM) is a cellular receptor for measles virus. In this study, we examined whether canine distemper and rinderpest viruses also use canine and bovine SLAMs, respectively, as cellular receptors. The Onderstepoort vaccine strain and two B95a (marmoset B cell line)-isolated strains of canine distemper virus caused extensive cytopathic effects in normally resistant CHO (Chinese hamster ovary) cells after expression of canine SLAM. The Ako vaccine strain of rinderpest virus produced strong cytopathic effects in bovine SLAM-expressing CHO cells. The data on entry with vesicular stomatitis virus pseudotypes bearing measles, canine distemper, or rinderpest virus envelope proteins were consistent with development of cytopathic effects in SLAM-expressing CHO cell clones after infection with the respective viruses, confirming that SLAM acts at the virus entry step (as a cellular receptor). Furthermore, most measles, canine distemper, and rinderpest virus strains examined could any use of the human, canine, and bovine SLAMs to infect cells. Our findings suggest that the use of SLAM as a cellular receptor may be a property common to most, if not all, morbilliviruses and explain the lymphotropism and immunosuppressive nature of morbilliviruses. PMID:11390585

  5. Activation of cellular signaling by 8-oxoguanine DNA glycosylase-1-initiated DNA base excision repair.

    PubMed

    German, Peter; Szaniszlo, Peter; Hajas, Gyorgy; Radak, Zsolt; Bacsi, Attila; Hazra, Tapas K; Hegde, Muralidhar L; Ba, Xueqing; Boldogh, Istvan

    2013-10-01

    Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.

  6. Activation of Wnt Signaling by Chemically Induced Dimerization of LRP5 Disrupts Cellular Homeostasis

    PubMed Central

    Pond, Adam C.; Seethammagari, Mamatha; Chiou, Shin-Heng; Cho, Kyucheol; Carstens, Julienne L.; Decker, William K.; McCrea, Pierre D.; Ittmann, Michael M.; Rosen, Jeffrey M.; Spencer, David M.

    2012-01-01

    Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion. PMID:22303459

  7. Special issue: redox active natural products and their interaction with cellular signalling pathways.

    PubMed

    Jacob, Claus

    2014-11-26

    During the last decade, research into natural products has experienced a certain renaissance. The urgent need for more and more effective antibiotics in medicine, the demand for ecologically friendly plant protectants in agriculture, "natural" cosmetics and the issue of a sustainable and healthy nutrition in an ageing society have fuelled research into Nature's treasure chest of "green gold". Here, redox active secondary metabolites from plants, fungi, bacteria and other (micro-)organisms often have been at the forefront of the most interesting developments. These agents provide powerful means to interfere with many, probably most cellular signaling pathways in humans, animals and lower organisms, and therefore can be used to protect, i.e., in form of antioxidants, and to frighten off or even kill, i.e., in form of repellants, antibiotics, fungicides and selective, often catalytic "sensor/effector" anticancer agents. Interestingly, whilst natural product research dates back many decades, in some cases even centuries, and compounds such as allicin and various flavonoids have been investigated thoroughly in the past, it has only recently become possible to investigate their precise interactions and mode(s) of action inside living cells. Here, fluorescent staining and labelling on the one side, and appropriate detection, either qualitatively under the microscope or quantitatively in flow cytometers and plate readers, on the other, enable researchers to obtain the various pieces of information necessary to construct a fairly complete puzzle of how such compounds act and interact in living cells. Complemented by the more traditional activity assays and Western Blots, and increasingly joined by techniques such as proteomics, chemogenetic screening and mRNA profiling, these cell based bioanalytical techniques form a powerful platform for "intracellular diagnostics". In the case of redox active compounds, especially of Reactive Sulfur Species (RSS), such techniques have

  8. Sleep Loss Activates Cellular Inflammation and Signal Transducer and Activator of Transcription (STAT) Family Proteins in Humans

    PubMed Central

    Irwin, Michael R.; Witarama, Tuff; Caudill, Marissa; Olmstead, Richard; Breen, Elizabeth Crabb

    2014-01-01

    Sleep disturbance and short sleep duration are associated with inflammation and related disorders including cardiovascular disease, arthritis, diabetes mellitus, and certain cancers. This study was undertaken to test the effects of experimental sleep loss on spontaneous cellular inflammation and activation of signal transducer and activator of transcription (STAT) family proteins, which together promote an inflammatory microenvironment. In 24 healthy adults (16 females; 8 males), spontaneous production of IL-6 and TNF in monocytes and spontaneous intranuclear expression of activated STAT1, STAT3, and STAT5 in peripheral blood mononuclear cells (PBMC), monocyte-, and lymphocyte populations were measured in the morning after uninterrupted baseline sleep, partial sleep deprivation (PSD, sleep period from 3 a.m. to 7 a.m.), and recovery sleep. Relative to baseline, spontaneous monocytic expression of IL-6 and TNF-α was significantly greater after PSD (P<0.02) and after recovery sleep (P<0.01). Relative to baseline, spontaneous monocytic expression of activated STAT 1 and STAT 5 was significantly greater after recovery sleep (P<0.007P<0.02, respectively) but not STAT 3 (P=0.09). No changes in STAT1, STAT3, or STAT5 were found in lymphocyte populations. Sleep loss induces activation of spontaneous cellular innate immunity and of STAT family proteins, which together map the dynamics of sleep loss on the molecular signaling pathways that regulate inflammatory and other immune responses. Treatments that target short sleep duration have the potential to constrain inflammation and reduce the risk for inflammatory disorders and some cancers in humans. PMID:25451613

  9. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    SciTech Connect

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-12-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.

  10. Optimal temporal patterns for dynamical cellular signaling

    NASA Astrophysics Data System (ADS)

    Hasegawa, Yoshihiko

    2016-11-01

    Cells use temporal dynamical patterns to transmit information via signaling pathways. As optimality with respect to the environment plays a fundamental role in biological systems, organisms have evolved optimal ways to transmit information. Here, we use optimal control theory to obtain the dynamical signal patterns for the optimal transmission of information, in terms of efficiency (low energy) and reliability (low uncertainty). Adopting an activation-deactivation decoding network, we reproduce several dynamical patterns found in actual signals, such as steep, gradual, and overshooting dynamics. Notably, when minimizing the energy of the input signal, the optimal signals exhibit overshooting, which is a biphasic pattern with transient and steady phases; this pattern is prevalent in actual dynamical patterns. We also identify conditions in which these three patterns (steep, gradual, and overshooting) confer advantages. Our study shows that cellular signal transduction is governed by the principle of minimizing free energy dissipation and uncertainty; these constraints serve as selective pressures when designing dynamical signaling patterns.

  11. Reactive nitrogen species in cellular signaling

    PubMed Central

    Adams, Levi; Franco, Maria C

    2015-01-01

    The transduction of cellular signals occurs through the modification of target molecules. Most of these modifications are transitory, thus the signal transduction pathways can be tightly regulated. Reactive nitrogen species are a group of compounds with different properties and reactivity. Some reactive nitrogen species are highly reactive and their interaction with macromolecules can lead to permanent modifications, which suggested they were lacking the specificity needed to participate in cell signaling events. However, the perception of reactive nitrogen species as oxidizers of macromolecules leading to general oxidative damage has recently evolved. The concept of redox signaling is now well established for a number of reactive oxygen and nitrogen species. In this context, the post-translational modifications introduced by reactive nitrogen species can be very specific and are active participants in signal transduction pathways. This review addresses the role of these oxidative modifications in the regulation of cell signaling events. PMID:25888647

  12. Structure-activity relations of leucine derivatives reveal critical moieties for cellular uptake and activation of mTORC1-mediated signaling.

    PubMed

    Nagamori, Shushi; Wiriyasermkul, Pattama; Okuda, Suguru; Kojima, Naoto; Hari, Yoshiyuki; Kiyonaka, Shigeki; Mori, Yasuo; Tominaga, Hideyuki; Ohgaki, Ryuichi; Kanai, Yoshikatsu

    2016-04-01

    Among amino acids, leucine is a potential signaling molecule to regulate cell growth and metabolism by activating mechanistic target of rapamycin complex 1 (mTORC1). To reveal the critical structures of leucine molecule to activate mTORC1, we examined the structure-activity relationships of leucine derivatives in HeLa S3 cells for cellular uptake and for the induction of phosphorylation of p70 ribosomal S6 kinase 1 (p70S6K), a downstream effector of mTORC1. The activation of mTORC1 by leucine and its derivatives was the consequence of two successive events: the cellular uptake by L-type amino acid transporter 1 (LAT1) responsible for leucine uptake in HeLa S3 cells and the activation of mTORC1 following the transport. The structural requirement for the recognition by LAT1 was to have carbonyl oxygen, alkoxy oxygen of carboxyl group, amino group and hydrophobic side chain. In contrast, the requirement for mTORC1 activation was more rigorous. It additionally required fixed distance between carbonyl oxygen and alkoxy oxygen of carboxyl group, and amino group positioned at α-carbon. L-Configuration in chirality and appropriate length of side chain with a terminal isopropyl group were also important. This confirmed that LAT1 itself is not a leucine sensor. Some specialized leucine sensing mechanism with rigorous requirement for agonistic structures should exist inside the cells because leucine derivatives not transported by LAT1 did not activate mTORC1. Because LAT1-mTOR axis is involved in the regulation of cell growth and cancer progression, the results from this study may provide a new insight into therapeutics targeting both LAT1 and leucine sensor.

  13. Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

    SciTech Connect

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R.; Katzenellenbogen, Rachel A.

    2015-04-15

    High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.

  14. Proteinase-activated receptor 2 (PAR(2)) in cholangiocarcinoma (CCA) cells: effects on signaling and cellular level.

    PubMed

    Kaufmann, Roland; Hascher, Alexander; Mussbach, Franziska; Henklein, Petra; Katenkamp, Kathrin; Westermann, Martin; Settmacher, Utz

    2012-12-01

    In this study, we demonstrate functional expression of the proteinase-activated receptor 2 (PAR(2)), a member of a G-protein receptor subfamily in primary cholangiocarcinoma (PCCA) cell cultures. Treatment of PCCA cells with the serine proteinase trypsin and the PAR(2)-selective activating peptide, furoyl-LIGRLO-NH(2), increased migration across a collagen membrane barrier. This effect was inhibited by a PAR(2)-selective pepducin antagonist peptide (P2pal-18S) and it was also blocked with the Met receptor tyrosine kinase (Met) inhibitors SU 11274 and PHA 665752, the MAPKinase inhibitors PD 98059 and SL 327, and the Stat3 inhibitor Stattic. The involvement of Met, p42/p44 MAPKinases and Stat3 in PAR(2)-mediated PCCA cell signaling was further supported by the findings that trypsin and the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated activating phosphorylation of these signaling molecules in cholangiocarcinoma cells. With our results, we provide a novel signal transduction module in cholangiocarcinoma cell migration involving PAR(2)-driven activation of Met, p42/p44 MAPKinases and Stat3.

  15. Cellular prion protein transduces neuroprotective signals

    PubMed Central

    Chiarini, Luciana B.; Freitas, Adriana R.O.; Zanata, Silvio M.; Brentani, Ricardo R.; Martins, Vilma R.; Linden, Rafael

    2002-01-01

    To test for a role for the cellular prion protein (PrPc) in cell death, we used a PrPc-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrPc-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106–126, with certain antibodies to PrPc or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrPc that increased cAMP also induced neuroprotection. Thus, engagement of PrPc transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrPc may function as a trophic receptor, the activation of which leads to a neuroprotective state. PMID:12093733

  16. SOD Therapeutics: Latest Insights into Their Structure-Activity Relationships and Impact on the Cellular Redox-Based Signaling Pathways

    PubMed Central

    Tovmasyan, Artak; Roberts, Emily R. H.; Vujaskovic, Zeljko; Leong, Kam W.; Spasojevic, Ivan

    2014-01-01

    Abstract Significance: Superoxide dismutase (SOD) enzymes are indispensable and ubiquitous antioxidant defenses maintaining the steady-state levels of O2·−; no wonder, thus, that their mimics are remarkably efficacious in essentially any animal model of oxidative stress injuries thus far explored. Recent Advances: Structure-activity relationship (half-wave reduction potential [E1/2] versus log kcat), originally reported for Mn porphyrins (MnPs), is valid for any other class of SOD mimics, as it is dominated by the superoxide reduction and oxidation potential. The biocompatible E1/2 of ∼+300 mV versus normal hydrogen electrode (NHE) allows powerful SOD mimics as mild oxidants and antioxidants (alike O2·−) to readily traffic electrons among reactive species and signaling proteins, serving as fine mediators of redox-based signaling pathways. Based on similar thermodynamics, both SOD enzymes and their mimics undergo similar reactions, however, due to vastly different sterics, with different rate constants. Critical Issues: Although log kcat(O2·−) is a good measure of therapeutic potential of SOD mimics, discussions of their in vivo mechanisms of actions remain mostly of speculative character. Most recently, the therapeutic and mechanistic relevance of oxidation of ascorbate and glutathionylation and oxidation of protein thiols by MnP-based SOD mimics and subsequent inactivation of nuclear factor κB has been substantiated in rescuing normal and killing cancer cells. Interaction of MnPs with thiols seems to be, at least in part, involved in up-regulation of endogenous antioxidative defenses, leading to the healing of diseased cells. Future Directions: Mechanistic explorations of single and combined therapeutic strategies, along with studies of bioavailability and translational aspects, will comprise future work in optimizing redox-active drugs. Antioxid. Redox Signal. 20, 2372–2415. PMID:23875805

  17. Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

    PubMed

    Wang, H D; Trivedi, A; Johnson, D L

    1997-12-01

    Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

  18. Repeated low-dose 17β-estradiol treatment prevents activation of apoptotic signaling both in the synaptosomal and cellular fraction in rat prefrontal cortex following cerebral ischemia.

    PubMed

    Stanojlović, Miloš; Zlatković, Jelena; Guševac, Ivana; Grković, Ivana; Mitrović, Nataša; Zarić, Marina; Horvat, Anica; Drakulić, Dunja

    2015-01-01

    Disturbance in blood circulation is associated with numerous pathological conditions characterized by cognitive decline and neurodegeneration. Activation of pro-apoptotic signaling previously detected in the synaptosomal fraction may underlie neurodegeneration in the prefrontal cortex of rats submitted to permanent bilateral common carotid arteries occlusion (two-vessel occlusion, 2VO). 17β-Estradiol (E) exerts potent neuroprotective effects in the brain affecting, among other, ischemia-induced pathological changes. As most significant changes in rats submitted to 2VO were observed on 7th day following the insult, of interest was to examine whether 7 day treatment with low dose of E (33.3 µg/kg/day) prevents formerly reported neurodegeneration and may represent additional therapy during the early post-ischemic period. Role of E treatment on apoptotic pathway was monitored on Bcl-2 family members, cytochrome c, caspase 3 and PARP protein level in the synaptosomal (P2) fraction of the prefrontal cortex. Furthermore, changes of these proteins were examined in the cytosolic, mitochondrial and nuclear fraction, with the emphasis on potential involvement of extracellular signal-regulated kinases (ERK) and protein kinase B (Akt) activation and their role in nuclear translocation of transcriptional nuclear factor kappa B (NF-kB) associated with alteration of Bax and Bcl-2 gene expression. The extent of cellular damage was determined using DNA fragmentation and Fluoro-Jade B staining. The absence of activation of apoptotic cascade both in the P2 and cell accompanied with decreased DNA fragmentation and number of degenerating neurons clearly indicates that E treatment ensures the efficient protection against ischemic insult. Moreover, E-mediated modulation of pro-apoptotic signaling in the cortical cellular fractions involves cooperative activation of ERK and Akt, which may be implicated in the observed prevention of neurodegenerative changes.

  19. ATR inhibition rewires cellular signaling networks induced by replication stress.

    PubMed

    Wagner, Sebastian A; Oehler, Hannah; Voigt, Andrea; Dalic, Denis; Freiwald, Anja; Serve, Hubert; Beli, Petra

    2016-02-01

    The slowing down or stalling of replication forks is commonly known as replication stress and arises from multiple causes such as DNA lesions, nucleotide depletion, RNA-DNA hybrids, and oncogene activation. The ataxia telangiectasia and Rad3-related kinase (ATR) plays an essential role in the cellular response to replication stress and inhibition of ATR has emerged as therapeutic strategy for the treatment of cancers that exhibit high levels of replication stress. However, the cellular signaling induced by replication stress and the substrate spectrum of ATR has not been systematically investigated. In this study, we employed quantitative MS-based proteomics to define the cellular signaling after nucleotide depletion-induced replication stress and replication fork collapse following ATR inhibition. We demonstrate that replication stress results in increased phosphorylation of a subset of proteins, many of which are involved in RNA splicing and transcription and have previously not been associated with the cellular replication stress response. Furthermore, our data reveal the ATR-dependent phosphorylation following replication stress and discover novel putative ATR target sites on MCM6, TOPBP1, RAD51AP1, and PSMD4. We establish that ATR inhibition rewires cellular signaling networks induced by replication stress and leads to the activation of the ATM-driven double-strand break repair signaling.

  20. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

    PubMed Central

    2010-01-01

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity. "One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity." Josiah Willard Gibbs (1839-1903) PMID:20534114

  1. Ligands Binding to Cell Surface Ganglioside GD2 Cause Src-Dependent Activation of N-Methyl-D-Aspartate Receptor Signaling and Changes in Cellular Morphology

    PubMed Central

    Gagnon, Martin; Saragovi, H. Uri

    2015-01-01

    Ganglioside GD2 is a plasma membrane glycosphinogolipid. In healthy adults it is expressed at low levels, but it is over-expressed in many cancers. For cancer therapy, GD2 is targeted with anti-GD2 monoclonal antibodies (mAbs), and one adverse side effect is severe visceral pain. Pain is not neuropathic, cannot be blocked with morphine, and stops on discontinuation of mAb therapy. Here, we provide evidence that ligand binding to cell surface GD2 induces rapid and transient activation of Src-family kinases, followed by Src-dependent phosphorylation of NMDA-receptor NR2B subunits selectively, activation of Ca++ fluxes, production of cAMP, and changes in cellular morphology. These GD2-ligand activated signals differ in kinetics and in pharmacology from activation of the same signals in the same cells by BDNF, the growth factor agonist of the TrkB receptor, suggesting biological specificity. Hence, cell surface GD2 regulates pathways that can be associated with neoplasia and with morphine-intractable pain; and this can explain why expression of GD2 correlates with these two pathologies. PMID:26252487

  2. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades.

    PubMed

    Sancho, Veronica; Berna, Marc J; Thill, Michelle; Jensen, R T

    2011-12-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth).

  3. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades

    PubMed Central

    Sancho, Veronica; Berna, Marc J.; Thill, Michelle; Jensen, R. T.

    2011-01-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal(GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin(CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA were present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ’s direct association with AKT, RafA, RafC and Lyn. These results show for the first time PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth). PMID:21810446

  4. Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling

    PubMed Central

    Lovelace, Erica S.; Wagoner, Jessica; MacDonald, James; Bammler, Theo; Bruckner, Jacob; Brownell, Jessica; Beyer, Richard; Zink, Erika M.; Kim, Young-Mo; Kyle, Jennifer E.; Webb-Robertson, Bobbie-Jo; Waters, Katrina M.; Metz, Thomas O.; Farin, Federico; Oberlies, Nicholas H.; Polyak, Stephen J.

    2016-01-01

    Silymarin, a characterized extract of the seeds of milk thistle (Silybum marianum), suppresses cellular inflammation. To define how this occurs, transcriptional profiling, metabolomics, and signaling studies were performed in human liver and T cell lines. Cellular stress and metabolic pathways were modulated within 4 h of silymarin treatment: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed silymarin suppression of glycolytic, tricarboxylic acid (TCA) cycle, and amino acid metabolism. Anti-inflammatory effects arose with prolonged (i.e. 24 h) silymarin exposure, with suppression of multiple pro-inflammatory mRNAs and signaling pathways including nuclear factor kappa B (NF-κB) and forkhead box O (FOXO). Studies with murine knock out cells revealed that silymarin inhibition of both mTOR and NF-κB was partially AMPK dependent, while silymarin inhibition of mTOR required DDIT4. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Thus, natural products activate stress and repair responses that culminate in an anti-inflammatory cellular phenotype. Natural products like silymarin may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation. PMID:26186142

  5. Studying Cellular Signal Transduction with OMIC Technologies

    PubMed Central

    Landry, Benjamin D.; Clarke, David C.; Lee, Michael J.

    2016-01-01

    In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly nonlinear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and “OMIC” approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the “signal” can take different forms in different situations. Signals are encoded in diverse ways such as protein-protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the “right” experiments and the “right” modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology. PMID:26244521

  6. Systematic quantitative characterization of cellular responses induced by multiple signals

    PubMed Central

    2011-01-01

    Background Cells constantly sense many internal and environmental signals and respond through their complex signaling network, leading to particular biological outcomes. However, a systematic characterization and optimization of multi-signal responses remains a pressing challenge to traditional experimental approaches due to the arising complexity associated with the increasing number of signals and their intensities. Results We established and validated a data-driven mathematical approach to systematically characterize signal-response relationships. Our results demonstrate how mathematical learning algorithms can enable systematic characterization of multi-signal induced biological activities. The proposed approach enables identification of input combinations that can result in desired biological responses. In retrospect, the results show that, unlike a single drug, a properly chosen combination of drugs can lead to a significant difference in the responses of different cell types, increasing the differential targeting of certain combinations. The successful validation of identified combinations demonstrates the power of this approach. Moreover, the approach enables examining the efficacy of all lower order mixtures of the tested signals. The approach also enables identification of system-level signaling interactions between the applied signals. Many of the signaling interactions identified were consistent with the literature, and other unknown interactions emerged. Conclusions This approach can facilitate development of systems biology and optimal drug combination therapies for cancer and other diseases and for understanding key interactions within the cellular network upon treatment with multiple signals. PMID:21624115

  7. Signalling Pathways Controlling Cellular Actin Organization.

    PubMed

    Steffen, Anika; Stradal, Theresia E B; Rottner, Klemens

    2017-01-01

    The actin cytoskeleton is essential for morphogenesis and virtually all types of cell shape changes. Reorganization is per definition driven by continuous disassembly and re-assembly of actin filaments, controlled by major, ubiquitously operating machines. These are specifically employed by the cell to tune its activities in accordance with respective environmental conditions or to satisfy specific needs.Here we sketch some fundamental signalling pathways established to contribute to the reorganization of specific actin structures at the plasma membrane. Rho-family GTPases are at the core of these pathways, and dissection of their precise contributions to actin reorganization in different cell types and tissues will thus continue to improve our understanding of these important signalling nodes. Furthermore, we will draw your attention to the emerging theme of actin reorganization on intracellular membranes, its functional relation to Rho-GTPase signalling, and its relevance for the exciting phenomenon autophagy.

  8. Silymarin Suppresses Cellular Inflammation By Inducing Reparative Stress Signaling

    SciTech Connect

    Lovelace, Erica S.; Wagoner, Jessica; MacDonald, James; Bammler, Theo; Bruckner, Jacob; Brownell, Jessica; Beyer, Richard; Zink, Erika M.; Kim, Young-Mo; Kyle, Jennifer E.; Webb-Robertson, Bobbie-Jo M.; Waters, Katrina M.; Metz, Thomas O.; Farin, Federico; Oberlies, Nicholas H.; Polyak, Steve

    2015-08-28

    Silymarin (SM), a natural product, is touted as a liver protectant and preventer of both chronic inflammation and diseases. To define how SM elicits these effects at a systems level, we performed transcriptional profiling, metabolomics, and signaling studies in human liver and T cell lines. Multiple pathways associated with cellular stress and metabolism were modulated by SM treatment within 0.5 to four hours: activation of Activating Transcription Factor 4 (ATF-4) and adenosine monophosphate protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) signaling, the latter being associated with induction of DNA-damage-inducible transcript 4 (DDIT4). Metabolomics analyses revealed suppression of glycolytic, TCA cycle, and amino acid metabolism by SM treatment. Antiinflammatory effects arose with prolonged (i.e. 24 hours) SM exposure, with suppression of multiple proinflammatory mRNAs and nuclear factor kappa B (NF-κB) and forkhead box O (FOXO) signaling. Studies with murine knock out cells revealed that SM inhibition of both mTOR and NF-κB was partially AMPK dependent, while SM inhibition of the mTOR pathway in part required DDIT4. Thus, SM activates stress and repair responses that culminate in an anti-inflammatory phenotype. Other natural products induced similar stress responses, which correlated with their ability to suppress inflammation. Therefore, natural products like SM may be useful as tools to define how metabolic, stress, and repair pathways regulate cellular inflammation.

  9. Optimal Prediction by Cellular Signaling Networks

    NASA Astrophysics Data System (ADS)

    Becker, Nils B.; Mugler, Andrew; ten Wolde, Pieter Rein

    2015-12-01

    Living cells can enhance their fitness by anticipating environmental change. We study how accurately linear signaling networks in cells can predict future signals. We find that maximal predictive power results from a combination of input-noise suppression, linear extrapolation, and selective readout of correlated past signal values. Single-layer networks generate exponential response kernels, which suffice to predict Markovian signals optimally. Multilayer networks allow oscillatory kernels that can optimally predict non-Markovian signals. At low noise, these kernels exploit the signal derivative for extrapolation, while at high noise, they capitalize on signal values in the past that are strongly correlated with the future signal. We show how the common motifs of negative feedback and incoherent feed-forward can implement these optimal response functions. Simulations reveal that E. coli can reliably predict concentration changes for chemotaxis, and that the integration time of its response kernel arises from a trade-off between rapid response and noise suppression.

  10. A Cellular System for Spatial Signal Decoding in Chemical Gradients.

    PubMed

    Hegemann, Björn; Unger, Michael; Lee, Sung Sik; Stoffel-Studer, Ingrid; van den Heuvel, Jasmin; Pelet, Serge; Koeppl, Heinz; Peter, Matthias

    2015-11-23

    Directional cell growth requires that cells read and interpret shallow chemical gradients, but how the gradient directional information is identified remains elusive. We use single-cell analysis and mathematical modeling to define the cellular gradient decoding network in yeast. Our results demonstrate that the spatial information of the gradient signal is read locally within the polarity site complex using double-positive feedback between the GTPase Cdc42 and trafficking of the receptor Ste2. Spatial decoding critically depends on low Cdc42 activity, which is maintained by the MAPK Fus3 through sequestration of the Cdc42 activator Cdc24. Deregulated Cdc42 or Ste2 trafficking prevents gradient decoding and leads to mis-oriented growth. Our work discovers how a conserved set of components assembles a network integrating signal intensity and directionality to decode the spatial information contained in chemical gradients.

  11. ROS and ROS-Mediated Cellular Signaling

    PubMed Central

    Zhang, Jixiang; Wang, Xiaoli; Vikash, Vikash; Ye, Qing; Wu, Dandan; Liu, Yulan; Dong, Weiguo

    2016-01-01

    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. PMID:26998193

  12. Only signaling modules that discriminate sharply between stimulatory and nonstimulatory inputs require basal signaling for fast cellular responses

    NASA Astrophysics Data System (ADS)

    Artomov, Mykyta; Kardar, Mehran; Chakraborty, Arup K.

    2010-09-01

    In many types of cells, binding of molecules to their receptors enables cascades of intracellular chemical reactions to take place (signaling). However, a low level of signaling also occurs in most unstimulated cells. Such basal signaling in resting cells can have many functions, one of which is that it is thought to be required for fast cellular responses to external stimuli. A mechanistic understanding of why this is true and which features of cellular signaling networks make basal signaling necessary for fast responses is unknown. We address this issue by obtaining the time required for activation of common types of cell signaling modules with and without basal signaling. Our results show that the absence of basal signaling does not have any dramatic effects on the response time for signaling modules that exhibit a graded response to increasing stimulus levels. In sharp contrast, signaling modules that exhibit sharp dose-response curves which discriminate sensitively between stimuli to which the cell needs to respond and low-grade inputs (or stochastic noise) require basal signaling for fast cellular responses. In such cases, we find that an optimal level of basal signaling balances the requirements for fast cellular responses while minimizing spurious activation without appropriate stimulation.

  13. Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

    PubMed Central

    Herman, Sarah E. M.; Gordon, Amber L.; Wagner, Amy J.; Heerema, Nyla A.; Zhao, Weiqiang; Flynn, Joseph M.; Jones, Jeffrey; Andritsos, Leslie; Puri, Kamal D.; Lannutti, Brian J.; Giese, Neill A.; Zhang, Xiaoli; Wei, Lai; Byrd, John C.

    2010-01-01

    Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders. PMID:20522708

  14. Pneumolysin induces cellular senescence by increasing ROS production and activation of MAPK/NF-κB signal pathway in glial cells.

    PubMed

    Kwon, Ii-Seul; Kim, Jinwook; Rhee, Dong-Kwon; Kim, Byung-Oh; Pyo, Suhkneung

    2017-02-20

    Senescence is an irreversible proliferation arrest that is induced by various stress stimuli including genotoxin. Pneumolysin (PLY) is a pathogenicity factor unique to Streptococcus pneumoniae that is important in pneumococcal-induced diseases such as otitis media, meningitis and pneumonia. However, the cell fate response to the toxin is mechanistically unclear. We investigated the effect of PLY on cellular senescence in BV-2 microglial cells. Exposure to PLY resulted in changes in the expression of phospho-p53, p21, p16, pRb and CDK2 and increased the number of senescence associated β-gal positive cells. PLY-treatment also increased PAI-1 expression and cell proliferation arrest in concentration- and time-dependent manners. PLY induced NF-κB activation and phosphorylation of SIRT-1, ERK1/2, JNK, and p38 MAPK. In addition, PLY increased the production of reactive oxygen species. Overall, the results suggest that PLY regulates microglial cellular senescence by enhancing production of reactive oxygen species, activation of MAPK and NF-κB, and phosphorylation of SIRT-1.

  15. Targeting cancer by binding iron: Dissecting cellular signaling pathways

    PubMed Central

    Lui, Goldie Y.L.; Kovacevic, Zaklina; Richardson, Vera; Merlot, Angelica M.; Kalinowski, Danuta S.; Richardson, Des R.

    2015-01-01

    Newer and more potent therapies are urgently needed to effectively treat advanced cancers that have developed resistance and metastasized. One such strategy is to target cancer cell iron metabolism, which is altered compared to normal cells and may facilitate their rapid proliferation. This is supported by studies reporting the anti-neoplastic activities of the clinically available iron chelators, desferrioxamine and deferasirox. More recently, ligands of the di-2-pyridylketone thiosemicarbazone (DpT) class have demonstrated potent and selective anti-proliferative activity across multiple cancer-types in vivo, fueling studies aimed at dissecting their molecular mechanisms of action. In the past five years alone, significant advances have been made in understanding how chelators not only modulate cellular iron metabolism, but also multiple signaling pathways implicated in tumor progression and metastasis. Herein, we discuss recent research on the targeting of iron in cancer cells, with a focus on the novel and potent DpT ligands. Several key studies have revealed that iron chelation can target the AKT, ERK, JNK, p38, STAT3, TGF-β, Wnt and autophagic pathways to subsequently inhibit cellular proliferation, the epithelial-mesenchymal transition (EMT) and metastasis. These developments emphasize that these novel therapies could be utilized clinically to effectively target cancer. PMID:26125440

  16. Glycosylation regulates prestin cellular activity.

    PubMed

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  17. Extra-cellular signal-regulated kinase 1/2 (ERK1/2) activated in the hippocampal CA1 neurons is critical for retrieval of auditory trace fear memory.

    PubMed

    Huang, Ching-Hsun; Chiang, Yu-Wei; Liang, Keng-Chen; Thompson, Richard F; Liu, Ingrid Y

    2010-04-22

    The brain regions involved with trace fear conditioning (TFC) and delayed fear conditioning (DFC) are well-characterized, but little is known about the cellular representation subsuming these types of classical conditioning. Previous evidence has shown that activation of the amygdala is required for both TFC and DFC, while TFC also involves the hippocampus for forming conditioned response to tone. Lesions of the hippocampus did not affect tone learning in DFC, but it impaired learning in TFC. Synaptic plasticity in the hippocampus, underlying a cellular representation subsuming learning and memory, is in part modulated by extra-cellular signal-regulated kinase (ERK) signaling pathway. ERK1/2 activation is required for both TFC and DFC during memory formation, but whether this pathway is involved in memory retrieval of TFC is still unknown. In the present study, we investigated changes in ERK1/2 phosphorylation after memory retrieval in groups of mice that received TFC, DFC, tone-shock un-paired conditioning, and naïve control. Our results showed that ERK1/2 phosphorylation was elevated in the hippocampal CA1 region after retrieval of all conditioned fear responses. In particular, in the TFC group, immunohistochemistry indicated higher level of ERK1/2 phosphorylation in the hippocampal pyramidal neurons 30min after tone testing. Inhibition of the ERK1/2 signaling pathway diminished fear memory elicited by a tone in TFC. Together these results suggest that the memory retrieval process in TFC is more dependent on ERK1/2 signaling pathway than that in DFC. ERK1/2 signaling is critical for retrieval associative memory of temporally noncontiguous stimuli.

  18. RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

    PubMed Central

    Wu, Jun; Pei, Rongjuan; Xu, Yang; Yang, Dongliang; Roggendorf, Michael; Lu, Mengji

    2013-01-01

    Background & Aims Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi. Methods Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting. Results In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2′-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction. Conclusions RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo. PMID:23700487

  19. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex

    PubMed Central

    Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay AA

    2014-01-01

    Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships, but require efficient methods for cell capture and mRNA sequencing1–4. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths5, the limitations of shallow sequencing have not been directly investigated. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In developing cortex we identify diverse cell types including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells. PMID:25086649

  20. SEM++: A particle model of cellular growth, signaling and migration

    NASA Astrophysics Data System (ADS)

    Milde, Florian; Tauriello, Gerardo; Haberkern, Hannah; Koumoutsakos, Petros

    2014-06-01

    We present a discrete particle method to model biological processes from the sub-cellular to the inter-cellular level. Particles interact through a parametrized force field to model cell mechanical properties, cytoskeleton remodeling, growth and proliferation as well as signaling between cells. We discuss the guiding design principles for the selection of the force field and the validation of the particle model using experimental data. The proposed method is integrated into a multiscale particle framework for the simulation of biological systems.

  1. Proanthocyanidins from the American Cranberry (Vaccinium macrocarpon) inhibit matrix metalloproteinase-2 and matrix metalloproteinase-9 activity in human prostate cancer cells via alterations in multiple cellular signalling pathways.

    PubMed

    Déziel, Bob A; Patel, Kunal; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert A R

    2010-10-15

    Prostate cancer is one of the most common cancers in the Western world, and it is believed that an individual's diet affects his risk of developing cancer. There has been an interest in examining phytochemicals, the secondary metabolites of plants, in order to determine their potential anti-cancer activities in vitro and in vivo. In this study we document the effects of proanthocyanidins (PACs) from the American Cranberry (Vaccinium macrocarpon) on matrix metalloproteinase (MMP) activity in DU145 human prostate cancer cells. Cranberry PACs decreased cellular viability of DU145 cells at a concentration of 25 µg/ml by 30% after 6 h of treatment. Treatment of DU145 cells with PACs resulted in an inhibition of both MMPs 2 and 9 activity. PACs increased the expression of TIMP-2, a known inhibitor of MMP activity, and decreased the expression of EMMPRIN, an inducer of MMP expression. PACs decreased the expression of PI-3 kinase and AKT proteins, and increased the phosphorylation of both p38 and ERK1/2. Cranberry PACs also decreased the translocation of the NF-κB p65 protein to the nucleus. Cranberry PACs increased c-jun and decreased c-fos protein levels. These results suggest that cranberry PACs decreases MMP activity through the induction and/or inhibition of specific temporal MMP regulators, and by affecting either the phosphorylation status and/or expression of MAP kinase, PI-3 kinase, NF-κB and AP-1 pathway proteins. This study further demonstrates that cranberry PACs are a strong candidate for further research as novel anti-cancer agents.

  2. Human gonadotropin-releasing hormone receptor-activated cellular functions and signaling pathways in extra-pituitary tissues and cancer cells (Review).

    PubMed

    Aguilar-Rojas, Arturo; Huerta-Reyes, Maira

    2009-11-01

    Human gonadotropin-releasing hormone receptor (GnRHR) and its natural ligand human gonadotropin-releasing hormone (GnRH) were initially described as signaling complexes that play a key role in reproductive functions. By binding to specific receptors present on pituitary gonadotropes, GnRH regulates the sperm and ovum maturation, as well as steroidogenesis within the context of the hypothalamus-hypophysis axis. The expression of GnRH and its receptor has clearly been established in many extra-pituitary organs. Some of them are tumors from non-reproductive tissues such as liver, larynx, pancreas, colon, lymphoma, kidney, skin, blood and brain as well as tissues from reproductive track, for example ovary, endometrium, prostate and breast or tumors derived from these organs. Expression of GnRH and its receptor in these organs has gained much attention and several research groups have established their role during cell proliferation and cell motility. Although the signaling pathways and their effector proteins in these samples remain unclear, the molecular mechanism employed for GnRH and its receptor in extra-pituitary tissues could be related with non-classical GnRHR-signaling pathways. In the present review, we explore the vast literature reported on GnRH and GnRHR principally in tumors, describing how cross-talk between GnRHR and growth factor receptor, the coupling between GnRHR and many G proteins depending on cell context, and the regulation of several proteins associated with cell proliferation and cell motility are employed by GnRHR/GnRH to regulate their extra-pituitary activities.

  3. The community structure of human cellular signaling network.

    PubMed

    Diao, Yuanbo; Li, Menglong; Feng, Zinan; Yin, Jiajian; Pan, Yi

    2007-08-21

    Living cell is highly responsive to specific chemicals in its environment, such as hormones and molecules in food or aromas. The reason is ascribed to the existence of widespread and diverse signal transduction pathways, between which crosstalks usually exist, thus constitute a complex signaling network. Evidently, knowledge of topology characteristic of this network could contribute a lot to the understanding of diverse cellular behaviors and life phenomena thus come into being. In this presentation, signal transduction data is extracted from KEGG to construct a cellular signaling network of Homo sapiens, which has 931 nodes and 6798 links in total. Computing the degree distribution, we find it is not a random network, but a scale-free network following a power-law of P(K) approximately K(-gamma), with gamma approximately equal to 2.2. Among three graph partition algorithms, the Guimera's simulated annealing method is chosen to study the details of topology structure and other properties of this cellular signaling network, as it shows the best performance. To reveal the underlying biological implications, further investigation is conducted on ad hoc community and sketch map of individual community is drawn accordingly. The involved experiment data can be found in the supplementary material.

  4. 1,4-naphthoquinones: from oxidative damage to cellular and inter-cellular signaling.

    PubMed

    Klotz, Lars-Oliver; Hou, Xiaoqing; Jacob, Claus

    2014-09-17

    Naphthoquinones may cause oxidative stress in exposed cells and, therefore, affect redox signaling. Here, contributions of redox cycling and alkylating properties of quinones (both natural and synthetic, such as plumbagin, juglone, lawsone, menadione, methoxy-naphthoquinones, and others) to cellular and inter-cellular signaling processes are discussed: (i) naphthoquinone-induced Nrf2-dependent modulation of gene expression and its potentially beneficial outcome; (ii) the modulation of receptor tyrosine kinases, such as the epidermal growth factor receptor by naphthoquinones, resulting in altered gap junctional intercellular communication. Generation of reactive oxygen species and modulation of redox signaling are properties of naphthoquinones that render them interesting leads for the development of novel compounds of potential use in various therapeutic settings.

  5. Selective targeting of c-Abl via a cryptic mitochondrial targeting signal activated by cellular redox status in leukemic and breast cancer cells

    PubMed Central

    Constance, Jonathan E.; Despres, Samuel D.; Nishida, Akemi; Lim, Carol S.

    2012-01-01

    Purpose The tyrosine kinase c-Abl localizes to the mitochondria under cell stress conditions and promotes apoptosis. However, c-Abl has not been directly targeted to the mitochondria. Fusing c-Abl to a mitochondrial translocation signal (MTS) that is activated by reactive oxygen species (ROS) will selectively target the mitochondria of cancer cells exhibiting an elevated ROS phenotype. Mitochondrially targeted c-Abl will thereby induce malignant cell death. Methods Confocal microscopy was used to determine mitochondrial colocalization of ectopically expressed c-Abl-EGFP/cMTS fusion across three cell lines (K562, Cos-7, and 1471.1) with varying levels of basal (and pharmacologically modulated) ROS. ROS were quantified by indicator dye assay. The functional consequences of mitochondrial c-Abl were assessed by DNA accessibility to 7-AAD using flow cytometry. Results The cMTS and cMTS/c-Abl fusions colocalized to the mitochondria in leukemic (K562) and breast (1471.1) cancer phenotypes (but not Cos-7 fibroblasts) in a ROS and PKC dependent manner. Conclusions We confirm and extend oxidative stress activated translocation of the cMTS by demonstrating that the cMTS and Abl/cMTS fusion selectively target the mitochondria of K562 leukemia and mammary adenocarcinoma 1471.1 cells. c-Abl induced K562 leukemia cell death when targeted to the matrix but not the outer membrane of the mitochondria. PMID:22549737

  6. In search of cellular control: signal transduction in context

    NASA Technical Reports Server (NTRS)

    Ingber, D.

    1998-01-01

    The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

  7. Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: implications for cellular Aβ metabolism.

    PubMed

    Leal, María C; Surace, Ezequiel I; Holgado, María P; Ferrari, Carina C; Tarelli, Rodolfo; Pitossi, Fernando; Wisniewski, Thomas; Castaño, Eduardo M; Morelli, Laura

    2012-02-01

    Cerebral amyloid β (Aβ) accumulation is pathogenically associated with sporadic Alzheimer's disease (SAD). BACE-1 is involved in Aβ generation while insulin-degrading enzyme (IDE) partakes in Aβ proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aβ accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at -379/-372 and -310-303 from the first translation start site in the -575/-19 (556 bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aβ metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD.

  8. Quiescence and activation of stem and precursor cell populations in the subependymal zone of the mammalian brain are associated with distinct cellular and extracellular matrix signals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The subependymal zone (SEZ) of the lateral ventricles is one of the areas of the adult brain where new neurons are continuously generated from neural stem cells (NSCs), via rapidly dividing precursors. This neurogenic niche is a complex cellular and extracellular microenvironment, highly vascularize...

  9. Magnetic fields, radicals and cellular activity.

    PubMed

    Montoya, Ryan D

    2017-01-01

    Some effects of low-intensity magnetic fields on the concentration of radicals and their influence on cellular functions are reviewed. These fields have been implicated as a potential modulator of radical recombination rates. Experimental evidence has revealed a tight coupling between cellular function and radical pair chemistry from signaling pathways to damaging oxidative processes. The effects of externally applied magnetic fields on biological systems have been extensively studied, and the observed effects lack sufficient mechanistic understanding. Radical pair chemistry offers a reasonable explanation for some of the molecular effects of low-intensity magnetic fields, and changes in radical concentrations have been observed to modulate specific cellular functions. Applied external magnetic fields have been shown to induce observable cellular changes such as both inhibiting and accelerating cell growth. These and other mechanisms, such as cell membrane potential modulation, are of great interest in cancer research due to the variations between healthy and deleterious cells. Radical concentrations demonstrate similar variations and are indicative of a possible causal relationship. Radicals, therefore, present a possible mechanism for the modulation of cellular functions such as growth or regression by means of applied external magnetic fields.

  10. PACRG, a protein linked to ciliary motility, mediates cellular signaling.

    PubMed

    Loucks, Catrina M; Bialas, Nathan J; Dekkers, Martijn P J; Walker, Denise S; Grundy, Laura J; Li, Chunmei; Inglis, P Nick; Kida, Katarzyna; Schafer, William R; Blacque, Oliver E; Jansen, Gert; Leroux, Michel R

    2016-07-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan.

  11. PACRG, a protein linked to ciliary motility, mediates cellular signaling

    PubMed Central

    Loucks, Catrina M.; Bialas, Nathan J.; Dekkers, Martijn P. J.; Walker, Denise S.; Grundy, Laura J.; Li, Chunmei; Inglis, P. Nick; Kida, Katarzyna; Schafer, William R.; Blacque, Oliver E.; Jansen, Gert; Leroux, Michel R.

    2016-01-01

    Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon–associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan. PMID:27193298

  12. The Aryl Hydrocarbon Receptor Relays Metabolic Signals to Promote Cellular Regeneration

    PubMed Central

    2016-01-01

    While sensing the cell environment, the aryl hydrocarbon receptor (AHR) interacts with different pathways involved in cellular homeostasis. This review summarizes evidence suggesting that cellular regeneration in the context of aging and diseases can be modulated by AHR signaling on stem cells. New insights connect orphaned observations into AHR interactions with critical signaling pathways such as WNT to propose a role of this ligand-activated transcription factor in the modulation of cellular regeneration by altering pathways that nurture cellular expansion such as changes in the metabolic efficiency rather than by directly altering cell cycling, proliferation, or cell death. Targeting the AHR to promote regeneration might prove to be a useful strategy to avoid unbalanced disruptions of homeostasis that may promote disease and also provide biological rationale for potential regenerative medicine approaches. PMID:27563312

  13. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2004-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  14. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

    Bearman, Gregory H. (Inventor); Fraser, Scott E. (Inventor); Lansford, Russell D. (Inventor)

    2002-01-01

    A system and method for monitoring cellular activity in a cellular specimen. According to one embodiment, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into respective wavelength bands. The respective bands of fluorescence are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.

  15. Cellular force signal integration through vector logic gates.

    PubMed

    Steward, Robert L; Tan, Cheemeng; Cheng, Chao-Min; LeDuc, Philip R

    2015-02-26

    The multi-signal mechanical environment mammalian cells experience is often unaccounted for in current mechanical stimulation studies. To address this we developed a novel technique to induce dual integrated force inputs, uniaxial stretch and fluid shear stress and present here for the first time a vector logic-gate framework to characterize cellular response as a function of cytoskeletal reorganization. Using this framework we found that under fluid shear stress and uniaxial stretch NIH 3T3 fibroblasts responded by the Stretch OR Shear vector logic-gate and HUVECs responded by the NOT Stretch OR Shear vector logic-gate. We further developed a parsimonious model of cellular response to multiple mechanical stimuli, which provides a unifying model that captured the experimental response of both cell types.

  16. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  17. Cellular Metabolic and Autophagic Pathways: Traffic Control by Redox Signaling

    PubMed Central

    Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

    2013-01-01

    It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality, and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function. PMID:23702245

  18. Cellular metabolic and autophagic pathways: traffic control by redox signaling.

    PubMed

    Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

    2013-10-01

    It has been established that the key metabolic pathways of glycolysis and oxidative phosphorylation are intimately related to redox biology through control of cell signaling. Under physiological conditions glucose metabolism is linked to control of the NADH/NAD redox couple, as well as providing the major reductant, NADPH, for thiol-dependent antioxidant defenses. Retrograde signaling from the mitochondrion to the nucleus or cytosol controls cell growth and differentiation. Under pathological conditions mitochondria are targets for reactive oxygen and nitrogen species and are critical in controlling apoptotic cell death. At the interface of these metabolic pathways, the autophagy-lysosomal pathway functions to maintain mitochondrial quality and generally serves an important cytoprotective function. In this review we will discuss the autophagic response to reactive oxygen and nitrogen species that are generated from perturbations of cellular glucose metabolism and bioenergetic function.

  19. A bead-based western for high-throughput cellular signal transduction analyses

    PubMed Central

    Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

    2016-01-01

    Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

  20. Cadmium and cellular signaling cascades: To be or not to be?

    SciTech Connect

    Thevenod, Frank

    2009-08-01

    The cellular effects of the toxic metal cadmium (Cd) are manifold. A large proportion of the cellular reactions affected by ionic Cd{sup 2+} are mediated by cellular signaling cascades. The aim of this review is to provide a principal understanding of the known physiological signaling cascades, which are recruited by Cd{sup 2+}, and to highlight the fact that Cd{sup 2+}, similarly to other toxic metals, disrupts physiological signal transduction. In principle, second messengers are generated at the time of receptor activation, are short-lived, and act specifically in space and time through non-covalent binding on effectors to transiently alter their activity. Signaling dysregulation induced by Cd{sup 2+} is reflected by a permanent disruption of transducing modules, resulting in low and/or elevated and constant levels of second messengers, which overwhelm the control mechanisms of signaling. This disturbs physiological cellular functions, gene transcription and regulation and may result in cell death and/or stress-induced adaptation and survival as well as carcinogenesis. The impact of Cd{sup 2+} on Ca{sup 2+}-, cAMP-, NO-, ROS-, MAP-kinase-, PKB/Akt-, nuclear factor-kappa B-, and developmental signaling is critically discussed. The hierarchical as well as cooperative and integrative character of signaling cascades activated by Cd{sup 2+} is illustrated in the kidney proximal tubule, a major target of Cd{sup 2+} toxicity. This review also aspires to pinpoint new avenues of research that may contribute to a more differentiated view of the complex mechanisms underlying Cd{sup 2+} toxicity in target tissues and eventually lead to rationales and strategies for prevention and therapy of Cd{sup 2+} toxicity.

  1. Rapid Turnover of Extracellular Signal-Regulated Kinase 3 by the Ubiquitin-Proteasome Pathway Defines a Novel Paradigm of Mitogen-Activated Protein Kinase Regulation during Cellular Differentiation

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Pelletier, Stéphane; Pellerin, Johanne; Meloche, Sylvain

    2003-01-01

    Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G1 arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability. PMID:12808096

  2. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  3. JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection.

    PubMed

    Yang, Hairu; Kronhamn, Jesper; Ekström, Jens-Ola; Korkut, Gül Gizem; Hultmark, Dan

    2015-12-01

    The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. Here, we show that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduced the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduced the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection.

  4. Regulation of organismal proteostasis by trans-cellular chaperone signaling

    PubMed Central

    van Oosten-Hawle, Patricija; Porter, Robert S.; Morimoto, Richard I.

    2013-01-01

    Summary A major challenge for metazoans is to ensure that different tissues each expressing distinctive proteomes are, nevertheless, well protected at an organismal level from proteotoxic stress. We have examined this and show that expression of endogenous metastable protein sensors in muscle cells induces a systemic stress response throughout multiple tissues of C. elegans. Suppression of misfolding in muscle cells can be achieved not only by enhanced expression of HSP90 in muscle cells, but as effective by elevated expression of HSP90 in intestine or neuronal cells. This cell-non-autonomous control of HSP90 expression relies upon transcriptional feedback between somatic tissues that is regulated by the FoxA transcription factor PHA-4. This trans-cellular chaperone signaling response maintains organismal proteostasis when challenged by a local tissue imbalance in folding and provides the basis for a novel form of organismal stress sensing surveillance. PMID:23746847

  5. Modeling Cellular Noise Underlying Heterogeneous Cell Responses in the Epidermal Growth Factor Signaling Pathway

    PubMed Central

    Iwamoto, Kazunari; Shindo, Yuki; Takahashi, Koichi

    2016-01-01

    Cellular heterogeneity, which plays an essential role in biological phenomena, such as drug resistance and migration, is considered to arise from intrinsic (i.e., reaction kinetics) and extrinsic (i.e., protein variability) noise in the cell. However, the mechanistic effects of these types of noise to determine the heterogeneity of signal responses have not been elucidated. Here, we report that the output of epidermal growth factor (EGF) signaling activity is modulated by cellular noise, particularly by extrinsic noise of particular signaling components in the pathway. We developed a mathematical model of the EGF signaling pathway incorporating regulation between extracellular signal-regulated kinase (ERK) and nuclear pore complex (NPC), which is necessary for switch-like activation of the nuclear ERK response. As the threshold of switch-like behavior is more sensitive to perturbations than the graded response, the effect of biological noise is potentially critical for cell fate decision. Our simulation analysis indicated that extrinsic noise, but not intrinsic noise, contributes to cell-to-cell heterogeneity of nuclear ERK. In addition, we accurately estimated variations in abundance of the signal proteins between individual cells by direct comparison of experimental data with simulation results using Apparent Measurement Error (AME). AME was constant regardless of whether the protein levels varied in a correlated manner, while covariation among proteins influenced cell-to-cell heterogeneity of nuclear ERK, suppressing the variation. Simulations using the estimated protein abundances showed that each protein species has different effects on cell-to-cell variation in the nuclear ERK response. In particular, variability of EGF receptor, Ras, Raf, and MEK strongly influenced cellular heterogeneity, while others did not. Overall, our results indicated that cellular heterogeneity in response to EGF is strongly driven by extrinsic noise, and that such heterogeneity

  6. Cellular defense processes regulated by pathogen-elicited receptor signaling

    NASA Astrophysics Data System (ADS)

    Wu, Rongcong; Goldsipe, Arthur; Schauer, David B.; Lauffenburger, Douglas A.

    2011-06-01

    Vertebrates are constantly threatened by the invasion of microorganisms and have evolved systems of immunity to eliminate infectious pathogens in the body. Initial sensing of microbial agents is mediated by the recognition of pathogens by means of molecular structures expressed uniquely by microbes of a given type. So-called 'Toll-like receptors' are expressed on host epithelial barrier cells play an essential role in the host defense against microbial pathogens by inducing cell responses (e.g., proliferation, death, cytokine secretion) via activation of intracellular signaling networks. As these networks, comprising multiple interconnecting dynamic pathways, represent highly complex multi-variate "information processing" systems, the signaling activities particularly critical for governing the host cell responses are poorly understood and not easily ascertained by a priori theoretical notions. We have developed over the past half-decade a "data-driven" computational modeling approach, on a 'cue-signal-response' combined experiment/computation paradigm, to elucidate key multi-variate signaling relationships governing the cell responses. In an example presented here, we study how a canonical set of six kinase pathways combine to effect microbial agent-induced apoptotic death of a macrophage cell line. One modeling technique, partial least-squares regression, yielded the following key insights: {a} signal combinations most strongly correlated to apoptotic death are orthogonal to those most strongly correlated with release of inflammatory cytokines; {b} the ratio of two key pathway activities is the most powerful predictor of microbe-induced macrophage apoptotic death; {c} the most influential time-window of this signaling activity ratio is surprisingly fast: less than one hour after microbe stimulation.

  7. Cellular chromophores and signaling in low level light therapy

    NASA Astrophysics Data System (ADS)

    Hamblin, Michael R.; Demidova-Rice, Tatiana N.

    2007-02-01

    particular, signaling cascades are initiated via cyclic adenosine monophosphate (cAMP) and nuclear factor kappa B (NF-κB). These signal transduction pathways in turn lead to increased cell proliferation and migration (particularly by fibroblasts), modulation in levels of cytokines, growth factors and inflammatory mediators, and increases in anti-apoptotic proteins. The results of these biochemical and cellular changes in animals and patients include such benefits as increased healing in chronic wounds, improvements in sports injuries and carpal tunnel syndrome, pain reduction in arthritis and neuropathies, and amelioration of damage after heart attacks, stroke, nerve injury and retinal toxicity.

  8. AGCVIII Kinases: at the crossroads of cellular signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

  9. Magnetogenetics: Remote Control of Cellular Signaling with Magnetic Fields

    NASA Astrophysics Data System (ADS)

    Sauer, Jeremy P.

    Means for temporally regulating gene expression and cellular activity are invaluable for elucidating the underlying physiological processes and have therapeutic implications. Here we report the development of a system for remote regulation of gene expression by low frequency radiowaves (RF) or by a static magnetic field. We accomplished this by first adding iron oxide nanoparticles - either exogenously or as genetically encoded ferritin/ferric oxyhydroxide particle. These particles have been designed with affinity to the plasma membrane ion channel Transient Receptor Potential Vanilloid 1 (TRPV1) by a conjugated antibody. Application of a magnetic field stimulates the particle to gate the ion channel and this, in turn, initiates calcium-dependent transgene expression. We first demonstrated in vitro that TRPV1 can be actuated to cause calcium flux into the cell by directly applying a localized magnetic field. In mice expressing these genetically encoded components, application of external magnetic field caused remote stimulation of insulin transgene expression and significantly lowered blood glucose. In addition, we are investigating mechanisms by which iron oxide nanoparticles can absorb RF, and transduce this energy to cause channel opening. This robust, repeatable method for remote cellular regulation in vivo may ultimately have applications in basic science, as well as in technology and therapeutics.

  10. Modulation of cellular signaling by herpesvirus-encoded G protein-coupled receptors

    PubMed Central

    de Munnik, Sabrina M.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Human herpesviruses (HHVs) are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, HHVs have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies. PMID:25805993

  11. Perturbation biology: inferring signaling networks in cellular systems.

    PubMed

    Molinelli, Evan J; Korkut, Anil; Wang, Weiqing; Miller, Martin L; Gauthier, Nicholas P; Jing, Xiaohong; Kaushik, Poorvi; He, Qin; Mills, Gordon; Solit, David B; Pratilas, Christine A; Weigt, Martin; Braunstein, Alfredo; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

    2013-01-01

    We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology.

  12. Cellular insulin resistance disrupts leptin-mediated control of neuronal signaling and transcription.

    PubMed

    Nazarians-Armavil, Anaies; Menchella, Jonathan A; Belsham, Denise D

    2013-06-01

    Central resistance to the actions of insulin and leptin is associated with the onset of obesity and type 2 diabetes mellitus, whereas leptin and insulin signaling is essential for both glucose and energy homeostasis. Although it is known that leptin resistance can lead to attenuated insulin signaling, whether insulin resistance can lead to or exacerbate leptin resistance is unknown. To investigate the molecular events underlying crosstalk between these signaling pathways, immortalized hypothalamic neuronal models, rHypoE-19 and mHypoA-2/10, were used. Prolonged insulin exposure was used to induce cellular insulin resistance, and thereafter leptin-mediated regulation of signal transduction and gene expression was assessed. Leptin directly repressed agouti-related peptide mRNA levels but induced urocortin-2, insulin receptor substrate (IRS)-1, IRS2, and IR transcription, through leptin-mediated phosphatidylinositol 3-kinase/Akt activation. Neuronal insulin resistance, as assessed by attenuated Akt phosphorylation, blocked leptin-mediated signal transduction and agouti-related peptide, urocortin-2, IRS1, IRS2, and insulin receptor synthesis. Insulin resistance caused a substantial decrease in insulin receptor protein levels, forkhead box protein 1 phosphorylation, and an increase in suppressor of cytokine signaling 3 protein levels. Cellular insulin resistance may cause or exacerbate neuronal leptin resistance and, by extension, obesity. It is essential to unravel the effects of neuronal insulin resistance given that both peripheral, as well as the less widely studied central insulin resistance, may contribute to the development of metabolic, reproductive, and cardiovascular disorders. This study provides improved understanding of the complex cellular crosstalk between insulin-leptin signal transduction that is disrupted during neuronal insulin resistance.

  13. Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses.

    PubMed

    Barth, Kenneth; Genco, Caroline Attardo

    2016-10-04

    The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses.

  14. Microbial Degradation of Cellular Kinases Impairs Innate Immune Signaling and Paracrine TNFα Responses

    PubMed Central

    Barth, Kenneth; Genco, Caroline Attardo

    2016-01-01

    The NFκB and MAPK signaling pathways are critical components of innate immunity that orchestrate appropriate immune responses to control and eradicate pathogens. Their activation results in the induction of proinflammatory mediators, such as TNFα a potent bioactive molecule commonly secreted by recruited inflammatory cells, allowing for paracrine signaling at the site of an infection. In this study we identified a novel mechanism by which the opportunistic pathogen Porphyromonas gingivalis dampens innate immune responses by disruption of kinase signaling and degradation of inflammatory mediators. The intracellular immune kinases RIPK1, TAK1, and AKT were selectively degraded by the P. gingivalis lysine-specific gingipain (Kgp) in human endothelial cells, which correlated with dysregulated innate immune signaling. Kgp was also observed to attenuate endothelial responsiveness to TNFα, resulting in a reduction in signal flux through AKT, ERK and NFκB pathways, as well as a decrease in downstream proinflammatory mRNA induction of cytokines, chemokines and adhesion molecules. A deficiency in Kgp activity negated decreases to host cell kinase protein levels and responsiveness to TNFα. Given the essential role of kinase signaling in immune responses, these findings highlight a unique mechanism of pathogen-induced immune dysregulation through inhibition of cell activation, paracrine signaling, and dampened cellular proinflammatory responses. PMID:27698456

  15. The Wnt signaling pathway in cellular proliferation and differentiation: A tale of two coactivators.

    PubMed

    Teo, Jia-Ling; Kahn, Michael

    2010-09-30

    Wnt signaling pathways play divergent roles during development, normal homeostasis and disease. The responses that result from the activation of the pathway control both proliferation and differentiation. Tight regulation and controlled coordination of the Wnt signaling cascade is required to maintain the balance between proliferation and differentiation. The non-redundant roles of the coactivator proteins CBP and p300, within the context of Wnt signaling are discussed. We highlight their roles as integrators of the various inputs that a cell receives to elicit the correct and coordinated response. We propose that essentially all cellular information - i.e. from other signaling pathways, nutrient levels, etc. - is funneled down into a choice of coactivators usage, either CBP or p300, by their interacting partner beta-catenin (or catenin-like molecules in the absence of beta-catenin) to make the critical decision to either remain quiescent, or once entering cycle to proliferate without differentiation or to initiate the differentiation process.

  16. Cadmium and cellular signaling cascades: interactions between cell death and survival pathways.

    PubMed

    Thévenod, Frank; Lee, Wing-Kee

    2013-10-01

    Cellular stress elicited by the toxic metal Cd(2+) does not coerce the cell into committing to die from the onset. Rather, detoxification and adaptive processes are triggered concurrently, allowing survival until normal function is restored. With high Cd(2+), death pathways predominate. However, if sublethal stress levels affect cells for prolonged periods, as in chronic low Cd(2+) exposure, adaptive and survival mechanisms may deregulate, such that tumorigenesis ensues. Hence, death and malignancy are the two ends of a continuum of cellular responses to Cd(2+), determined by magnitude and duration of Cd(2+) stress. Signaling cascades are the key factors affecting cellular reactions to Cd(2+). This review critically surveys recent literature to outline major features of death and survival signaling pathways as well as their activation, interactions and cross talk in cells exposed to Cd(2+). Under physiological conditions, receptor activation generates 2nd messengers, which are short-lived and act specifically on effectors through their spatial and temporal dynamics to transiently alter effector activity. Cd(2+) recruits physiological 2nd messenger systems, in particular Ca(2+) and reactive oxygen species (ROS), which control key Ca(2+)- and redox-sensitive molecular switches dictating cell function and fate. Severe ROS/Ca(2+) signals activate cell death effectors (ceramides, ASK1-JNK/p38, calpains, caspases) and/or cause irreversible damage to vital organelles, such as mitochondria and endoplasmic reticulum (ER), whereas low localized ROS/Ca(2+) levels act as 2nd messengers promoting cellular adaptation and survival through signal transduction (ERK1/2, PI3K/Akt-PKB) and transcriptional regulators (Ref1-Nrf2, NF-κB, Wnt, AP-1, bestrophin-3). Other cellular proteins and processes targeted by ROS/Ca(2+) (metallothioneins, Bcl-2 proteins, ubiquitin-proteasome system, ER stress-associated unfolded protein response, autophagy, cell cycle) can evoke death or survival

  17. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

    PubMed Central

    Mckenzie, Grahame; Ward, George; Stallwood, Yvette; Briend, Emmanuel; Papadia, Sofia; Lennard, Andrew; Turner, Martin; Champion, Brian; Hardingham, Giles E

    2006-01-01

    Background Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. Results We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. Conclusion The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses. PMID:16507111

  18. Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

    PubMed Central

    Hellesøy, Monica; Lorens, James B.

    2015-01-01

    The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

  19. Modulation of 17β-Estradiol Signaling on Cellular Proliferation by Caveolin-2.

    PubMed

    Totta, Pierangela; Gionfra, Fabio; Busonero, Claudia; Acconcia, Filippo

    2016-06-01

    The sex hormone 17β-estradiol (E2) exerts pleiotropic effects by binding to the ligand-activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects (i.e., estrogen responsive element [ERE]-based gene transcription) and non-transcriptional membrane-initiated effects (i.e., the activation of extra-nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω-shaped membrane sub-domains that are composed of scaffold proteins named caveolins (i.e., caveolin-1, caveolin-2, and caveolin-3). Although caveolin-3 is exclusively expressed in muscles, caveolin-1 and caveolin-2 are co-expressed in all human tissues. From a functional point of view, caveolin-2 can operate both dependently on and independently of caveolin-1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin-1 and ERα has been reported in the control of E2-induced physiological effects, the role of caveolin-2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA-mediated caveolin-2 depletion in breast ductal carcinoma cells (MCF-7) reduces E2-induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα-mediated extra-nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin-1 depletion on ERα signaling in MCF-7 cells is shown to be similar to that elicited by siRNA-mediated caveolin-2 depletion. Altogether, these data demonstrate that caveolin-2 expression is necessary for the control of E2-dependent cellular proliferation.

  20. Sub-cellular and Multi-cellular Signaling Mechanisms Revealed by Quantitative Laser Microscopies

    NASA Astrophysics Data System (ADS)

    Piston, David

    2005-03-01

    Newly developed instrumentation and optical probes allows us to image quantitatively dynamic processes within ever more complicated biological systems. Using methods such as fluorescence recovery after photobleaching (FRAP) and Förster resonance energy transfer (FRET) of GFPs fused to the glucose sensing enzyme glucokinase (GK), we have discovered that the location and activity of beta cell GK is acutely regulated by insulin. These findings provide a mechanism whereby the glucose sensing ability of the beta cell is tightly coupled to insulin signaling. We have also measured pancreatic β-cell metabolism during glucose stimulation by quantitative two-photon NAD(P)H imaging. We have developed methods to delineate quantitatively the NAD(P)H signals from the cytoplasm and mitochondria, and show that the metabolic response of these two compartments are differentially stimulated by glucose and other metabolites. Absolute levels of NAD(P)H were determined using two-photon excited fluorescence lifetime imaging (FLIM). These findings elucidate the relative contributions of glycolytic and citric acid cycle metabolism in normal and diabetic cells.

  1. Signal Destruction Tunes the Zone of Activation in Spatially Distributed Signaling Networks.

    PubMed

    Silva, Kalinga Pavan; Chellamuthu, Prithiviraj; Boedicker, James Q

    2017-03-14

    Diverse microbial communities coordinate group behaviors through signal exchange, such as the exchange of acyl-homoserine lactones (AHLs) by Gram-negative bacteria. Cellular communication is prone to interference by neighboring microbes. One mechanism of interference is signal destruction through the production of an enzyme that cleaves the signaling molecule. Here we examine the ability of one such interference enzyme, AiiA, to modulate signal propagation in a spatially distributed system of bacteria. We have developed an experimental assay to measure signal transduction and implement a theoretical model of signaling dynamics to predict how the system responds to interference. We show that titration of an interfering strain into a signaling network tunes the spatial range of activation over the centimeter length scale, quantifying the robustness of the signaling network to signal destruction and demonstrating the ability to program systems-level responses of spatially heterogeneous cellular networks.

  2. Cellular signal mechanisms of reward-related plasticity in the hippocampus.

    PubMed

    Isokawa, Masako

    2012-01-01

    The hippocampus has the extraordinary capacity to process and store information. Consequently, there is an intense interest in the mechanisms that underline learning and memory. Synaptic plasticity has been hypothesized to be the neuronal substrate for learning. Ca(2+) and Ca(2+)-activated kinases control cellular processes of most forms of hippocampal synapse plasticity. In this paper, I aim to integrate our current understanding of Ca(2+)-mediated synaptic plasticity and metaplasticity in motivational and reward-related learning in the hippocampus. I will introduce two representative neuromodulators that are widely studied in reward-related learning (e.g., ghrelin and endocannabinoids) and show how they might contribute to hippocampal neuron activities and Ca(2+)-mediated signaling processes in synaptic plasticity. Additionally, I will discuss functional significance of these two systems and their signaling pathways for its relevance to maladaptive reward learning leading to addiction.

  3. Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

    SciTech Connect

    Zhdanov, Alexander V.; Waters, Alicia H.C.; Golubeva, Anna V.; Papkovsky, Dmitri B.

    2015-01-01

    Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP, produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.

  4. Cellular signaling: aspects for tumor diagnosis and therapy.

    PubMed

    Wolf, Bernhard; Brischwein, Martin; Lob, Volker; Ressler, Johann; Wiest, Joachim

    2007-02-01

    Cells are organic microsystems with functional compartments interconnected by complex signal chains. Intracellular signaling routes and signal reception from the extracellular environment are characterized by redundancy, i.e., parallel pathways exist. If a cell is exposed to an external "signal input", the signal processing elements within the cell provide a response that will be a pattern of reactions manifest as a metabolic, morphologic or electric "signal output". Cell-chip hybrid structures are miniaturized analytical systems with the capability to monitor such cell responses in real time and under continuous control of the environmental conditions. A system analysis approach gives an idea of how the biological component of these hybrid structures works. This is exemplified by the putative role of the microenvironmental pH as a parameter of the utmost importance for the malignant "mode" of tumor cells, which can be monitored and modeled on such hybrid structures.

  5. Sensitivity control through attenuation of signal transfer efficiency by negative regulation of cellular signalling.

    PubMed

    Toyoshima, Yu; Kakuda, Hiroaki; Fujita, Kazuhiro A; Uda, Shinsuke; Kuroda, Shinya

    2012-03-13

    Sensitivity is one of the hallmarks of biological and pharmacological responses. However, the principle of controlling sensitivity remains unclear. Here we theoretically analyse a simple biochemical reaction and find that the signal transfer efficiency of the transient peak amplitude attenuates depending on the strength of negative regulation. We experimentally find that many signalling pathways in various cell lines, including the Akt and ERK pathways, can be approximated by simple biochemical reactions and that the same property of the attenuation of signal transfer efficiency was observed for such pathways. Because of this property, a downstream molecule should show higher sensitivity to an activator and lower sensitivity to an inhibitor than an upstream molecule. Indeed, we experimentally verify that S6, which lies downstream of Akt, shows lower sensitivity to an epidermal growth factor receptor inhibitor than Akt. Thus, cells can control downstream sensitivity through the attenuation of signal transfer efficiency by changing the expression level of negative regulators.

  6. ECM Signaling Regulates Collective Cellular Dynamics to Control Pancreas Branching Morphogenesis.

    PubMed

    Shih, Hung Ping; Panlasigui, Devin; Cirulli, Vincenzo; Sander, Maike

    2016-01-12

    During pancreas development, epithelial buds undergo branching morphogenesis to form an exocrine and endocrine gland. Proper morphogenesis is necessary for correct lineage allocation of pancreatic progenitors; however, the cellular events underlying pancreas morphogenesis are unknown. Here, we employed time-lapse microscopy and fluorescent labeling of cells to analyze cell behaviors associated with pancreas morphogenesis. We observed that outer bud cells adjacent to the basement membrane are pleomorphic and rearrange frequently; additionally, they largely remain in the outer cell compartment even after mitosis. These cell behaviors and pancreas branching depend on cell contacts with the basement membrane, which induce actomyosin cytoskeleton remodeling via integrin-mediated activation of FAK/Src signaling. We show that integrin signaling reduces E-cadherin-mediated cell-cell adhesion in outer cells and provide genetic evidence that this regulation is necessary for initiation of branching. Our study suggests that regulation of cell motility and adhesion by local niche cues initiates pancreas branching morphogenesis.

  7. LPA signaling through LPA receptors regulates cellular functions of endothelial cells treated with anticancer drugs.

    PubMed

    Mori, Shiori; Araki, Mutsumi; Ishii, Shuhei; Hirane, Miku; Fukushima, Kaori; Tomimatsu, Ayaka; Takahashi, Kaede; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2015-10-01

    Lysophosphatidic acid (LPA) signaling via LPA receptors provides a variety of cellular functions, including angiogenesis. In this study, to assess an involvement of LPA receptors in cell motile activities of endothelial cells during chemotherapy, F-2 cells were treated with cisplatin (CDDP) and doxorubicin (DOX) at a concentration of 0.01 μM every 24 h for at least 1 month. The treatment of CDDP and DOX inhibited the expression levels of the LPA receptor-1 (Lpar1), Lpar2, and Lpar3 genes in F-2 cells. The cell motile activities of CDDP and DOX treated cells were relatively lower than those of untreated cells. Next, we investigated whether cancer cells could stimulate the cell motile activities of F-2 cells treated with CDDP and DOX. For cell motility assay, CDDP- and DOX-treated cells were co-cultured with pancreatic cancer PANC-1 cells. The cell motile activities of CDDP- and DOX-treated cells were significantly enhanced by the existence of PANC-1 cells, correlating with the LPA receptor expressions. In addition, the elevated cell motile activities were suppressed by the pretreatment of an autotaxin inhibitor S32826. These results suggest that LPA signaling via LPA receptors may regulate the cell motile activities of F-2 cells treated with anticancer drugs.

  8. Neurophysiological, metabolic and cellular compartments that drive neurovascular coupling and neuroimaging signals

    PubMed Central

    Moreno, Andrea; Jego, Pierrick; de la Cruz, Feliberto; Canals, Santiago

    2013-01-01

    Complete understanding of the mechanisms that coordinate work and energy supply of the brain, the so called neurovascular coupling, is fundamental to interpreting brain energetics and their influence on neuronal coding strategies, but also to interpreting signals obtained from brain imaging techniques such as functional magnetic resonance imaging. Interactions between neuronal activity and cerebral blood flow regulation are largely compartmentalized. First, there exists a functional compartmentalization in which glutamatergic peri-synaptic activity and its electrophysiological events occur in close proximity to vascular responses. Second, the metabolic processes that fuel peri-synaptic activity are partially segregated between glycolytic and oxidative compartments. Finally, there is cellular segregation between astrocytic and neuronal compartments, which has potentially important implications on neurovascular coupling. Experimental data is progressively showing a tight interaction between the products of energy consumption and neurotransmission-driven signaling molecules that regulate blood flow. Here, we review some of these issues in light of recent findings with special attention to the neuron-glia interplay on the generation of neuroimaging signals. PMID:23543907

  9. Dial 9-1-1 for p53: Mechanisms of p53 Activation by Cellular Stress

    PubMed Central

    Ljungman, Mats

    2000-01-01

    Abstract The tumor suppressor protein, p53, is part of the cell's emergency team that is called upon following cellular insult. How do cells sense DNA damage and other cellular stresses and what signal transduction pathways are used to alert p53? How is the resulting nuclear accumulation of p53 accomplished and what determines the outcome of p53 induction? Many posttranslational modifications of p53, such as phosphorylation, dephosphorylation, acetylation and ribosylation, have been shown to occur following cellular stress. Some of these modifications may activate the p53 protein, interfere with MDM2 binding and/or dictate cellular localization of p53. This review will focus on recent findings about how the p53 response may be activated following cellular stress. PMID:10935507

  10. Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

    PubMed

    O'Clock, George D

    2016-08-01

    Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

  11. Signaling pathways involved in PDGF-evoked cellular responses in human RPE cells

    SciTech Connect

    Hollborn, Margrit . E-mail: hollbm@medizin.uni-leipzig.de; Bringmann, Andreas; Faude, Frank; Wiedemann, Peter; Kohen, Leon

    2006-06-09

    We examined whether PDGF may directly stimulate the expression of VEGF by retinal pigment epithelial (RPE) cells in vitro, and the involvement of three signal transduction pathways in the regulation of PDGF-evoked cell proliferation, migration, and production of VEGF-A was investigated. PDGF stimulated the gene and protein expression of VEGF-A by RPE cells, and increased cell proliferation and chemotaxis. PDGF activated all signaling pathways investigated, as determined by increased phosphorylation levels of ERK1/2, p38, and Akt proteins. The three signaling pathways were involved in the mediation of PDGF-evoked cell proliferation, while p38 and PI3K mediated cell migration, and PI3K mediated secretion of VEGF-A. In addition to VEGF-A, the cells expressed mRNAs for various members of the VEGF family and for their receptors, including VEGF-B, -C, -D, flt-1, and KDR. The data indicate that PDGF selectively stimulates the expression of VEGF-A in RPE cells. PDGF evokes at least three signal transduction pathways which are differentially involved in various cellular responses.

  12. Driving Cellular Plasticity and Survival Through the Signal Transduction Pathways of Metabotropic Glutamate Receptors

    PubMed Central

    Maiese, Kenneth; Chong, Zhao Zhong; Li, Faqi

    2008-01-01

    Metabotropic glutamate receptors (mGluRs) share a common molecular morphology with other G protein–linked receptors, but there expression throughout the mammalian nervous system places these receptors as essential mediators not only for the initial development of an organism, but also for the vital determination of a cell’s fate during many disorders in the nervous system that include amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, Multiple Sclerosis, epilepsy, trauma, and stroke. Given the ubiquitous distribution of these receptors, the mGluR system impacts upon neuronal, vascular, and glial cell function and is activated by a wide variety of stimuli that includes neurotransmitters, peptides, hormones, growth factors, ions, lipids, and light. Employing signal transduction pathways that can modulate both excitatory and inhibitory responses, the mGluR system drives a spectrum of cellular pathways that involve protein kinases, endonucleases, cellular acidity, energy metabolism, mitochondrial membrane potential, caspases, and specific mitogen-activated protein kinases. Ultimately these pathways can converge to regulate genomic DNA degradation, membrane phosphatidylserine (PS) residue exposure, and inflammatory microglial activation. As we continue to push the envelope for our understanding of this complex and critical family of metabotropic receptors, we should be able to reap enormous benefits for both clinical disease as well as our understanding of basic biology in the nervous system. PMID:16375723

  13. A nexus for cellular homeostasis: the interplay between metabolic and signal transduction pathways.

    PubMed

    Gomes, Ana P; Blenis, John

    2015-08-01

    In multicellular organisms, individual cells have evolved to sense external and internal cues in order to maintain cellular homeostasis and survive under different environmental conditions. Cells efficiently adjust their metabolism to reflect the abundance of nutrients, energy and growth factors. The ability to rewire cellular metabolism between anabolic and catabolic processes is crucial for cells to thrive. Thus, cells have developed, through evolution, metabolic networks that are highly plastic and tightly regulated to meet the requirements necessary to maintain cellular homeostasis. The plasticity of these cellular systems is tightly regulated by complex signaling networks that integrate the intracellular and extracellular information. The coordination of signal transduction and metabolic pathways is essential in maintaining a healthy and rapidly responsive cellular state.

  14. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  15. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  16. Stable cellular senescence is associated with persistent DDR activation.

    PubMed

    Fumagalli, Marzia; Rossiello, Francesca; Mondello, Chiara; d'Adda di Fagagna, Fabrizio

    2014-01-01

    The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.

  17. Neu1 sialidase and matrix metalloproteinase-9 cross-talk regulates nucleic acid-induced endosomal TOLL-like receptor-7 and -9 activation, cellular signaling and pro-inflammatory responses.

    PubMed

    Abdulkhalek, Samar; Szewczuk, Myron R

    2013-11-01

    The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G-protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B GPCR, all of which form a tripartite complex with TLR-7 and -9. siRNA silencing Neu1, MMP-9 and neuromedin-B GPCR in RAW-blue macrophage cells significantly reduced TLR7 imiquimod- and TLR9 ODN1826-induced NF-κB (NF-κB-pSer(536)) activity. Tamiflu, specific MMP-9 inhibitor, neuromedin B receptor specific antagonist BIM23127, and the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 significantly block nucleic acid-induced TLR-7 and -9 MyD88 recruitment, NF-κB activation and proinflammatory TNFα and MCP-1 cytokine responses. For the first time, Neu1 clearly plays a central role in mediating nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR-MMP9-Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses.

  18. Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

    PubMed Central

    Billaud, Marie; Lohman, Alexander W.; Johnstone, Scott R.; Biwer, Lauren A.; Mutchler, Stephanie; Isakson, Brant E.

    2014-01-01

    It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

  19. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen.

  20. REGgamma modulates p53 activity by regulating its cellular localization.

    PubMed

    Liu, Jian; Yu, Guowu; Zhao, Yanyan; Zhao, Dengpan; Wang, Ying; Wang, Lu; Liu, Jiang; Li, Lei; Zeng, Yu; Dang, Yongyan; Wang, Chuangui; Gao, Guang; Long, Weiwen; Lonard, David M; Qiao, Shanlou; Tsai, Ming-Jer; Zhang, Bianhong; Luo, Honglin; Li, Xiaotao

    2010-12-01

    The proteasome activator REGγ mediates a shortcut for the destruction of intact mammalian proteins. The biological roles of REGγ and the underlying mechanisms are not fully understood. Here we provide evidence that REGγ regulates cellular distribution of p53 by facilitating its multiple monoubiquitylation and subsequent nuclear export and degradation. We also show that inhibition of p53 tetramerization by REGγ might further enhance cytoplasmic relocation of p53 and reduce active p53 in the nucleus. Furthermore, multiple monoubiquitylation of p53 enhances its physical interaction with HDM2 and probably facilitates subsequent polyubiquitylation of p53, suggesting that monoubiquitylation can act as a signal for p53 degradation. Depletion of REGγ sensitizes cells to stress-induced apoptosis, validating its crucial role in the control of apoptosis, probably through regulation of p53 function. Using a mouse xenograft model, we show that REGγ knockdown results in a significant reduction of tumor growth, suggesting an important role for REGγ in tumor development. Our study therefore demonstrates that REGγ-mediated inactivation of p53 is one of the mechanisms involved in cancer progression.

  1. Reporters to monitor cellular MMP12 activity

    NASA Astrophysics Data System (ADS)

    Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten

    2010-02-01

    Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.

  2. Misconstrued versatility of Ganoderma lucidum: a key player in multi-targeted cellular signaling.

    PubMed

    Gill, Balraj Singh; Sharma, Prateek; Kumar, Raj; Kumar, Sanjeev

    2016-03-01

    A Basidiomycetes fungus belonging to polypore family of mushrooms, Ganoderma lucidum (GL), has been known since a long time for their myriad therapeutic indications. Renowned as an invaluable resource of cardinal mycoconstituents they encompass numerous terpenoids polysaccharides and proteins. Possessing the therapeutically potent lanosteroidal skeleton, terpenoids are upheld for their invariable participation in therapeutically diverse bioactivities. Polysaccharides and proteins exhibiting distinguishable bioactivities provide this oriental mushroom with additional edges over immune function and anti-cancer potential. This review is a concerted effort to throw light upon the therapeutic versatility of the fungus, shadowed by various other natural products. An effort has been made towards conglomerating the mycoconstituents decisive for the many activities portrayed by this fungus. More importantly, this review seeks to fathom the inextricable role played by derivatives in modulating signaling cascades such as downregulation of various mitogenic pathways, inhibiting growth factors, or upregulating certain pathways enhancing cellular integrity.

  3. Influence of marihuana on cellular structures and biochemical activities.

    PubMed

    Tahir, S K; Zimmerman, A M

    1991-11-01

    Cannabinoids are known to affect a number of cellular systems and functions, but the basis for their action is unclear. In this paper we review the current evidence describing cannabinoid effects on various levels of cellular structure and activity and we present our current studies on the influence of delta-9-tetrahydrocannabinol, cannabidiol and cannabinol on one cellular system, the cytoskeleton. The organization of two cytoskeletal structures, microtubules and microfilaments, were examined and the mRNA levels of tubulin and actin, the major protein components of microtubules and microfilaments, respectively, were analysed.

  4. Modeling earthquake activity using a memristor-based cellular grid

    NASA Astrophysics Data System (ADS)

    Vourkas, Ioannis; Sirakoulis, Georgios Ch.

    2013-04-01

    Earthquakes are absolutely among the most devastating natural phenomena because of their immediate and long-term severe consequences. Earthquake activity modeling, especially in areas known to experience frequent large earthquakes, could lead to improvements in infrastructure development that will prevent possible loss of lives and property damage. An earthquake process is inherently a nonlinear complex system and lately scientists have become interested in finding possible analogues of earthquake dynamics. The majority of the models developed so far were based on a mass-spring model of either one or two dimensions. An early approach towards the reordering and the improvement of existing models presenting the capacitor-inductor (LC) analogue, where the LC circuit resembles a mass-spring system and simulates earthquake activity, was also published recently. Electromagnetic oscillation occurs when energy is transferred between the capacitor and the inductor. This energy transformation is similar to the mechanical oscillation that takes place in the mass-spring system. A few years ago memristor-based oscillators were used as learning circuits exposed to a train of voltage pulses that mimic environment changes. The mathematical foundation of the memristor (memory resistor), as the fourth fundamental passive element, has been expounded by Leon Chua and later extended to a more broad class of memristors, known as memristive devices and systems. This class of two-terminal passive circuit elements with memory performs both information processing and storing of computational data on the same physical platform. Importantly, the states of these devices adjust to input signals and provide analog capabilities unavailable in standard circuit elements, resulting in adaptive circuitry and providing analog parallel computation. In this work, a memristor-based cellular grid is used to model earthquake activity. An LC contour along with a memristor is used to model seismic activity

  5. Coordinated modulation of cellular signaling through ligand-gated ion channels in Hydra vulgaris (Cnidaria, Hydrozoa).

    PubMed

    Pierobon, Paola

    2012-01-01

    Cnidarians lack well developed organs, but they have evolved the molecular and cellular components needed to assemble a nervous system. The apparent 'simplicity' of the cnidarian nervous net does not occur at the cellular level, but rather in the organisation of conducting systems. Cnidarian neurons are in fact electrically excitable, show the typical extended morphology and are connected by chemical synapses or gap junctions. They have been regarded as peptidergic, given the wealth of neuropeptides generally distributed along neurites and in cell bodies, supporting the hypothesis of a modulatory role in neurotransmission. However, the presence of clear-cored, as well as dense-cored synaptic vesicles in cnidarian neurons suggests both fast and slow synaptic transmission mechanisms. In fact, biochemical and functional evidence indicates that classical neurotransmitters and their metabolic partners are present in cnidarian tissues, where they are involved in coordinating motility and behavior. We have identified and characterized in Hydra tissues receptors to the inhibitory and excitatory amino acid neurotransmitters, GABA, glycine and NMDA, that are similar to mammalian ionotropic receptors in terms of their biochemical and pharmacological properties. These receptors appear to regulate pacemaker activities and their physiological correlates; in the live animal, they also affect feeding behavior, namely the duration and termination of the response elicited by reduced glutathione, with opposite actions of GABA and glycine or NMDA, respectively. These results suggest that modulation of cellular signaling through ligand-gated-ion channels is an ancient characteristic in the animal kingdom, and that the pharmacological properties of these receptors have been highly conserved during evolution.

  6. Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

    PubMed Central

    Shi, Xiaohong; Botting, Catherine H.; Li, Ping; Niglas, Mark; Brennan, Benjamin; Shirran, Sally L.; Szemiel, Agnieszka M.; Elliott, Richard M.

    2016-01-01

    The M genome segment of Bunyamwera virus (BUNV)—the prototype of both the Bunyaviridae family and the Orthobunyavirus genus—encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SPNSm and NSm domain V as SPGc. Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SPGc) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17–312 or nearby residues; NSm, 332–477; and Gc, 478–1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies. PMID:27439867

  7. Dynamic Simulation of 1D Cellular Automata in the Active aTAM.

    PubMed

    Jonoska, Nataša; Karpenko, Daria; Seki, Shinnosuke

    2015-07-01

    The Active aTAM is a tile based model for self-assembly where tiles are able to transfer signals and change identities according to the signals received. We extend Active aTAM to include deactivation signals and thereby allow detachment of tiles. We show that the model allows a dynamic simulation of cellular automata with assemblies that do not record the entire computational history but only the current updates of the states, and thus provide a way for (a) algorithmic dynamical structural changes in the assembly and (b) reusable space in self-assembly. The simulation is such that at a given location the sequence of tiles that attach and detach corresponds precisely to the sequence of states the synchronous cellular automaton generates at that location.

  8. Dynamic Simulation of 1D Cellular Automata in the Active aTAM

    PubMed Central

    Jonoska, Nataša; Karpenko, Daria; Seki, Shinnosuke

    2016-01-01

    The Active aTAM is a tile based model for self-assembly where tiles are able to transfer signals and change identities according to the signals received. We extend Active aTAM to include deactivation signals and thereby allow detachment of tiles. We show that the model allows a dynamic simulation of cellular automata with assemblies that do not record the entire computational history but only the current updates of the states, and thus provide a way for (a) algorithmic dynamical structural changes in the assembly and (b) reusable space in self-assembly. The simulation is such that at a given location the sequence of tiles that attach and detach corresponds precisely to the sequence of states the synchronous cellular automaton generates at that location. PMID:27789918

  9. Reverse Signaling by Semaphorin-6A Regulates Cellular Aggregation and Neuronal Morphology

    PubMed Central

    Perez-Branguli, Francesc; Zagar, Yvrick; Shanley, Daniel K.; Graef, Isabella A.; Chédotal, Alain; Mitchell, Kevin J.

    2016-01-01

    The transmembrane semaphorin, Sema6A, has important roles in axon guidance, cell migration and neuronal connectivity in multiple regions of the nervous system, mediated by context-dependent interactions with plexin receptors, PlxnA2 and PlxnA4. Here, we demonstrate that Sema6A can also signal cell-autonomously, in two modes, constitutively, or in response to higher-order clustering mediated by either PlxnA2-binding or chemically induced multimerisation. Sema6A activation stimulates recruitment of Abl to the cytoplasmic domain of Sema6A and phos¡phorylation of this cytoplasmic tyrosine kinase, as well as phosphorylation of additional cytoskeletal regulators. Sema6A reverse signaling affects the surface area and cellular complexity of non-neuronal cells and aggregation and neurite formation of primary neurons in vitro. Sema6A also interacts with PlxnA2 in cis, which reduces binding by PlxnA2 of Sema6A in trans but not vice versa. These experiments reveal the complex nature of Sema6A biochemical functions and the molecular logic of the context-dependent interactions between Sema6A and PlxnA2. PMID:27392094

  10. Acylcarnitines activate proinflammatory signaling pathways.

    PubMed

    Rutkowsky, Jennifer M; Knotts, Trina A; Ono-Moore, Kikumi D; McCoin, Colin S; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Adams, Sean H; Hwang, Daniel H

    2014-06-15

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed D,L isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant L-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. L-C14 carnitine (5-25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, L-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, L-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified.

  11. Acylcarnitines activate proinflammatory signaling pathways

    PubMed Central

    Rutkowsky, Jennifer M.; Knotts, Trina A.; Ono-Moore, Kikumi D.; McCoin, Colin S.; Huang, Shurong; Schneider, Dina; Singh, Shamsher; Hwang, Daniel H.

    2014-01-01

    Incomplete β-oxidation of fatty acids in mitochondria is a feature of insulin resistance and type 2 diabetes mellitus (T2DM). Previous studies revealed that plasma concentrations of medium- and long-chain acylcarnitines (by-products of incomplete β-oxidation) are elevated in T2DM and insulin resistance. In a previous study, we reported that mixed d,l isomers of C12- or C14-carnitine induced an NF-κB-luciferase reporter gene in RAW 264.7 cells, suggesting potential activation of proinflammatory pathways. Here, we determined whether the physiologically relevant l-acylcarnitines activate classical proinflammatory signaling pathways and if these outcomes involve pattern recognition receptor (PRR)-associated pathways. Acylcarnitines induced the expression of cyclooxygenase-2 in a chain length-dependent manner in RAW 264.7 cells. l-C14 carnitine (5–25 μM), used as a representative acylcarnitine, stimulated the expression and secretion of proinflammatory cytokines in a dose-dependent manner. Furthermore, l-C14 carnitine induced phosphorylation of JNK and ERK, common downstream components of many proinflammatory signaling pathways including PRRs. Knockdown of MyD88, a key cofactor in PRR signaling and inflammation, blunted the proinflammatory effects of acylcarnitine. While these results point to potential involvement of PRRs, l-C14 carnitine promoted IL-8 secretion from human epithelial cells (HCT-116) lacking Toll-like receptors (TLR)2 and -4, and did not activate reporter constructs in TLR overexpression cell models. Thus, acylcarnitines have the potential to activate inflammation, but the specific molecular and tissue target(s) involved remain to be identified. PMID:24760988

  12. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

    PubMed

    Kalo, Alon; Kanter, Itamar; Shraga, Amit; Sheinberger, Jonathan; Tzemach, Hadar; Kinor, Noa; Singer, Robert H; Lionnet, Timothée; Shav-Tal, Yaron

    2015-04-21

    The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  13. Lysophosphatidic acid signaling via LPA1 and LPA3 regulates cellular functions during tumor progression in pancreatic cancer cells.

    PubMed

    Fukushima, Kaori; Takahashi, Kaede; Yamasaki, Eri; Onishi, Yuka; Fukushima, Nobuyuki; Honoki, Kanya; Tsujiuchi, Toshifumi

    2017-03-01

    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors exhibits a variety of biological effects, such as cell proliferation, motility and differentiation. The aim of this study was to evaluate the roles of LPA1 and LPA3 in cellular functions during tumor progression in pancreatic cancer cells. LPA1 and LPA3 knockdown cells were generated from PANC-1 cells. The cell motile and invasive activities of PANC-1 cells were inhibited by LPA1 and LPA3 knockdown. In gelatin zymography, LPA1 and LPA3 knockdown cells indicated the low activation of matrix metalloproteinase-2 (MMP-2) in the presence of LPA. Next, to assess whether LPA1 and LPA3 regulate cellular functions induced by anticancer drug, PANC-1 cells were treated with cisplatin (CDDP) for approximately 6 months. The cell motile and invasive activities of long-term CDDP treated cells were markedly higher than those of PANC-1 cells, correlating with the expression levels of LPAR1 and LPAR3 genes. In soft agar assay, the long-term CDDP treated cells formed markedly large sized colonies. In addition, the cell motile and invasive activities enhanced by CDDP were significantly suppressed by LPA1 and LPA3 knockdown as well as colony formation. These results suggest that LPA signaling via LPA1 and LPA3 play an important role in the regulation of cellular functions during tumor progression in PANC-1 cells.

  14. Modeling the effect of microscopic driving behaviors on Kerner's time-delayed traffic breakdown at traffic signal using cellular automata

    NASA Astrophysics Data System (ADS)

    Wang, Yang; Chen, Yan-Yan

    2016-12-01

    The signalized traffic is considerably complex due to the fact that various driving behaviors have emerged to respond to traffic signals. However, the existing cellular automaton models take the signal-vehicle interactions into account inadequately, resulting in a potential risk that vehicular traffic flow dynamics may not be completely explored. To remedy this defect, this paper proposes a more realistic cellular automaton model by incorporating a number of the driving behaviors typically observed when the vehicles are approaching a traffic light. In particular, the anticipatory behavior proposed in this paper is realized with a perception factor designed by considering the vehicle speed implicitly and the gap to its preceding vehicle explicitly. Numerical simulations have been performed based on a signal controlled road which is partitioned into three sections according to the different reactions of drivers. The effects of microscopic driving behaviors on Kerner's time-delayed traffic breakdown at signal (Kerner 2011, 2013) have been investigated with the assistance of spatiotemporal pattern and trajectory analysis. Furthermore, the contributions of the driving behaviors on the traffic breakdown have been statistically examined. Finally, with the activation of the anticipatory behavior, the influences of the other driving behaviors on the formation of platoon have been investigated in terms of the number of platoons, the averaged platoon size, and the averaged flow rate.

  15. Modeling the sub-cellular signaling pathways involved in reinforcement learning at the striatum.

    PubMed

    Wanjerkhede, Shesharao M; Bapi, Raju S

    2008-01-01

    A general discussion of various levels of models in computational neuroscience is presented. A detailed case study of modeling at the sub-cellular level is undertaken. The process of learning actions by reward or punishment is called 'Instrumental Conditioning' or 'Reinforcement Learning' (RL). Temporal difference learning (TDL) is a mathematical framework for RL. Houk et al. (1995) proposed a cellular signaling model for interaction of dopamine (DA) and glutamate activities at the striatum that forms the basis for TDL. In the model, glutamatergic input generates a membrane depolarization through N-methyl-d-aspartate (NMDA), alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA), metabotropic glutamate receptors (mGluR), and opens calcium two plus ion (Ca(2+)) channels resulting in the influx of Ca(2+) into the dendritic spine. This raises the postsynaptic calcium concentration in the dendritic spine leading to the autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). The timely arrival of the DA input at the neck of the spine head generates a cascade of reactions which then leads to the prolongation of long-term potentiation (LTP) generated by the autophosphorylation of CaMKII. Since no simulations were done so far to support this proposal, we undertook the task of computational verification of the model. During the simulations it was found that there was enhancement and prolongation of autophosphorylation of CaMKII. This result verifies Houk's proposal for LTP in the striatum. Our simulation results are generally in line with the known biological experimental data and also suggest predictions for future experimental verification.

  16. RACK1/Asc1p, a Ribosomal Node in Cellular Signaling

    PubMed Central

    Rachfall, Nicole; Schmitt, Kerstin; Bandau, Susanne; Smolinski, Nadine; Ehrenreich, Armin; Valerius, Oliver; Braus, Gerhard H.

    2013-01-01

    RACK1/Asc1p and its essential orthologues in higher eukaryotes, such as RACK1 in metazoa, are involved in several distinct cellular signaling processes. The implications of a total deletion have never been assessed in a comprehensive manner. This study reveals the major cellular processes affected in a Saccharomyces cerevisiae Δasc1 deletion background via de novo proteome and transcriptome analysis, as well as subsequent phenotypical characterizations. The deletion of ASC1 reduces iron uptake and causes nitrosative stress, both known indicators for hypoxia, which manifests in a shift of energy metabolism from respiration to fermentation in the Δasc1 strain. Asc1p further impacts cellular metabolism through its regulative role in the MAP kinase signal transduction pathways of invasive/filamentous growth and cell wall integrity. In the Δasc1 mutant strain, aberrations from the expected cellular response, mediated by these pathways, can be observed and are linked to changes in protein abundances of pathway-targeted transcription factors. Evidence of the translational regulation of such transcription factors suggests that ribosomal Asc1p is involved in signal transduction pathways and controls the biosynthesis of the respective final transcriptional regulators. PMID:23071099

  17. Shedding of glycan-modifying enzymes by signal peptide peptidase-like 3 (SPPL3) regulates cellular N-glycosylation

    PubMed Central

    Voss, Matthias; Künzel, Ulrike; Higel, Fabian; Kuhn, Peer-Hendrik; Colombo, Alessio; Fukumori, Akio; Haug-Kröper, Martina; Klier, Bärbel; Grammer, Gudula; Seidl, Andreas; Schröder, Bernd; Obst, Reinhard; Steiner, Harald; Lichtenthaler, Stefan F; Haass, Christian; Fluhrer, Regina

    2014-01-01

    Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, β-1,3 N-acetylglucosaminyltransferase 1 and β-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes. PMID:25354954

  18. Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level*

    PubMed Central

    Han, Yumiao; Yuan, Zuo-Fei; Molden, Rosalynn C.; Garcia, Benjamin A.

    2016-01-01

    Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP1 approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set

  19. p21-activated kinase signaling in breast cancer

    PubMed Central

    Gururaj, Anupama E; Rayala, Suresh K; Kumar, Rakesh

    2005-01-01

    The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials. PMID:15642175

  20. Cellular Signaling Networks Function as Generalized Wiener-Kolmogorov Filters to Suppress Noise

    NASA Astrophysics Data System (ADS)

    Hinczewski, Michael; Thirumalai, D.

    2014-10-01

    Cellular signaling involves the transmission of environmental information through cascades of stochastic biochemical reactions, inevitably introducing noise that compromises signal fidelity. Each stage of the cascade often takes the form of a kinase-phosphatase push-pull network, a basic unit of signaling pathways whose malfunction is linked with a host of cancers. We show that this ubiquitous enzymatic network motif effectively behaves as a Wiener-Kolmogorov optimal noise filter. Using concepts from umbral calculus, we generalize the linear Wiener-Kolmogorov theory, originally introduced in the context of communication and control engineering, to take nonlinear signal transduction and discrete molecule populations into account. This allows us to derive rigorous constraints for efficient noise reduction in this biochemical system. Our mathematical formalism yields bounds on filter performance in cases important to cellular function—such as ultrasensitive response to stimuli. We highlight features of the system relevant for optimizing filter efficiency, encoded in a single, measurable, dimensionless parameter. Our theory, which describes noise control in a large class of signal transduction networks, is also useful both for the design of synthetic biochemical signaling pathways and the manipulation of pathways through experimental probes such as oscillatory input.

  1. Label free detection of optogenetically stimulated cellular activity by low coherence interferometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Satpathy, Sarmishtha; Batabyal, Subrata; Dave, Digant P.; Mohanty, Samarendra K.

    2016-03-01

    Detecting cellular activity in sub-millisecond timescale and micrometer resolution without using invasive means has been a long standing goal in the study of cellular networks. Here, we have employed phase sensitive low coherence interferometry for detecting optogenetically stimulated activity of cells. Nanoscale changes in optical path length (due to change in refractive index and changes in cell thickness) occur when cells are activated, which we aim to detect by phase sensitive low coherence interferometry. A low coherence interferometry and patch-clamp electrophysiology systems were integrated with an inverted fluorescence microscope. Blue laser beam was coupled to the electrophysiology-interferometric detection system for optogenetic stimulation. The phase-sensitive measurements were carried out on Channelrhodopsin-2 sensitized cells (identified by YFP fluorescence) as well as control cells in reflection mode for different intensities and exposures of optogenetic stimulation beam. This method offers good temporal and spatial resolution without using exogenous labeling. Results of studies on all optical stimulation and detection of cellular activity will be presented. Interpretation of the optical activity signals will be discussed in context with changes in cell physiology during stimulation. We will also discuss the potential sources of various artifacts in optical/electrical detection of cellular activity during optical stimulation.

  2. Detection of recombinant and cellular MALT1 paracaspase activity.

    PubMed

    Nagel, Daniel; Krappmann, Daniel

    2015-01-01

    MALT1 (mucosa-associated lymphoid tissue protein 1) is a key regulator of antigen-induced NF-κB activation in the adaptive immune response. Activation of proteolytic activity of the MALT1 paracaspase was shown to boost the immune response. Additionally, MALT1 proteolytic activity is essential for the survival of MALT1-dependent lymphoma, such as the activated B-cell type (ABC) of diffuse large B-cell lymphoma (DLBCL) or MALT lymphoma. The functional impact of MALT1 paracaspase on T-cell activation and lymphomagenesis suggests that MALT1 is a promising therapeutic target for the treatment of autoimmune diseases and distinct lymphoma entities. To evaluate the requirement of MALT1 in further detail, direct measurement of its activity status is of great importance. We have established a fluorogenic cleavage assay which can be used to measure activity of recombinant and cellular MALT1. Here we describe the basis of the cleavage assay and include a detailed protocol for recombinant production of MALT1 and also the cellular immunoprecipitation of endogenous MALT1 to determine its proteolytic activity.

  3. Signals for the lysosome: a control center for cellular clearance and energy metabolism

    PubMed Central

    Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L.

    2015-01-01

    Preface For a long time lysosomes were considered merely to be cellular “incinerators” involved in the degradation and recycling of cellular waste. However, there is now compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metabolism. Furthermore, the essential role of lysosomes in the autophagic pathway puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master gene, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy, has revealed how the lysosome adapts to environmental cues, such as starvation, and suggests novel therapeutic strategies for modulating lysosomal function in human disease. PMID:23609508

  4. Molecular Signaling Network Motifs Provide a Mechanistic Basis for Cellular Threshold Responses

    PubMed Central

    Bhattacharya, Sudin; Conolly, Rory B.; Clewell, Harvey J.; Kaminski, Norbert E.; Andersen, Melvin E.

    2014-01-01

    Background: Increasingly, there is a move toward using in vitro toxicity testing to assess human health risk due to chemical exposure. As with in vivo toxicity testing, an important question for in vitro results is whether there are thresholds for adverse cellular responses. Empirical evaluations may show consistency with thresholds, but the main evidence has to come from mechanistic considerations. Objectives: Cellular response behaviors depend on the molecular pathway and circuitry in the cell and the manner in which chemicals perturb these circuits. Understanding circuit structures that are inherently capable of resisting small perturbations and producing threshold responses is an important step towards mechanistically interpreting in vitro testing data. Methods: Here we have examined dose–response characteristics for several biochemical network motifs. These network motifs are basic building blocks of molecular circuits underpinning a variety of cellular functions, including adaptation, homeostasis, proliferation, differentiation, and apoptosis. For each motif, we present biological examples and models to illustrate how thresholds arise from specific network structures. Discussion and Conclusion: Integral feedback, feedforward, and transcritical bifurcation motifs can generate thresholds. Other motifs (e.g., proportional feedback and ultrasensitivity)produce responses where the slope in the low-dose region is small and stays close to the baseline. Feedforward control may lead to nonmonotonic or hormetic responses. We conclude that network motifs provide a basis for understanding thresholds for cellular responses. Computational pathway modeling of these motifs and their combinations occurring in molecular signaling networks will be a key element in new risk assessment approaches based on in vitro cellular assays. Citation: Zhang Q, Bhattacharya S, Conolly RB, Clewell HJ III, Kaminski NE, Andersen ME. 2014. Molecular signaling network motifs provide a

  5. Optical Tools to Investigate Cellular Activity in the Intestinal Wall

    PubMed Central

    Boesmans, Werend; Hao, Marlene M; Berghe, Pieter Vanden

    2015-01-01

    Live imaging has become an essential tool to investigate the coordinated activity and output of cellular networks. Within the last decade, 2 Nobel prizes have been awarded to recognize innovations in the field of imaging: one for the discovery, use, and optimization of the green fluorescent protein (2008) and the second for the development of super-resolved fluorescence microscopy (2014). New advances in both optogenetics and microscopy now enable researchers to record and manipulate activity from specific populations of cells with better contrast and resolution, at higher speeds, and deeper into live tissues. In this review, we will discuss some of the recent developments in microscope technology and in the synthesis of fluorescent probes, both synthetic and genetically encoded. We focus on how live imaging of cellular physiology has progressed our understanding of the control of gastrointestinal motility, and we discuss the hurdles to overcome in order to apply the novel tools in the field of neurogastroenterology and motility. PMID:26130630

  6. Wnt-Frizzled/Planar Cell Polarity Signaling: Cellular Orientation by Facing the Wind (Wnt)

    PubMed Central

    Yang, Yingzi; Mlodzik, Marek

    2015-01-01

    The establishment of planar cell polarity (PCP) in epithelial and mesenchymal cells is a critical, evolutionarily conserved process during development and organogenesis. Analyses in Drosophila and several vertebrate model organisms have contributed a wealth of information on the regulation of PCP. A key conserved pathway regulating PCP, the so-called core Wnt-Frizzled PCP (Fz/PCP) signaling pathway, was initially identified through genetic studies of Drosophila. PCP studies in vertebrates, most notably mouse and zebrafish, have identified novel factors in PCP signaling and have also defined cellular features requiring PCP signaling input. These studies have shifted focus to the role of Van Gogh (Vang)/Vangl genes in this molecular system. This review focuses on new insights into the core Fz/Vangl/PCP pathway and recent advances in Drosophila and vertebrate PCP studies. We attempt to integrate these within the existing core Fz/Vangl/PCP signaling framework. PMID:26566118

  7. Methods of analysis for chemicals that disrupt cellular signaling pathways: risk assessment for potential endocrine disruptors.

    PubMed

    Umezawa, Yoshio; Ozawa, Takeaki; Sato, Moritoshi; Inadera, Hidekuni; Kaneko, Shuichi; Kunimoto, Manabu; Hashimoto, Shin-ichi

    2005-01-01

    Here we present a basic concept and several examples of methods of analysis for chemicals that disrupt cellular signaling pathways, in view of risk assessment for potential endocrine disrupting chemicals (EDCs). The key cellular signaling pathways include 1) ER/coactivator interaction, 2) AR translocation into the nucleus, 3) ER/NO/sGC/cGMP, 4) ER/Akt, 5) ER/Src, 6)ER/Src/Grb2, and 7) ER/Ca2+/CaM/CaMK pathways. These were visualized in relevant live cells using newly developed fluorescent and bioluminescent probes. Changes in cellular signals were thereby observed in nongenomic pathways of steroid hormones upon treatment of the target cells with steroid hormones and related chemicals. This method of analysis appears to be a rational approach to high-throughput prescreening (HTPS) of biohazardous chemicals, EDCs, in particular. Also described was the screening of gene expression by serial analysis of gene expression and gene chips upon applying EDCs to breast cancer cells, mouse livers, and human neuroblastoma NB-1 cells.

  8. Osteoporosis and alzheimer pathology: Role of cellular stress response and hormetic redox signaling in aging and bone remodeling

    PubMed Central

    Cornelius, Carolin; Koverech, Guido; Crupi, Rosalia; Di Paola, Rosanna; Koverech, Angela; Lodato, Francesca; Scuto, Maria; Salinaro, Angela T.; Cuzzocrea, Salvatore; Calabrese, Edward J.; Calabrese, Vittorio

    2014-01-01

    Alzheimer’s disease (AD) and osteoporosis are multifactorial progressive degenerative disorders. Increasing evidence shows that osteoporosis and hip fracture are common complication observed in AD patients, although the mechanisms underlying this association remain poorly understood. Reactive oxygen species (ROS) are emerging as intracellular redox signaling molecules involved in the regulation of bone metabolism, including receptor activator of nuclear factor-κB ligand-dependent osteoclast differentiation, but they also have cytotoxic effects that include lipoperoxidation and oxidative damage to proteins and DNA. ROS generation, which is implicated in the regulation of cellular stress response mechanisms, is an integrated, highly regulated, process under control of redox sensitive genes coding for redox proteins called vitagenes. Vitagenes, encoding for proteins such as heat shock proteins (Hsps) Hsp32, Hsp70, the thioredoxin, and the sirtuin protein, represent a systems controlling a complex network of intracellular signaling pathways relevant to life span and involved in the preservation of cellular homeostasis under stress conditions. Consistently, nutritional anti-oxidants have demonstrated their neuroprotective potential through a hormetic-dependent activation of vitagenes. The biological relevance of dose–response affects those strategies pointing to the optimal dosing to patients in the treatment of numerous diseases. Thus, the heat shock response has become an important hormetic target for novel cytoprotective strategies focusing on the pharmacological development of compounds capable of modulating stress response mechanisms. Here we discuss possible signaling mechanisms involved in the activation of vitagenes which, relevant to bone remodeling and through enhancement of cellular stress resistance provide a rationale to limit the deleterious consequences associated to homeostasis disruption with consequent impact on the aging process. PMID:24959146

  9. Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

    SciTech Connect

    Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario . E-mail: chiariel@unina.it

    2006-05-05

    Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes.

  10. Cellular and Molecular Mechanisms Underpinning Macrophage Activation during Remyelination

    PubMed Central

    Lloyd, Amy F.; Miron, Veronique E.

    2016-01-01

    Remyelination is an example of central nervous system (CNS) regeneration, whereby myelin is restored around demyelinated axons, re-establishing saltatory conduction and trophic/metabolic support. In progressive multiple sclerosis, remyelination is limited or fails altogether which is considered to contribute to axonal damage/loss and consequent disability. Macrophages have critical roles in both CNS damage and regeneration, such as remyelination. This diverse range in functions reflects the ability of macrophages to acquire tissue microenvironment-specific activation states. This activation is dynamically regulated during efficient regeneration, with a switch from pro-inflammatory to inflammation-resolution/pro-regenerative phenotypes. Although, some molecules and pathways have been implicated in the dynamic activation of macrophages, such as NFκB, the cellular and molecular mechanisms underpinning plasticity of macrophage activation are unclear. Identifying mechanisms regulating macrophage activation to pro-regenerative phenotypes may lead to novel therapeutic strategies to promote remyelination in multiple sclerosis. PMID:27446913

  11. Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

    PubMed

    Willis, Angelica N; Dean, Shirley E Bradley; Habbouche, Joe A; Kempers, Brian T; Ludwig, Megan L; Sayfie, Aaron D; Lewis, Steven P; Harrier, Stephanie; DeBruine, Zachary J; Garrett, Richard; Burnatowska-Hledin, Maria A

    2017-04-01

    VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 ((640)PKLKRQ(646)) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence ((S730A)VACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in (S730A)VACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in (S730A)VACM-1/CUL5 sequences, is critical for their control of cell proliferation.

  12. Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor-suppressor activities.

    PubMed

    Kagawa, S; Natsuizaka, M; Whelan, K A; Facompre, N; Naganuma, S; Ohashi, S; Kinugasa, H; Egloff, A M; Basu, D; Gimotty, P A; Klein-Szanto, A J; Bass, A J; Wong, K-K; Diehl, J A; Rustgi, A K; Nakagawa, H

    2015-04-30

    Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor-suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor-suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16(INK4A)-Rb pathway. Loss of p16(INK4A) or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as transforming growth factor-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor-suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.

  13. Chromium picolinate enhances skeletal muscle cellular insulin signaling in vivo in obese, insulin-resistant JCR:LA-cp rats.

    PubMed

    Wang, Zhong Q; Zhang, Xian H; Russell, James C; Hulver, Matthew; Cefalu, William T

    2006-02-01

    Chromium is one of the few trace minerals for which a specific cellular mechanism of action has not been identified. Recent in vitro studies suggest that chromium supplementation may improve insulin sensitivity by enhancing insulin receptor signaling, but this has not been demonstrated in vivo. We investigated the effect of chromium supplementation on insulin receptor signaling in an insulin-resistant rat model, the JCR:LA-corpulent rat. Male JCR:LA-cp rats (4 mo of age) were randomly assigned to receive chromium picolinate (CrPic) (obese n=6, lean n=5) or vehicle (obese n=5, lean n=5) for 3 mo. The CrPic was provided in the water, and based on calculated water intake, rats randomized to CrPic received 80 microg/(kg.d). At the end of the study, skeletal muscle (vastus lateralis) biopsies were obtained at baseline and at 5, 15, and 30 min postinsulin stimulation to assess insulin signaling. Obese rats treated with CrPic had significantly improved glucose disposal rates and demonstrated a significant increase in insulin-stimulated phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidylinositol (PI)-3 kinase activity in skeletal muscle compared with obese controls. The increase in cellular signaling was not associated with increased protein levels of the IRS proteins, PI-3 kinase or Akt. However, protein tyrosine phosphatase 1B (PTP1B) levels were significantly lower in obese rats administered CrPic than obese controls. When corrected for protein content, PTP1B activity was also significantly lower in obese rats administered CrPic than obese controls. Our data suggest that chromium supplementation of obese, insulin-resistant rats may improve insulin action by enhancing intracellular signaling.

  14. Anchoring Dipalmitoyl Phosphoethanolamine to Nanoparticles Boosts Cellular Uptake and Fluorine-19 Magnetic Resonance Signal

    NASA Astrophysics Data System (ADS)

    Waiczies, Sonia; Lepore, Stefano; Sydow, Karl; Drechsler, Susanne; Ku, Min-Chi; Martin, Conrad; Lorenz, Dorothea; Schütz, Irene; Reimann, Henning M.; Purfürst, Bettina; Dieringer, Matthias A.; Waiczies, Helmar; Dathe, Margitta; Pohlmann, Andreas; Niendorf, Thoralf

    2015-02-01

    Magnetic resonance (MR) methods to detect and quantify fluorine (19F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with 19F nanoparticles, introduced into living organisms and tracked by 19F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of 19F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of 19F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of 19F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching 19F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting 19F signal per cell in order to overcome some of the limitations related to 19F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

  15. Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

    PubMed Central

    Ryu, Shin-Young; Jhun, Bong Sook; Hurst, Stephen

    2014-01-01

    Abstract Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006. PMID:24180309

  16. Monocyte Activation in Immunopathology: Cellular Test for Development of Diagnostics and Therapy

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

    Several highly prevalent human diseases are associated with immunopathology. Alterations in the immune system are found in such life-threatening disorders as cancer and atherosclerosis. Monocyte activation followed by macrophage polarization is an important step in normal immune response to pathogens and other relevant stimuli. Depending on the nature of the activation signal, macrophages can acquire pro- or anti-inflammatory phenotypes that are characterized by the expression of distinct patterns of secreted cytokines and surface antigens. This process is disturbed in immunopathologies resulting in abnormal monocyte activation and/or bias of macrophage polarization towards one or the other phenotype. Such alterations could be used as important diagnostic markers and also as possible targets for the development of immunomodulating therapy. Recently developed cellular tests are designed to analyze the phenotype and activity of living cells circulating in patient's bloodstream. Monocyte/macrophage activation test is a successful example of cellular test relevant for atherosclerosis and oncopathology. This test demonstrated changes in macrophage activation in subclinical atherosclerosis and breast cancer and could also be used for screening a panel of natural agents with immunomodulatory activity. Further development of cellular tests will allow broadening the scope of their clinical implication. Such tests may become useful tools for drug research and therapy optimization. PMID:26885534

  17. Thioredoxin-dependent Redox Regulation of Cellular Signaling and Stress Response through Reversible Oxidation of Methionines

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2011-06-01

    Generation of reactive oxygen species (ROS) is a common feature of many forms of stress to which plants are exposed. Successful adaptation to changing environmental conditions requires sensitive sensors of ROS such as protein-bound methionines that are converted to their corresponding methionine sulfoxides, which in turn can influence cellular signaling pathways. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress response in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the sensitivity of individual methionines within plant and animal calmodulin to ROS, the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes.

  18. Study of Stevia rebaudiana Bertoni antioxidant activities and cellular properties.

    PubMed

    Bender, Cecilia; Graziano, Sara; Zimmermann, Benno F

    2015-01-01

    The aim of our study was to determine the antioxidant activities, cytotoxicity and proliferative properties in Stevia rebaudiana leaves and stems. Leaves extracts exhibited a higher antioxidant activity than stems extract, through oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Stevioside and rebaudioside A, the main sweetening metabolites in stevia leaves, exhibited a low ORAC value in comparison with plant extracts, while did not elicit any CAA. Stevia rebaudiana did not exhibit toxicity against HepG2 (hepatocellular carcinoma) human cells. No proliferative nor catalase modulations were observed in cells treated with such extracts. Our findings support the promising role of stevia that, apart from its sweetness, can act as a source of antioxidants, even at the intracellular level. This activity makes S. rebaudiana crude extract an interesting resource of natural sweetness with antioxidant properties which may find numerous applications in foods and nutritional supplements industries.

  19. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer’s disease

    PubMed Central

    Haas, Laura T.; Salazar, Santiago V.; Kostylev, Mikhail A.; Um, Ji Won; Kaufman, Adam C.

    2016-01-01

    Alzheimer’s disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer’s disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer’s disease transgenes or by human Alzheimer’s disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp–Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer’s disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer’s disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

  20. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

  1. MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways.

    PubMed

    Hu, Zhaoyong; Klein, Janet D; Mitch, William E; Zhang, Liping; Martinez, Ivan; Wang, Xiaonan H

    2014-03-01

    The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

  2. MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways

    PubMed Central

    Hu, Zhaoyong; Klein, Janet D.; Mitch, William E.; Zhang, Liping; Martinez, Ivan; Wang, Xiaonan H.

    2014-01-01

    The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia. PMID:24659628

  3. Knowledge-guided fuzzy logic modeling to infer cellular signaling networks from proteomic data

    PubMed Central

    Liu, Hui; Zhang, Fan; Mishra, Shital Kumar; Zhou, Shuigeng; Zheng, Jie

    2016-01-01

    Modeling of signaling pathways is crucial for understanding and predicting cellular responses to drug treatments. However, canonical signaling pathways curated from literature are seldom context-specific and thus can hardly predict cell type-specific response to external perturbations; purely data-driven methods also have drawbacks such as limited biological interpretability. Therefore, hybrid methods that can integrate prior knowledge and real data for network inference are highly desirable. In this paper, we propose a knowledge-guided fuzzy logic network model to infer signaling pathways by exploiting both prior knowledge and time-series data. In particular, the dynamic time warping algorithm is employed to measure the goodness of fit between experimental and predicted data, so that our method can model temporally-ordered experimental observations. We evaluated the proposed method on a synthetic dataset and two real phosphoproteomic datasets. The experimental results demonstrate that our model can uncover drug-induced alterations in signaling pathways in cancer cells. Compared with existing hybrid models, our method can model feedback loops so that the dynamical mechanisms of signaling networks can be uncovered from time-series data. By calibrating generic models of signaling pathways against real data, our method supports precise predictions of context-specific anticancer drug effects, which is an important step towards precision medicine. PMID:27774993

  4. Knowledge-guided fuzzy logic modeling to infer cellular signaling networks from proteomic data

    NASA Astrophysics Data System (ADS)

    Liu, Hui; Zhang, Fan; Mishra, Shital Kumar; Zhou, Shuigeng; Zheng, Jie

    2016-10-01

    Modeling of signaling pathways is crucial for understanding and predicting cellular responses to drug treatments. However, canonical signaling pathways curated from literature are seldom context-specific and thus can hardly predict cell type-specific response to external perturbations; purely data-driven methods also have drawbacks such as limited biological interpretability. Therefore, hybrid methods that can integrate prior knowledge and real data for network inference are highly desirable. In this paper, we propose a knowledge-guided fuzzy logic network model to infer signaling pathways by exploiting both prior knowledge and time-series data. In particular, the dynamic time warping algorithm is employed to measure the goodness of fit between experimental and predicted data, so that our method can model temporally-ordered experimental observations. We evaluated the proposed method on a synthetic dataset and two real phosphoproteomic datasets. The experimental results demonstrate that our model can uncover drug-induced alterations in signaling pathways in cancer cells. Compared with existing hybrid models, our method can model feedback loops so that the dynamical mechanisms of signaling networks can be uncovered from time-series data. By calibrating generic models of signaling pathways against real data, our method supports precise predictions of context-specific anticancer drug effects, which is an important step towards precision medicine.

  5. Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization

    PubMed Central

    Smalheiser, Neil R.

    2008-01-01

    For many microRNAs, in many normal tissues and in cancer cells, the cellular levels of mature microRNAs are not simply determined by transcription of microRNA genes. This mini-review will discuss how microRNA biogenesis and function can be regulated by various nuclear and cytoplasmic processing events, including emerging evidence that microRNA pathway components can be selectively regulated by control of their subcellular localization and by modifications that occur during dynamic cellular signaling. Finally, I will briefly summarize studies of microRNAs in synaptic fractions of adult mouse forebrain, which may serve as a model for other cell types as well. PMID:18433727

  6. Liposome-Mediated Cellular Delivery of Active gp91phox

    PubMed Central

    Marques, Bruno; Liguori, Lavinia; Paclet, Marie-Hélène; Villegas-Mendéz, Ana; Rothe, Romy; Morel, Françoise; Lenormand, Jean-Luc

    2007-01-01

    Background Gp91phox is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. Methodology Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. Conclusions Using this system, we over-express truncated forms of the gp91phox protein under soluble form in the presence of detergents or lipids resulting in active proteins with a “native-like” conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67phox, p47phox, p40phox and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91phox protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91phox proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91phox protein. PMID:17848987

  7. NMDA and PACAP Receptor Signaling Interact to Mediate Retinal-Induced SCN Cellular Rhythmicity in the Absence of Light

    PubMed Central

    Webb, Ian C.; Coolen, Lique M.; Lehman, Michael N.

    2013-01-01

    The “core” region of the suprachiasmatic nucleus (SCN), a central clock responsible for coordinating circadian rhythms, shows a daily rhythm in phosphorylation of extracellular regulated kinase (pERK). This cellular rhythm persists under constant darkness and, despite the absence of light, is dependent upon inputs from the eye. The neural signals driving this rhythmicity remain unknown and here the roles of glutamate and PACAP are examined. First, rhythmic phosphorylation of the NR1 NMDA receptor subunit (pNR1, a marker for receptor activation) was shown to coincide with SCN core pERK, with a peak at circadian time (CT) 16. Enucleation and intraocular TTX administration attenuated the peak in the pERK and pNR1 rhythms, demonstrating that activation of the NMDA receptor and ERK in the SCN core at CT16 are dependent on retinal inputs. In contrast, ERK and NR1 phosphorylation in the SCN shell region were unaffected by these treatments. Intraventricular administration of the NMDA receptor antagonist MK-801 also attenuated the peak in SCN core pERK, indicating that ERK phosphorylation in this region requires NMDA receptor activation. As PACAP is implicated in photic entrainment and is known to modulate glutamate signaling, the effects of a PAC1 receptor antagonist (PACAP 6-38) on SCN core pERK and pNR1 also were examined. PACAP 6-38 administration attenuated SCN core pERK and pNR1, suggesting that PACAP induces pERK directly, and indirectly via a modulation of NMDA receptor signaling. Together, these data indicate that, in the absence of light, retinal-mediated NMDA and PAC1 receptor activation interact to induce cellular rhythms in the SCN core. These results highlight a novel function for glutamate and PACAP release in the hamster SCN apart from their well-known roles in the induction of photic circadian clock resetting. PMID:24098484

  8. On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

    PubMed Central

    Luo, Xin; Kraus, W. Lee

    2012-01-01

    Cellular stress responses are mediated through a series of regulatory processes that occur at the genomic, transcriptional, post-transcriptional, translational, and post-translational levels. These responses require a complex network of sensors and effectors from multiple signaling pathways, including the abundant and ubiquitous nuclear enzyme poly(ADP-ribose) (PAR) polymerase-1 (PARP-1). PARP-1 functions at the center of cellular stress responses, where it processes diverse signals and, in response, directs cells to specific fates (e.g., DNA repair vs. cell death) based on the type and strength of the stress stimulus. Many of PARP-1's functions in stress response pathways are mediated by its regulated synthesis of PAR, a negatively charged polymer, using NAD+ as a donor of ADP-ribose units. Thus, PARP-1's functions are intimately tied to nuclear NAD+ metabolism and the broader metabolic profile of the cell. Recent studies in cell and animal models have highlighted the roles of PARP-1 and PAR in the response to a wide variety of extrinsic and intrinsic stress signals, including those initiated by oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, and metabolic stresses. These responses underlie pathological conditions, including cancer, inflammation-related diseases, and metabolic dysregulation. The development of PARP inhibitors is being pursued as a therapeutic approach to these conditions. In this review, we highlight the newest findings about PARP-1's role in stress responses in the context of the historical data. PMID:22391446

  9. Elevated levels of alpha-synuclein blunt cellular signal transduction downstream of Gq protein-coupled receptors.

    PubMed

    Volta, Mattia; Lavdas, Alexandros A; Obergasteiger, Julia; Überbacher, Christa; Picard, Anne; Pramstaller, Peter P; Hicks, Andrew A; Corti, Corrado

    2017-01-01

    Alpha-synuclein is central to Parkinson's disease pathogenesis and pathology, however its precise functions are still unclear. It has been shown to bind both PLCβ1 and MAPKs, but how this property influences the downstream signaling of Gq protein-coupled receptors has not been elucidated. Here we show that recombinant expression of alpha-synuclein in human neuroblastoma cells enhances cellular levels of PLCβ1 but blunts its signaling pathway, preventing the agonist-dependent rise of cytoplasmic Ca(2+). In addition, overexpressing alpha-synuclein abolishes the activation of ERK1/2 upon agonist stimulation, indicating an upstream action in the signal transduction pathway. This data demonstrates that alpha-synuclein, when recombinantly expressed, interferes with the normal signaling of Gq-protein coupled receptors, which are then dysfunctional. Since many neurotransmitter systems utilize these receptor signaling pathways to mediate different abilities affected in Parkinson's disease, we argue this novel perspective might be helpful in designing treatment strategies for some of the non-motor symptoms in Parkinson's disease and synucleinopathies.

  10. A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time.

    PubMed

    Thongwichian, Rossukon; Kosten, Jonas; Benary, Uwe; Rose, Honor May; Stuiver, Marchel; Theillet, Francois-Xavier; Dose, Alexander; Koch, Birgit; Yokoyama, Hideki; Schwarzer, Dirk; Wolf, Jana; Selenko, Philipp

    2015-05-27

    Cell signaling is governed by dynamic changes in kinase and phosphatase activities, which are difficult to assess with discontinuous readout methods. Here, we introduce an NMR-based reporter approach to directly identify active kinases and phosphatases in complex physiological environments such as cell lysates and to measure their individual activities in a semicontinuous fashion. Multiplexed NMR profiling of reporter phosphorylation states provides unique advantages for kinase inhibitor studies and reveals reversible modulations of cellular enzyme activities under different metabolic conditions.

  11. The data-and-signals cellular automaton and its application to growing structures.

    PubMed

    Stauffer, André; Sipper, Moshe

    2004-01-01

    In a traditional cellular automaton (CA) a cell is implemented by a rule table defining its state at the next time step, given its present state and those of its neighbors. The cell thus deals only with states. We present a novel CA where the cell handles data and signals. The cell is designed as a digital system comprising a processing unit and a control unit. This allows the realization of various growing structures, including self-replicating loops and biomorphs. We also describe the hardware implementation of these structures within our electronic wall for bio-inspired applications, the BioWall.

  12. Energy-Efficient Crowdsensing of Human Mobility and Signal Levels in Cellular Networks

    PubMed Central

    Foremski, Paweł; Gorawski, Michał; Grochla, Krzysztof; Polys, Konrad

    2015-01-01

    The paper presents a practical application of the crowdsensing idea to measure human mobility and signal coverage in cellular networks. Currently, virtually everyone is carrying a mobile phone, which may be used as a sensor to gather research data by measuring, e.g., human mobility and radio signal levels. However, many users are unwilling to participate in crowdsensing experiments. This work begins with the analysis of the barriers for engaging people in crowdsensing. A survey showed that people who agree to participate in crowdsensing expect a minimum impact on their battery lifetime and phone usage habits. To address these requirements, this paper proposes an application for measuring the location and signal strength data based on energy-efficient GPS tracking, which allows one to perform the measurements of human mobility and radio signal levels with minimum energy utilization and without any engagement of the user. The method described combines measurements from the accelerometer with effective management of the GPS to monitor the user mobility with the decrease in battery lifetime by approximately 20%. To show the applicability of the proposed platform, the sample results of signal level distribution and coverage maps gathered for an LTE network and representing human mobility are shown. PMID:26340633

  13. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    PubMed

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  14. Activation of cellular immune response in acute pancreatitis.

    PubMed Central

    Mora, A; Pérez-Mateo, M; Viedma, J A; Carballo, F; Sánchez-Payá, J; Liras, G

    1997-01-01

    BACKGROUND: Inflammatory mediators have recently been implicated as potential markers of severity in acute pancreatitis. AIMS: To determine the value of neopterin and polymorphonuclear (PMN) elastase as markers of activation of cellular immunity and as early predictors of disease severity. PATIENTS: Fifty two non-consecutive patients classified according to their clinical outcome into mild (n = 26) and severe pancreatitis (n = 26). METHODS: Neopterin in serum and the PMN elastase/A1PI complex in plasma were measured during the first three days of hospital stay. RESULTS: Within three days after the onset of acute pancreatitis, PMN elastase was significantly higher in the severe pancreatitis group. Patients with severe disease also showed significantly higher values of neopterin on days 1 and 2 but not on day 3 compared with patients with mild disease. There was a significant correlation between PMN elastase and neopterin values on days 1 and 2. PMN elastase on day 1 predicted disease severity with a sensitivity of 76.7% and a specificity of 91.6%. Neopterin did not surpass PMN elastase in the probability of predicting disease severity. CONCLUSIONS: These data show that activation of cellular immunity is implicated in the pathogenesis of acute pancreatitis and may be a main contributory factor to disease severity. Neopterin was not superior to PMN elastase in the prediction of severity. PMID:9245935

  15. Sleep loss activates cellular markers of inflammation: sex differences.

    PubMed

    Irwin, Michael R; Carrillo, Carmen; Olmstead, Richard

    2010-01-01

    Sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. Given sex differences in the prevalence of inflammatory disorders with stronger associations in females, this study was undertaken to test the effects of sleep loss on cellular mechanisms that contribute to proinflammatory cytokine activity. In 26 healthy adults (11 females; 15 males), monocyte intracellular proinflammatory cytokine production was repeatedly assessed at 08:00, 12:00, 16:00, 20:00, and 23:00h during a baseline period and after partial sleep deprivation (awake from 23:00 to 3.00h). In the morning after a night of sleep loss, monocyte production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) differentially changed between the two sexes. Whereas both females and males showed a marked increase in the lipopolysaccharide (LPS) - stimulated production of IL-6 and TNF-alpha in the morning immediately after PSD, production of these cytokines during the early- and late evening was increased in the females as compared to decreases in the males. Sleep loss induces a functional alteration of monocyte proinflammatory cytokine responses with females showing greater cellular immune activation as compared to changes in males. These results have implications for understanding the role of sleep disturbance in the differential risk profile for inflammatory disorders between the sexes.

  16. Sleep Loss Activates Cellular Markers of Inflammation: Sex Differences

    PubMed Central

    Irwin, Michael R.; Carrillo, Carmen; Olmstead, Richard

    2009-01-01

    Sleep disturbance is associated with inflammation and related disorders including cardiovascular disease, arthritis, and diabetes mellitus. Given sex differences in the prevalence of inflammatory disorders with stronger associations in females, this study was undertaken to test the effects of sleep loss on cellular mechanisms that contribute to proinflammatory cytokine activity. In 26 healthy adults (11 females; 15 males), monocyte intracellular proinflammatory cytokine production was repeatedly assessed at 08:00, 12:00, 16:00, 20:00, and 23:00 h during a baseline period and after partial sleep deprivation (awake from 11 PM to 3 AM). In the morning after a night of sleep loss, monocyte production of interleukin 6 and tumor necrosis factor- α differentially changed between the two sexes. Whereas both females and males showed a marked increase in the lipopolysaccharide (LPS) - stimulated production of IL-6 and TNF-α in the morning immediately after PSD, production of these cytokines during the early- and late evening was increased in the females as compared to decreases in the males. Sleep loss induces a functional alteration of monocyte proinflammatory cytokine responses with females showing greater cellular immune activation as compared to changes in males. These results have implications for understanding the role of sleep disturbance in the differential risk profile for inflammatory disorders between the sexes. PMID:19520155

  17. Cellular automaton simulations of a four-leg intersection with two-phase signalization

    NASA Astrophysics Data System (ADS)

    Jin, Cheng-Jie; Wang, Wei; Jiang, Rui; Wang, Hao

    2014-10-01

    In this paper, we present a cellular automaton (CA) simulation of a signalized intersection. When there is no exclusive lane for left-turn vehicles, through vehicles and left-turn vehicles have to share one lane. Under such situation usually two-phase signalization is adopted, and the conflicts between the two traffic streams need to be analyzed. We use a refined configuration for the intersection simulation: the geometry of the intersection has been considered and vehicles are assumed to move along 1/4 circle arcs. We focus on the averaged travel times on left lanes and their distributions. The diagrams of intersection approach capacities (IACs) and the corresponding phase diagrams are also presented, which depend on the approach flow rates and the percentage of left-turn vehicles. Besides, we find that the minimum green time could be determined by finding out the critical value for the travel times.

  18. Signal processing for molecular and cellular biological physics: an emerging field

    PubMed Central

    Little, Max A.; Jones, Nick S.

    2013-01-01

    Recent advances in our ability to watch the molecular and cellular processes of life in action—such as atomic force microscopy, optical tweezers and Forster fluorescence resonance energy transfer—raise challenges for digital signal processing (DSP) of the resulting experimental data. This article explores the unique properties of such biophysical time series that set them apart from other signals, such as the prevalence of abrupt jumps and steps, multi-modal distributions and autocorrelated noise. It exposes the problems with classical linear DSP algorithms applied to this kind of data, and describes new nonlinear and non-Gaussian algorithms that are able to extract information that is of direct relevance to biological physicists. It is argued that these new methods applied in this context typify the nascent field of biophysical DSP. Practical experimental examples are supplied. PMID:23277603

  19. Cellular Links between Neuronal Activity and Energy Homeostasis

    PubMed Central

    Shetty, Pavan K.; Galeffi, Francesca; Turner, Dennis A.

    2012-01-01

    Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply. PMID:22470340

  20. Sigma-1 receptor chaperone at the ER-mitochondrion interface mediates the mitochondrion-ER-nucleus signaling for cellular survival.

    PubMed

    Mori, Tomohisa; Hayashi, Teruo; Hayashi, Eri; Su, Tsung-Ping

    2013-01-01

    The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.

  1. CPSF30 at the Interface of Alternative Polyadenylation and Cellular Signaling in Plants

    PubMed Central

    Chakrabarti, Manohar; Hunt, Arthur G.

    2015-01-01

    Post-transcriptional processing, involving cleavage of precursor messenger RNA (pre mRNA), and further incorporation of poly(A) tail to the 3' end is a key step in the expression of genetic information. Alternative polyadenylation (APA) serves as an important check point for the regulation of gene expression. Recent studies have shown widespread prevalence of APA in diverse systems. A considerable amount of research has been done in characterizing different subunits of so-called Cleavage and Polyadenylation Specificity Factor (CPSF). In plants, CPSF30, an ortholog of the 30 kD subunit of mammalian CPSF is a key polyadenylation factor. CPSF30 in the model plant Arabidopsis thaliana was reported to possess unique biochemical properties. It was also demonstrated that poly(A) site choice in a vast majority of genes in Arabidopsis are CPSF30 dependent, suggesting a pivotal role of this gene in APA and subsequent regulation of gene expression. There are also indications of this gene being involved in oxidative stress and defense responses and in cellular signaling, suggesting a role of CPSF30 in connecting physiological processes and APA. This review will summarize the biochemical features of CPSF30, its role in regulating APA, and possible links with cellular signaling and stress response modules. PMID:26061761

  2. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  3. Cellular Internalization of Fibroblast Growth Factor-12 Exerts Radioprotective Effects on Intestinal Radiation Damage Independently of FGFR Signaling

    SciTech Connect

    Nakayama, Fumiaki; Umeda, Sachiko; Yasuda, Takeshi; Fujita, Mayumi; Asada, Masahiro; Meineke, Viktor; Imamura, Toru; Imai, Takashi

    2014-02-01

    Purpose: Several fibroblast growth factors (FGFs) were shown to inhibit radiation-induced tissue damage through FGF receptor (FGFR) signaling; however, this signaling was also found to be involved in the pathogenesis of several malignant tumors. In contrast, FGF12 cannot activate any FGFRs. Instead, FGF12 can be internalized readily into cells using 2 cell-penetrating peptide domains (CPP-M, CPP-C). Therefore, this study focused on clarifying the role of FGF12 internalization in protection against radiation-induced intestinal injury. Methods and Materials: Each FGF or peptide was administered intraperitoneally to BALB/c mice in the absence of heparin 24 hours before or after total body irradiation with γ rays at 9 to 12 Gy. Several radioprotective effects were examined in the jejunum. Results: Administration of FGF12 after radiation exposure was as effective as pretreatment in significantly promoting intestinal regeneration, proliferation of crypt cells, and epithelial differentiation. Two domains, comprising amino acid residues 80 to 109 and 140 to 169 of FGF12B, were identified as being responsible for the radioprotective activity, so that deletion of both domains from FGF12B resulted in a reduction in activity. Interestingly, these regions included the CPP-M and CPP-C domains, respectively; however, CPP-C by itself did not show an antiapoptotic effect. In addition, FGF1, prototypic FGF, possesses a domain corresponding to CPP-M, whereas it lacks CPP-C, so the fusion of FGF1 with CPP-C (FGF1/CPP-C) enhanced cellular internalization and increased radioprotective activity. However, FGF1/CPP-C reduced in vitro mitogenic activity through FGFRs compared with FGF1, implying that FGFR signaling might not be essential for promoting the radioprotective effect of FGF1/CPP-C. In addition, internalized FGF12 suppressed the activation of p38α after irradiation, resulting in reduced radiation-induced apoptosis. Conclusions: These findings indicate that FGF12 can protect the

  4. Activity and cellular localization of amylases of rabbit cecal bacteria.

    PubMed

    Sirotek, K; Marounek, M; Suchorská, O

    2006-01-01

    Five 11-week-old rabbits, fed a commercial granulated feed, were slaughtered and cecal starch-degrading bacteria enumerated; total concentration of cultivable bacteria utilizing starch averaged 5.5 x 10(10) CFU/g. The activity and cellular localization of amylases was determined in 9 bacteria identified as Actinomyces israeli (strains AA2 and AD4), Bacteroides spp. (strain AA3), Dichelobacter nodosus (strain AA4), Mitsuokella multiacidus (strain AA6), Eubacterium spp. (strains AA7 and AB2), Clostridium spp. (strains AD1 and AA5). Four strains (AA3, AA4, AA5, AD4) produced extracellular amylases with an activity of 26-35 micromol of reducing sugars per h per mg of protein; in five strains (AA2, AA6, AA7, AB2, AD1) amylases were membrane-bound with an activity of 14-18 micromol of reducing sugars per h per mg of protein. All strains exhibited a low intracellular amylolytic activity. The pH optimum of amylases was 6.8-7.0. In strains producing extracellular amylases a substantial loss of viscosity was observed during incubations of cultivation supernatant with starch, similar to viscosity reduction in starch solutions treated with alpha-amylase; this indicates an endo-type (random cleavage) of extracellular amylase reaction in the bacteria under study. No strain possessed glucoamylase activity.

  5. CDPK Activation in PRR Signaling.

    PubMed

    Seybold, Heike; Boudsocq, Marie; Romeis, Tina

    2017-01-01

    Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.

  6. Linking cellular actin status with cAMP signaling in Candida albicans.

    PubMed

    Wang, Yue; Zou, Hao; Fang, Hao-Ming; Zhu, Yong

    2010-01-01

    The fungal pathogen Candida albicans has a remarkable ability to switch growth forms. Particularly, the yeast-to-hyphae switch is closely linked with its virulence. A range of chemicals and conditions can promote hyphal growth including serum, peptidoglycan, CO2, neutral pH, and elevated temperature. All these signals act essentially through the adenylyl cyclase Cyr1 that synthesizes cAMP. Cells lacking Cyr1 are completely defective in hyphal growth. Recently, cellular actin status is found to influence cAMP synthesis. However, how Cyr1 senses and processes multiple external and internal signals to produce a contextually proper level of cAMP remains unclear. We hypothesized that Cyr1 itself possesses multiple sensors for different signals and achieves signal integration through a combined allosteric effect on the catalytic center. To test this hypothesis, we affinity-purified a Cyr1-containing complex and found that it could enhance cAMP synthesis upon treatment with serum, peptidoglycan or CO2 in vitro. The data indicate that the complex is an essentially intact sensor/effector apparatus for cAMP synthesis. The complex contains two more subunits, the cyclase-associated protein Cap1 and G-actin. We discovered that G-actin plays a regulatory role, rendering cAMP synthesis responsive to actin dynamics. These findings shed new lights on the mechanisms that regulate cAMP-mediated responses in fungi.

  7. Active Cellular Mechanics and Information Processing in the Living Cell

    NASA Astrophysics Data System (ADS)

    Rao, M.

    2014-07-01

    I will present our recent work on the organization of signaling molecules on the surface of living cells. Using novel experimental and theoretical approaches we have found that many cell surface receptors are organized as dynamic clusters driven by active currents and stresses generated by the cortical cytoskeleton adjoining the cell surface. We have shown that this organization is optimal for both information processing and computation. In connecting active mechanics in the cell with information processing and computation, we bring together two of the seminal works of Alan Turing.

  8. Physicochemical properties of iron oxide nanoparticles that contribute to cellular ROS-dependent signaling and acellular production of hydroxyl radical.

    PubMed

    Vogel, Christoph F A; Charrier, Jessica G; Wu, Dalei; McFall, Alexander S; Li, Wen; Abid, Aamir; Kennedy, Ian M; Anastasio, Cort

    2016-01-01

    While nanoparticles (NPs) are increasingly used in a variety of consumer products and medical applications, some of these materials have potential health concerns. Macrophages are the primary responders to particles that initiate oxidative stress and inflammatory reactions. Here, we utilized six flame-synthesized, engineered iron oxide NPs with various physicochemical properties (e.g. Fe oxidation state and crystal size) to study their interactions with RAW 264.7 macrophages, their iron solubilities, and their abilities to produce hydroxyl radical in an acellular assay. Both iron solubility and hydroxyl radical production varied between NPs depending on crystalline diameter and surface area of the particles, but not on iron oxidation state. Macrophage treatment with the iron oxide NPs showed a dose-dependent increase of heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO-1). The nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (Nrf2) modulates the transcriptional activity of antioxidant response element (ARE)-driven genes, such as HO-1 and NQO-1. Here, we show that the iron oxide NPs activate Nrf2, leading to its increased nuclear accumulation and enhanced Nrf2 DNA-binding activity in NP-treated RAW 264.7 macrophages. Iron solubility and acellular hydroxyl radical generation depend on the physical properties of the NPs, especially crystalline diameter; however, these properties are weakly linked to the activation of cellular signaling of Nrf2 and the expression of oxidative stress markers. Overall, our work shows for the first time that iron oxide nanoparticles induce cellular marker genes of oxidative stress and that this effect is transcriptionally mediated through the Nrf2-ARE signaling pathway in macrophages.

  9. Cellular Signaling Circuits Interfaced with Synthetic, Post-Translational, Negating Boolean Logic Devices

    PubMed Central

    2014-01-01

    A negating functionality is fundamental to information processing of logic circuits within cells and computers. Aiming to adapt unutilized electronic concepts to the interrogation of signaling circuits in cells, we first took a bottom-up strategy whereby we created protein-based devices that perform negating Boolean logic operations such as NOT, NOR, NAND, and N-IMPLY. These devices function in living cells within a minute by precisely commanding the localization of an activator molecule among three subcellular spaces. We networked these synthetic gates to an endogenous signaling circuit and devised a physiological output. In search of logic functions in signal transduction, we next took a top–down approach and computationally screened 108 signaling pathways to identify commonalities and differences between these biological pathways and electronic circuits. This combination of synthetic and systems approaches will guide us in developing foundations for deconstruction of intricate cell signaling, as well as construction of biomolecular computers. PMID:25000210

  10. Critical roles of cellular glutathione homeostasis and jnk activation in andrographolide-mediated apoptotic cell death in human hepatoma cells.

    PubMed

    Ji, Lili; Shen, Kaikai; Jiang, Ping; Morahan, Grant; Wang, Zhengtao

    2011-08-01

    Andrographolide (ANDRO), isolated from the traditional herbal medicine Andrographis paniculata, is reported to have the potential therapeutic effects for hepatocellular carcinoma (HCC) in our previous reports. Here, we investigated the mechanism of ANDRO-mediated apoptotic cell death, focusing on the involvement of cellular reduced glutathione (GSH) homeostasis and c-Jun NH(2) -Terminal kinase (JNK). Buthionine sulfoximine (BSO), an inhibitor of cellular GSH biosynthesis, significantly augmented ANDRO-induced cytotoxicity in hepatoma Hep3B and HepG2 cells. BSO depleted cellular GSH, and augmented ANDRO-induced apoptosis, inhibition of colony formation and JNK activation in Hep3B cells. All these effects could be reversed by GSH monoethyl ester (GSH.EE), whose deacetylation replenishes cellular GSH. BSO also augmented ANDRO-induced activation of apoptosis signal-regulating kinase 1 (ASK1), mitogen-activated protein kinase kinase-4 (MKK4) and c-Jun, which are all up-stream or down-stream signals of JNK. Further results showed that JNK inhibitor SP600125 and 420116 both reversed ANDRO-induced cytotoxicity, and SP600125 also decreased ANDRO-increased intracellular GSH and GCL activity. Finally, we showed that in nude mice bearing xenografted Hep3B tumors, BSO improved the inhibition of tumor growth by ANDRO. Taken together, our results suggest that there is a crosstalk between JNK activation and cellular GSH homeostasis, and ANDRO targets this to induce cytotoxicity in hepatoma cells.

  11. A theoretical study on cellular antioxidant activity of selected flavonoids

    NASA Astrophysics Data System (ADS)

    Rong, Yuzhi; Wang, Zhengwu; Wu, Jinhong; Zhao, Bo

    The antioxidant capacities of the selected flavonoids quercetin, luteolin and taxifolin have been investigated at density functional level of theory with the aim of verifying the cellular antioxidant activity (CAA) values representative of experimental findings. The selected flavonoids were believed to act through the H-atom transfer mechanism. Their potentiality of hydrogen abstraction was evaluated by computing the Osbnd H bond dissociation enthalpy (BDE) in gas-phase and in dimethylsulfoxide solution. Results indicate that the order of antioxidant efficacies calculated in this work is in agreement with that reported by experimental results of CAA. Time-dependent density functional theory (TDDFT) calculations were also performed both in gas-phase and in dimethylsulfoxide to reproduce the electronic UV-vis spectra of the selected flavonoids.

  12. Stra13 regulates satellite cell activation by antagonizing Notch signaling

    PubMed Central

    Sun, Hong; Li, Li; Vercherat, Cécile; Gulbagci, Neriman Tuba; Acharjee, Sujata; Li, Jiali; Chung, Teng-Kai; Thin, Tin Htwe; Taneja, Reshma

    2007-01-01

    Satellite cells play a critical role in skeletal muscle regeneration in response to injury. Notch signaling is vital for satellite cell activation and myogenic precursor cell expansion but inhibits myogenic differentiation. Thus, precise spatial and temporal regulation of Notch activity is necessary for efficient muscle regeneration. We report that the basic helix-loop-helix transcription factor Stra13 modulates Notch signaling in regenerating muscle. Upon injury, Stra13−/− mice exhibit increased cellular proliferation, elevated Notch signaling, a striking regeneration defect characterized by degenerated myotubes, increased mononuclear cells, and fibrosis. Stra13−/− primary myoblasts also exhibit enhanced Notch activity, increased proliferation, and defective differentiation. Inhibition of Notch signaling ex vivo and in vivo ameliorates the phenotype of Stra13−/− mutants. We demonstrate in vitro that Stra13 antagonizes Notch activity and reverses the Notch-imposed inhibition of myogenesis. Thus, Stra13 plays an important role in postnatal myogenesis by attenuating Notch signaling to reduce myoblast proliferation and promote myogenic differentiation. PMID:17502421

  13. Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

    PubMed Central

    Seervi, M; Joseph, J; Sobhan, P K; Bhavya, B C; Santhoshkumar, T R

    2011-01-01

    Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators. PMID:21900958

  14. Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease.

    PubMed

    Quest, Andrew F G; Leyton, Lisette; Párraga, Mario

    2004-02-01

    Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol- and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.

  15. Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting

    PubMed Central

    Jacob, Thomas; Agarwal, Anupriya; Ramunno-Johnson, Damien; O’Hare, Thomas; Gönen, Mehmet; Tyner, Jeffrey W.; Druker, Brian J.; Vu, Tania Q.

    2016-01-01

    Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells. PMID:27320899

  16. Comparative study of protein tyrosine phosphatase-epsilon isoforms: membrane localization confers specificity in cellular signalling.

    PubMed Central

    Andersen, J N; Elson, A; Lammers, R; Rømer, J; Clausen, J T; Møller, K B; Møller, N P

    2001-01-01

    To study the influence of subcellular localization as a determinant of signal transduction specificity, we assessed the effects of wild-type transmembrane and cytoplasmic protein tyrosine phosphatase (PTP) epsilon on tyrosine kinase signalling in baby hamster kidney (BHK) cells overexpressing the insulin receptor (BHK-IR). The efficiency by which differently localized PTPepsilon and PTPalpha variants attenuated insulin-induced cell rounding and detachment was determined in a functional clonal-selection assay and in stable cell lines. Compared with the corresponding receptor-type PTPs, the cytoplasmic PTPs (cytPTPs) were considerably less efficient in generating insulin-resistant clones, and exceptionally high compensatory expression levels were required to counteract phosphotyrosine-based signal transduction. Targeting of cytPTPepsilon to the plasma membrane via the Lck-tyrosine kinase dual acylation motif restored high rescue efficiency and abolished the need for high cytPTPepsilon levels. Consistent with these results, expression levels and subcellular localization of PTPepsilon were also found to determine the phosphorylation level of cellular proteins including focal adhesion kinase (FAK). Furthermore, PTPepsilon stabilized binding of phosphorylated FAK to Src, suggesting this complex as a possible mediator of the PTPepsilon inhibitory response to insulin-induced cell rounding and detachment in BHK-IR cells. Taken together, the present localization-function study indicates that transcriptional control of the subcellular localization of PTPepsilon may provide a molecular mechanism that determines PTPepsilon substrate selectivity and isoform-specific function. PMID:11237862

  17. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes

    PubMed Central

    Kreienkamp, Ray; Croke, Monica; Neumann, Martin A.; Bedia-Diaz, Gonzalo; Graziano, Simona; Dusso, Adriana; Dorsett, Dale; Carlberg, Carsten; Gonzalo, Susana

    2016-01-01

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence. Here we uncover new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via 1α,25-dihydroxyvitamin D3 (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the 1,25D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. 1,25D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging. PMID:27145372

  18. Vitamin D receptor signaling improves Hutchinson-Gilford progeria syndrome cellular phenotypes.

    PubMed

    Kreienkamp, Ray; Croke, Monica; Neumann, Martin A; Bedia-Diaz, Gonzalo; Graziano, Simona; Dusso, Adriana; Dorsett, Dale; Carlberg, Carsten; Gonzalo, Susana

    2016-05-24

    Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating incurable premature aging disease caused by accumulation of progerin, a toxic lamin A mutant protein. HGPS patient-derived cells exhibit nuclear morphological abnormalities, altered signaling pathways, genomic instability, and premature senescence. Here we uncover new molecular mechanisms contributing to cellular decline in progeria. We demonstrate that HGPS cells reduce expression of vitamin D receptor (VDR) and DNA repair factors BRCA1 and 53BP1 with progerin accumulation, and that reconstituting VDR signaling via 1α,25-dihydroxyvitamin D3 (1,25D) treatment improves HGPS phenotypes, including nuclear morphological abnormalities, DNA repair defects, and premature senescence. Importantly, we discovered that the 1,25D/VDR axis regulates LMNA gene expression, as well as expression of DNA repair factors. 1,25D dramatically reduces progerin production in HGPS cells, while stabilizing BRCA1 and 53BP1, two key factors for genome integrity. Vitamin D/VDR axis emerges as a new target for treatment of HGPS and potentially other lamin-related diseases exhibiting VDR deficiency and genomic instability. Because progerin expression increases with age, maintaining vitamin D/VDR signaling could keep the levels of progerin in check during physiological aging.

  19. Trans-species learning of cellular signaling systems with bimodal deep belief networks

    PubMed Central

    Chen, Lujia; Cai, Chunhui; Chen, Vicky; Lu, Xinghua

    2015-01-01

    Motivation: Model organisms play critical roles in biomedical research of human diseases and drug development. An imperative task is to translate information/knowledge acquired from model organisms to humans. In this study, we address a trans-species learning problem: predicting human cell responses to diverse stimuli, based on the responses of rat cells treated with the same stimuli. Results: We hypothesized that rat and human cells share a common signal-encoding mechanism but employ different proteins to transmit signals, and we developed a bimodal deep belief network and a semi-restricted bimodal deep belief network to represent the common encoding mechanism and perform trans-species learning. These ‘deep learning’ models include hierarchically organized latent variables capable of capturing the statistical structures in the observed proteomic data in a distributed fashion. The results show that the models significantly outperform two current state-of-the-art classification algorithms. Our study demonstrated the potential of using deep hierarchical models to simulate cellular signaling systems. Availability and implementation: The software is available at the following URL: http://pubreview.dbmi.pitt.edu/TransSpeciesDeepLearning/. The data are available through SBV IMPROVER website, https://www.sbvimprover.com/challenge-2/overview, upon publication of the report by the organizers. Contact: xinghua@pitt.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25995230

  20. Synthesis of marmycin A and investigation into its cellular activity

    NASA Astrophysics Data System (ADS)

    Cañeque, Tatiana; Gomes, Filipe; Mai, Trang Thi; Maestri, Giovanni; Malacria, Max; Rodriguez, Raphaël

    2015-09-01

    Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective Diels-Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging Friedel-Crafts cyclization. Remarkably, fluorescence microscopy revealed that marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological and therapeutic applications.

  1. Cellular Interrogation: Exploiting Cell-to-Cell Variability to Discriminate Regulatory Mechanisms in Oscillatory Signalling

    PubMed Central

    Gibson, Daniel; Chang, Frederick; Gnad, Florian; Gunawardena, Jeremy

    2016-01-01

    The molecular complexity within a cell may be seen as an evolutionary response to the external complexity of the cell’s environment. This suggests that the external environment may be harnessed to interrogate the cell’s internal molecular architecture. Cells, however, are not only nonlinear and non-stationary, but also exhibit heterogeneous responses within a clonal, isogenic population. In effect, each cell undertakes its own experiment. Here, we develop a method of cellular interrogation using programmable microfluidic devices which exploits the additional information present in cell-to-cell variation, without requiring model parameters to be fitted to data. We focussed on Ca2+ signalling in response to hormone stimulation, which exhibits oscillatory spiking in many cell types and chose eight models of Ca2+ signalling networks which exhibit similar behaviour in simulation. We developed a nonlinear frequency analysis for non-stationary responses, which could classify models into groups under parameter variation, but found that this question alone was unable to distinguish critical feedback loops. We further developed a nonlinear amplitude analysis and found that the combination of both questions ruled out six of the models as inconsistent with the experimentally-observed dynamics and heterogeneity. The two models that survived the double interrogation were mathematically different but schematically identical and yielded the same unexpected predictions that we confirmed experimentally. Further analysis showed that subtle mathematical details can markedly influence non-stationary responses under parameter variation, emphasising the difficulty of finding a “correct” model. By developing questions for the pathway being studied, and designing more versatile microfluidics, cellular interrogation holds promise as a systematic strategy that can complement direct intervention by genetics or pharmacology. PMID:27367445

  2. Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

    PubMed

    Adan, Aysun; Baran, Yusuf

    2016-05-01

    Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

  3. Cellular Signaling Pathways and Posttranslational Modifications Mediated by Nematode Effector Proteins1

    PubMed Central

    Hewezi, Tarek

    2015-01-01

    Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. PMID:26315856

  4. Irreparable telomeric DNA damage and persistent DDR signalling as a shared causative mechanism of cellular senescence and ageing.

    PubMed

    Rossiello, Francesca; Herbig, Utz; Longhese, Maria Pia; Fumagalli, Marzia; d'Adda di Fagagna, Fabrizio

    2014-06-01

    The DNA damage response (DDR) orchestrates DNA repair and halts cell cycle. If damage is not resolved, cells can enter into an irreversible state of proliferative arrest called cellular senescence. Organismal ageing in mammals is associated with accumulation of markers of cellular senescence and DDR persistence at telomeres. Since the vast majority of the cells in mammals are non-proliferating, how do they age? Are telomeres involved? Also oncogene activation causes cellular senescence due to altered DNA replication and DDR activation in particular at the telomeres. Is there a common mechanism shared among apparently distinct types of cellular senescence? And what is the role of telomeric DNA damage?

  5. Differential redox regulation of ORAI ion channels: a mechanism to tune cellular calcium signaling.

    PubMed

    Bogeski, Ivan; Kummerow, Carsten; Al-Ansary, Dalia; Schwarz, Eva C; Koehler, Richard; Kozai, Daisuke; Takahashi, Nobuaki; Peinelt, Christine; Griesemer, Desiree; Bozem, Monika; Mori, Yasuo; Hoth, Markus; Niemeyer, Barbara A

    2010-03-30

    Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca(2+)) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca(2+) imaging, patch-clamp recordings, and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H(2)O(2)) on ORAI channel activity and human T helper lymphocyte (T(H) cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H(2)O(2). The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. T(H) cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector T(H) cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector T(H) cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector T(H) cells may tune cellular responses under oxidative stress.

  6. Platelet-activating factor: receptors and signal transduction.

    PubMed

    Chao, W; Olson, M S

    1993-06-15

    During the past two decades, studies describing the chemistry and biology of PAF have been extensive. This potent phosphoacylglycerol exhibits a wide variety of physiological and pathophysiological effects in various cells and tissues. PAF acts, through specific receptors and a variety of signal transduction systems, to elicit diverse biochemical responses. Several important future directions can be enumerated for the characterization of PAF receptors and their attendant signalling mechanisms. The recent cloning and sequence analysis of the gene for the PAF receptor will allow a number of important experimental approaches for characterizing the structure and analysing the function of the various domains of the receptor. Using molecular genetic and immunological technologies, questions relating to whether there is receptor heterogeneity, the precise mechanism(s) for the regulation of the PAF receptor, and the molecular details of the signalling mechanisms in which the PAF receptor is involved can be explored. Another area of major significance is the examination of the relationship between the signalling response(s) evoked by PAF binding to its receptor and signalling mechanisms activated by a myriad of other mediators, cytokines and growth factors. A very exciting recent development in which PAF receptors undoubtedly play a role is in the regulation of the function of various cellular adhesion molecules. Finally, there remain many incompletely characterized physiological and pathophysiological situations in which PAF and its receptor play a crucial signalling role. Our laboratory has been active in the elucidation of several tissue responses in which PAF exhibits major autocoid signalling responses, e.g. hepatic injury and inflammation, acute and chronic pancreatitis, and cerebral stimulation and/or trauma. As new experimental strategies are developed for characterizing the fine structure of the molecular mechanisms involved in tissue injury and inflammation, the

  7. Ligand-binding dynamics rewire cellular signaling via Estrogen Receptor-α

    PubMed Central

    Srinivasan, Sathish; Nwachukwu, Jerome C.; Parent, Alex A.; Cavett, Valerie; Nowak, Jason; Hughes, Travis S.; Kojetin, Douglas J.; Katzenellenbogen, John A.; Nettles, Kendall W.

    2013-01-01

    Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands displayed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways, and reveals a novel role of the DBD in allosteric control of ERα-mediated signaling. PMID:23524984

  8. Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways

    PubMed Central

    Mozolewski, Paweł; Moskot, Marta; Jakóbkiewicz-Banecka, Joanna; Węgrzyn, Grzegorz; Bocheńska, Katarzyna; Banecki, Bogdan; Gabig-Cimińska, Magdalena

    2017-01-01

    In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways. PMID:28240227

  9. Three Decades of Research on O-GlcNAcylation - A Major Nutrient Sensor That Regulates Signaling, Transcription and Cellular Metabolism.

    PubMed

    Hart, Gerald W

    2014-01-01

    Even though the dynamic modification of polypeptides by the monosaccharide, O-linked N-acetylglucosamine (O-GlcNAcylation) was discovered over 30 years ago, its physiological significance as a major nutrient sensor that regulates myriad cellular processes has only recently been more widely appreciated. O-GlcNAcylation, either on its own or by its interplay with other post-translational modifications, such as phosphorylation, ubiquitination, and others, modulates the activities of signaling proteins, regulates most components of the transcription machinery, affects cell cycle progression and regulates the targeting/turnover or functions of myriad other regulatory proteins, in response to nutrients. Acute increases in O-GlcNAcylation protect cells from stress-induced injury, while chronic deregulation of O-GlcNAc cycling contributes to the etiology of major human diseases of aging, such as diabetes, cancer, and neurodegeneration. Recent advances in tools to study O-GlcNAcylation at the individual site level and specific inhibitors of O-GlcNAc cycling have allowed more rapid progress toward elucidating the specific functions of O-GlcNAcylation in essential cellular processes.

  10. Inter-cellular signaling network reveals a mechanistic transition in tumor microenvironment

    PubMed Central

    Wu, Yu; Garmire, Lana X.; Fan, Rong

    2012-01-01

    We conducted inter-cellular cytokine correlation and network analysis based upon a stochastic population dynamics model that comprises five cell types and fifteen signaling molecules inter-connected through a large number of cell-cell communication pathways. We observed that the signaling molecules are tightly correlated even at very early stages (e.g. the first month) of human glioma, but such correlation rapidly diminishes when tumor grows to a size that can be clinically detected. Further analysis suggests that paracrine is shown to be the dominant force during tumor initiation and priming, while autocrine supersedes it and supports a robust tumor expansion. In correspondence, the cytokine correlation network evolves through an increasing to decreasing complexity. This study indicates a possible mechanistic transition from the microenvironment-controlled, paracrine-based regulatory mechanism to self-sustained rapid progression to fetal malignancy. It also reveals key nodes that are responsible for such transition and can be potentially harnessed for the design of new anti-cancer therapies. PMID:23080410

  11. Nanoporous noninvasive cellular electrical activity-based analysis devices.

    PubMed

    Prasad, Shalini; Quijano, Jorge

    2007-03-01

    In recent years, rapid advancements have been made in the biomedical applications of microtechnology and nanotechnology. While the focus of such technologies have been primarily on in vitro analytical and diagnostic tools, more recently in vivo therapeutic and sensing applications have gained attention. The long-term integration of cells with inorganic materials provides the basis for novel sensing platforms. The work presented here focuses on the ability to maintain cells long-term in nanoporous silicon-based microenvironments. This article describes the creation of nanoporous, biocompatible, alumina membranes as a platform for incorporation into a cell-based device targeted for in situ recording of cellular electrical activity variations due to the changes associated with the surrounding microenvironments. Studies described herein focus on the interaction of nanoporous alumina substrates embedded in silicon patterned with cells of interest. The fidelity of such a system is demonstrated in terms of viability, proliferation, and functionality. The capability of such microfabricated nanoporous membranes, as in vitro for cell-based assays for sensing and drug delivery applications, is also demonstrated. It has potential in vivo application for therapeutic immunoisolation.

  12. Active medulloblastoma enhancers reveal subgroup-specific cellular origins.

    PubMed

    Lin, Charles Y; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C; Ju, Bensheng; Orr, Brent A; Zeid, Rhamy; Polaski, Donald R; Segura-Wang, Maia; Waszak, Sebastian M; Jones, David T W; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V; Millen, Kathleen J; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O; Pfister, Stefan M; Bradner, James E; Northcott, Paul A

    2016-02-04

    Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

  13. Active medulloblastoma enhancers reveal subgroup-specific cellular origins

    PubMed Central

    Lin, Charles Y.; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J.; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C.; Ju, Bensheng; Orr, Brent A.; Zeid, Rhamy; Polaski, Donald R.; Segura-Wang, Maia; Waszak, Sebastian M.; Jones, David T.W.; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V.; Millen, Kathleen J.; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O.; Pfister, Stefan M.; Bradner, James E.; Northcott, Paul A.

    2016-01-01

    Summary Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-Seq, that are responsible for subgroup divergence and implicate candidate cells-of-origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins. PMID:26814967

  14. Scaffolds are 'active' regulators of signaling modules.

    PubMed

    Alexa, Anita; Varga, János; Reményi, Attila

    2010-11-01

    Signaling cascades, in addition to proteins with obvious signaling-relevant activities (e.g. protein kinases or receptors), also employ dedicated 'inactive' proteins whose functions appear to be the organization of the former components into higher order complexes through protein-protein interactions. The core function of signaling adaptors, anchors and scaffolds is the recruitment of proteins into one macromolecular complex. Several recent studies have demonstrated that the recruiter and the recruited molecules mutually influence each other in a scaffolded complex. This yields fundamentally novel properties for the signaling complex as a whole. Because these are not merely additive to the properties of the individual components, scaffolded signaling complexes may behave as functionally distinct modules.

  15. LOV-based optogenetic devices: light-driven modules to impart photoregulated control of cellular signaling

    PubMed Central

    Pudasaini, Ashutosh; El-Arab, Kaley K.; Zoltowski, Brian D.

    2015-01-01

    The Light-Oxygen-Voltage domain family of proteins is widespread in biology where they impart sensory responses to signal transduction domains. The small, light responsive LOV modules offer a novel platform for the construction of optogenetic tools. Currently, the design and implementation of these devices is partially hindered by a lack of understanding of how light drives allosteric changes in protein conformation to activate diverse signal transduction domains. Further, divergent photocycle properties amongst LOV family members complicate construction of highly sensitive devices with fast on/off kinetics. In the present review we discuss the history of LOV domain research with primary emphasis on tuning LOV domain chemistry and signal transduction to allow for improved optogenetic tools. PMID:25988185

  16. Why cellular communication during plant reproduction is particularly mediated by CRP signalling.

    PubMed

    Bircheneder, Susanne; Dresselhaus, Thomas

    2016-08-01

    Secreted cysteine-rich peptides (CRPs) represent one of the main classes of signalling peptides in plants. Whereas post-translationally modified small non-CRP peptides (psNCRPs) are mostly involved in signalling events during vegetative development and interactions with the environment, CRPs are overrepresented in reproductive processes including pollen germination and growth, self-incompatibility, gamete activation and fusion as well as seed development. In this opinion paper we compare the involvement of both types of peptides in vegetative and reproductive phases of the plant lifecycle. Besides their conserved cysteine pattern defining structural features, CRPs exhibit hypervariable primary sequences and a rapid evolution rate. As a result, CRPs represent a pool of highly polymorphic signalling peptides involved in species-specific functions during reproduction and thus likely represent key players to trigger speciation in plants by supporting reproductive isolation. In contrast, precursers of psNCRPs are proteolytically processed into small functional domains with high sequence conservation and act in more general processes. We discuss parallels in downstream processes of CRP signalling in both reproduction and defence against pathogenic fungi and alien pollen tubes, with special emphasis on the role of ROS and ion channels. In conclusion we suggest that CRP signalling during reproduction in plants has evolved from ancient defence mechanisms.

  17. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    PubMed

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells.

  18. Mechanisms in photodynamic therapy: part two—cellular signaling, cell metabolism and modes of cell death

    PubMed Central

    Castano, Ana P.; Demidova, Tatiana N.; Hamblin, Michael R.

    2013-01-01

    Summary Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In the second of a series of three reviews, we will discuss the mechanisms that operate in PDT on a cellular level. In Part I [Castano AP, Demidova TN, Hamblin MR. Mechanism in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization. Photodiagn Photodyn Ther 2004;1:279–93] it was shown that one of the most important factors governing the outcome of PDT, is how the photosensitizer (PS) interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. PS can localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes. An explosion of investigation and explorations in the field of cell biology have elucidated many of the pathways that mammalian cells undergo when PS are delivered in tissue culture and subsequently illuminated. There is an acute stress response leading to changes in calcium and lipid metabolism and production of cytokines and stress proteins. Enzymes particularly, protein kinases, are activated and transcription factors are expressed. Many of the cellular responses are centered on mitochondria. These effects frequently lead to induction of apoptosis either by the mitochondrial pathway involving caspases and release of cytochrome c, or by pathways involving ceramide or death receptors. However, under certain circumstances cells subjected to PDT die by necrosis. Although there have been many reports of DNA damage caused by PDT, this is not thought to be an important cell-death pathway. This mechanistic research is expected to lead to optimization of PDT as a tumor treatment, and to rational selection of combination therapies that include PDT as a component. PMID:25048553

  19. Biased signaling by peptide agonists of protease activated receptor 2.

    PubMed

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  20. Metabolic signals and innate immune activation in obesity and exercise.

    PubMed

    Ringseis, Robert; Eder, Klaus; Mooren, Frank C; Krüger, Karsten

    2015-01-01

    The combination of a sedentary lifestyle and excess energy intake has led to an increased prevalence of obesity which constitutes a major risk factor for several co-morbidities including type 2 diabetes and cardiovascular diseases. Intensive research during the last two decades has revealed that a characteristic feature of obesity linking it to insulin resistance is the presence of chronic low-grade inflammation being indicative of activation of the innate immune system. Recent evidence suggests that activation of the innate immune system in the course of obesity is mediated by metabolic signals, such as free fatty acids (FFAs), being elevated in many obese subjects, through activation of pattern recognition receptors thereby leading to stimulation of critical inflammatory signaling cascades, like IκBα kinase/nuclear factor-κB (IKK/NF- κB), endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) and NOD-like receptor P3 (NLRP3) inflammasome pathway, that interfere with insulin signaling. Exercise is one of the main prescribed interventions in obesity management improving insulin sensitivity and reducing obesity- induced chronic inflammation. This review summarizes current knowledge of the cellular recognition mechanisms for FFAs, the inflammatory signaling pathways triggered by excess FFAs in obesity and the counteractive effects of both acute and chronic exercise on obesity-induced activation of inflammatory signaling pathways. A deeper understanding of the effects of exercise on inflammatory signaling pathways in obesity is useful to optimize preventive and therapeutic strategies to combat the increasing incidence of obesity and its comorbidities.

  1. Antimicrobial activities and cellular responses to natural silicate clays and derivatives modified by cationic alkylamine salts.

    PubMed

    Hsu, Shan-Hui; Tseng, Hsiang-Jung; Hung, Huey-Shan; Wang, Ming-Chien; Hung, Chiung-Hui; Li, Pei-Ru; Lin, Jiang-Jen

    2009-11-01

    Nanometer-scale silicate platelet (NSP) materials were previously developed by increasing the interlayer space and exfoliation of layered silicate clays such as montmorillonite and synthetic fluorinated mica by the process of polyamine exfoliation. In this study, the antibacterial activity and cytotoxicity of these nanometer-scale silicate clays were evaluated. The derivatives of NSP (NSP-S) which were modified by C18-fatty amine salts via ionic exchange association exhibited the highest antibacterial activity in the aqueous state among all clays. The high antibacterial activity, however, was accompanied by elevated cytotoxicity. The variations of cell surface markers (CD29 and CD44) and type I collagen expression of fibroblasts treated with the clays were measured to clarify the mechanism of the silicate-induced cytotoxicity. The signal transduction pathway involved the downregulation of extracellular-signal-regulated kinase (ERK), which appeared to participate in silicate-induced cytotoxicity. This study helped to understand the antibacterial potential of NSP and the interaction of natural and modified clays with cellular activities.

  2. Adaptive Cellular Interactions in the Immune System: The Tunable Activation Threshold and the Significance of Subthreshold Responses

    NASA Astrophysics Data System (ADS)

    Grossman, Zvi; Paul, William E.

    1992-11-01

    A major challenge for immunologists is to explain how the immune system adjusts its responses to the microenvironmental context in which antigens are recognized. We propose that lymphocytes achieve this by tuning and updating their responsiveness to recurrent signals. In particular, cellular anergy in vivo is a dynamic state in which the threshold for a stereotypic mode of activation has been elevated. Anergy is associated with other forms of cellular activity, not paralysis. Cells engaged in such subthreshold interactions mediate functions such as maintenance of immunological memory and control of infections. In such interactions, patterns of signals are recognized and classified and evoke selective responses. The robust mechanism proposed for segregation of suprathreshold and subthreshold immune responses allows lymphocytes to use recognition of self-antigens in executing physiological functions. Autoreactivity is allowed where it is dissociated from uncontrolled aggression.

  3. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  4. Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions.

    PubMed

    Menchero, Sergio; Rayon, Teresa; Andreu, Maria Jose; Manzanares, Miguel

    2017-04-01

    The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc.

  5. Effects of Tetrahydrocurcumin on Tumor Growth and Cellular Signaling in Cervical Cancer Xenografts in Nude Mice

    PubMed Central

    Yoysungnoen, Bhornprom; Bhattarakosol, Parvapan; Changtam, Chatchawan; Patumraj, Suthiluk

    2016-01-01

    Tetrahydrocurcumin (THC) is a stable metabolite of curcumin (CUR) in physiological systems. The mechanism underlying the anticancer effect of THC is not completely understood. In the present study, we investigated the effects of THC on tumor growth and cellular signaling in cervical cancer xenografts in nude mice. Cervical cancer cells (CaSki) were subcutaneously injected in nude mice to establish tumors. One month after the injection, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. Relative tumor volume (RTV) was measured every 3-4 days. COX-2, EGFR, p-ERK1&2, p-AKT, and Ki-67 expressions were measured by immunohistochemistry whereas cell apoptosis was detected by TUNELS method. THC treatments at the doses of 100, 300, and 500 mg/kg statistically retarded the RTV by 70.40%, 76.41%, and 77.93%, respectively. The CaSki + vehicle group also showed significantly increased COX-2, EGFR, p-ERK1&2, and p-AKT; however they were attenuated by all treatments with THC. Ki-67 overexpression and a decreasing of cell apoptosis were found in CaSki + vehicle group, but these findings were reversed after the THC treatments. PMID:26881213

  6. Effects of Tetrahydrocurcumin on Tumor Growth and Cellular Signaling in Cervical Cancer Xenografts in Nude Mice.

    PubMed

    Yoysungnoen, Bhornprom; Bhattarakosol, Parvapan; Changtam, Chatchawan; Patumraj, Suthiluk

    2016-01-01

    Tetrahydrocurcumin (THC) is a stable metabolite of curcumin (CUR) in physiological systems. The mechanism underlying the anticancer effect of THC is not completely understood. In the present study, we investigated the effects of THC on tumor growth and cellular signaling in cervical cancer xenografts in nude mice. Cervical cancer cells (CaSki) were subcutaneously injected in nude mice to establish tumors. One month after the injection, mice were orally administered vehicle or 100, 300, and 500 mg/kg of THC daily for 30 consecutive days. Relative tumor volume (RTV) was measured every 3-4 days. COX-2, EGFR, p-ERK1&2, p-AKT, and Ki-67 expressions were measured by immunohistochemistry whereas cell apoptosis was detected by TUNELS method. THC treatments at the doses of 100, 300, and 500 mg/kg statistically retarded the RTV by 70.40%, 76.41%, and 77.93%, respectively. The CaSki + vehicle group also showed significantly increased COX-2, EGFR, p-ERK1&2, and p-AKT; however they were attenuated by all treatments with THC. Ki-67 overexpression and a decreasing of cell apoptosis were found in CaSki + vehicle group, but these findings were reversed after the THC treatments.

  7. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling.

    PubMed

    Martínez-Herrera, Alejandro; Aragón, Jorge; Bermúdez-Cruz, Rosa Ma; Bazán, Ma Luisa; Soid-Raggi, Gabriela; Ceja, Víctor; Santos Coy-Arechavaleta, Andrea; Alemán, Víctor; Depardón, Francisco; Montañez, Cecilia

    2015-09-01

    Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids (93)LEQEHNNLV(101) and (168)LLLHDSIQI(176) could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled "EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40" (J. Aragón et al. Neurosci. Lett. 600 (2015) 115-120) [1].

  8. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling

    PubMed Central

    Martínez-Herrera, Alejandro; Aragón, Jorge; Bermúdez-Cruz, Rosa Ma.; Bazán, Ma. Luisa; Soid-Raggi, Gabriela; Ceja, Víctor; Santos Coy-Arechavaleta, Andrea; Alemán, Víctor; Depardón, Francisco; Montañez, Cecilia

    2015-01-01

    Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015) 115–120) [1]. PMID:26217814

  9. Redox Modulation of Cellular Signaling and Metabolism Through Reversible Oxidation of Methionine Sensors in Calcium Regulatory Proteins

    SciTech Connect

    Bigelow, Diana J.; Squier, Thomas C.

    2005-01-17

    Adaptive responses associated with environmental stressors are critical to cell survival. These involve the modulation of central signaling protein functions through site-specific and enzymatically reversible oxidative modifications of methionines to coordinate cellular metabolism, energy utilization, and calcium signaling. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. Thus, depending on either the ecological niche of the organism or the cellular milieu of different organ systems, cellular metabolism can be fine-tuned to maintain optimal function in the face of variable amounts of collateral oxidative damage. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met{sup 144} or Met{sup 145} results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylationdependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in

  10. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    SciTech Connect

    Kim, Hag Dong; Jang, Chang-Young; Choe, Jeong Min; Sohn, Jeongwon; Kim, Joon

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  11. Transient Receptor Potential Ion Channel Function in Sensory Transduction and Cellular Signaling Cascades Underlying Visceral Hypersensitivity.

    PubMed

    Balemans, Dafne; Boeckxstaens, Guy E; Talavera, Karel; Wouters, Mira M

    2017-04-06

    Visceral hypersensitivity is an important mechanism underlying increased abdominal pain perception in functional gastrointestinal disorders (FGID) including functional dyspepsia, irritable bowel syndrome (IBS) and inflammatory bowel disease in remission. Although the exact pathophysiological mechanisms are poorly understood, recent studies described upregulation and altered functions of nociceptors and their signaling pathways in aberrant visceral nociception, in particular the transient receptor potential (TRP) channel family. A variety of TRP channels are present in the gastrointestinal tract (TRPV1, TRPV3, TRPV4, TRPA1, TRPM2, TRPM5 and TRPM8) and modulation of their function by increased activation or sensitization (decreased activation threshold) or altered expression in visceral afferents, have been reported in visceral hypersensitivity. TRP channels directly detect or transduce osmotic, mechanical, thermal and chemosensory stimuli. In addition, pro-inflammatory mediators released in tissue damage or inflammation can activate receptors of the G-protein coupled receptor (GPCR) superfamily leading to TRP channel sensitization and activation, which amplify pain and neurogenic inflammation. In this review, we highlight the current knowledge on the functional roles of neuronal TRP channels in visceral hypersensitivity and discuss the signaling pathways that underlie TRP channel modulation. We propose that a better understanding of TRP channels and their modulators may facilitate the development of more selective and effective therapies to treat visceral hypersensitivity.

  12. Receptor binding and cellular uptake studies of macrophage migration inhibitory factor (MIF): use of biologically active labeled MIF derivatives.

    PubMed

    Kleemann, Robert; Grell, Matthias; Mischke, Ralf; Zimmermann, Gudrun; Bernhagen, Jürgen

    2002-03-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine for which a receptor has not been identified. That MIF has intracellular functions has been suggested by its enzymatic activity and constitutive expression profile. The discovery of functional MIF-c-Jun activation domain binding protein 1 (JAB1) binding has confirmed this notion and indicated that nonreceptor-based signaling mechanisms are important for MIF function. Here, we have generated and tested several biologically active labeled MIF derivatives to further define target protein binding by MIF and its cellular uptake characteristics. (35)S-MIF, biotinylated MIF, and fluoresceinated MIF were demonstrated to exhibit full biologic activity. Neither by applying a standard iodinated MIF preparation nor by using the biologically active (35)S-MIF derivative in receptor-binding studies were we able to measure any receptor-binding activity on numerous cells, confirming that uptake of MIF into target cells and MIF signaling can occur by receptor-independent pathways. When MIF derivatives were applied in cellular uptake studies, MIF was found to be endocytosed into both immune and nonimmune cells and targeted to the cytosol and lysosomes. The entry of MIF was temperature and energy dependent and was inhibited by monodansylcadaverine but not by ouabain. Endocytosed biotin-MIF bound JAB1 not only in macrophages, as shown previously, but also in nonimmune cells. A tagged MIF construct, MIF-enhanced green fluorescent protein (EGFP), was shown to be a valuable tool, as EGFP constructs of critical MIF cysteine mutants exhibited identical cellular localization properties to those of wild-type MIF (wtMIF). Our results indicate that MIF membrane receptors are not widely expressed, if at all, and suggest that the cellular uptake of MIF occurs by nonreceptor-mediated endocytosis rather than penetration. All the derivatives investigated, except for iodinated MIF, represent valuable tools for further MIF target

  13. Four-phase or two-phase signal plan? A study on four-leg intersection by cellular automaton simulations

    NASA Astrophysics Data System (ADS)

    Jin, Cheng-Jie; Wang, Wei; Jiang, Rui

    2016-08-01

    The proper setting of traffic signals at signalized intersections is one of the most important tasks in traffic control and management. This paper has evaluated the four-phase traffic signal plans at a four-leg intersection via cellular automaton simulations. Each leg consists of three lanes, an exclusive left-turn lane, a through lane, and a through/right-turn lane. For a comparison, we also evaluate the two-phase signal plan. The diagram of the intersection states in the space of inflow rate versus turning ratio has been presented, which exhibits four regions: In region I/II/III, congestion will propagate upstream and laterally and result in queue spillover with both signal plans/two-phase signal plan/four-phase signal plan, respectively. Therefore, neither signal plan works in region I, and only the four-phase signal plan/two-phase signal plan works in region II/III. In region IV, both signal plans work, but two-phase signal plan performs better in terms of average delays of vehicles. Finally, we study the diagram of the intersection states and average delays in the asymmetrical configurations.

  14. Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

    PubMed

    Disanza, A; Steffen, A; Hertzog, M; Frittoli, E; Rottner, K; Scita, G

    2005-05-01

    Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

  15. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

    PubMed Central

    Urano, T; Emkey, R; Feig, L A

    1996-01-01

    Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation. Images PMID:8631302

  16. The Roles of Mitochondrial Reactive Oxygen Species in Cellular Signaling and Stress Response in Plants1[OPEN

    PubMed Central

    Millar, A. Harvey

    2016-01-01

    Mitochondria produce ATP via respiratory oxidation of organic acids and transfer of electrons to O2 via the mitochondrial electron transport chain. This process produces reactive oxygen species (ROS) at various rates that can impact respiratory and cellular function, affecting a variety of signaling processes in the cell. Roles in redox signaling, retrograde signaling, plant hormone action, programmed cell death, and defense against pathogens have been attributed to ROS generated in plant mitochondria (mtROS). The shortcomings of the black box-idea of mtROS are discussed in the context of mechanistic considerations and the measurement of mtROS. The overall aim of this update is to better define our current understanding of mtROS and appraise their potential influence on cellular function in plants. Furthermore, directions for future research are provided, along with suggestions to increase reliability of mtROS measurements. PMID:27021189

  17. Assessment of General Public Exposure to LTE signals compared to other Cellular Networks Present in Thessaloniki, Greece.

    PubMed

    Gkonis, Fotios; Boursianis, Achilles; Samaras, Theodoros

    2016-12-15

    To assess general public exposure to electromagnetic fields from Long Term Evolution (LTE) base stations, measurements at 10 sites in Thessaloniki, Greece were performed. Results are compared with other mobile cellular networks currently in use. All exposure values satisfy the guidelines for general public exposure of the International Commission on Non-Ionizing Radiation Protection (ICNIRP), as well as the reference levels by the Greek legislation at all sites. LTE electric field measurements were recorded up to 0.645 V/m. By applying the ICNIRP guidelines, the exposure ratio for all LTE signals is between 2.9 × 10(-5) and 2.8 × 10(-2) From the measurements results it is concluded that the average and maximum power density contribution of LTE downlink signals to the overall cellular networks signals are 7.8% and 36.7%, respectively.

  18. Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

    PubMed Central

    Garbers, Christoph; Kuck, Fabian; Aparicio-Siegmund, Samadhi; Konzak, Kirstin; Kessenbrock, Mareike; Sommerfeld, Annika; Häussinger, Dieter; Lang, Philipp A; Brenner, Dirk; Mak, Tak W.; Rose-John, Stefan; Essmann, Frank; Schulze-Osthoff, Klaus; Piekorz, Roland P; Scheller, Jürgen

    2013-01-01

    Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development. PMID:24047696

  19. Regulators of G-protein Signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity

    PubMed Central

    Lambert, Nevin A.; Johnston, Christopher A.; Cappell, Steven D.; Kuravi, Sudhakiranmayi; Kimple, Adam J.; Willard, Francis S.; Siderovski, David P.

    2010-01-01

    G-protein heterotrimers, composed of a guanine nucleotide-binding Gα subunit and an obligate Gβγ dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by Gα-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of “regulator of G-protein signaling” (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a Gα switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP·AlF4 −-bound conformation of the G202A mutant was found to be nearly identical to wild-type, Gαi1(G202A)·GDP assumed a divergent conformation more closely resembling the GDP·AlF4 −-bound state. When placed within Saccharomyces cerevisiae Gα subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose–response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus Gα-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein–coupled receptor (GPCR) signaling in a cellular context. PMID:20351284

  20. Sodium Methyldithiocarbamate Exerts Broad Inhibition of Cellular Signaling and Expression of Effector Molecules of Inflammation

    PubMed Central

    Pruett, Stephen B.

    2013-01-01

    Sodium methyldithiocarbamate (SMD) is one of the most abundantly used conventional pesticides in the United States. At dosages relevant to occupational exposure, it causes major effects on the immune system in mice, including a decreased resistance to sepsis. This lab has identified some of the mechanisms of action of this compound and some of the immunological parameters affected, but the global effects have not previously been assessed. The purpose of the present study was to conduct transcriptomic analysis of the effects of SMD on lipopolysaccharide-induced expression of mediators important in innate immunity and inflammation. The results revealed broad effects on expression of transcription factors in both branches of Toll-like receptor 4 (TLR4) signaling (MyD88 and TRIF). However, TLR3 and interferon signaling pathways were decreased to a greater extent, and assessment of the effects of SMD on polyinosinic polycytidylic acid–induced cytokine and chemokine production revealed that these responses mediated by TLR3 were indeed sensitive to the effects of SMD, with inhibition occurring at lower dosages than required to inhibit responses to other immunological stimuli tested in our previous studies. In the downstream signaling pathways of these TLRs, functional analysis also revealed that NF-κB activation was inhibited by SMD, as indicated by gene expression analysis and a reporter construct in mice. A previously unreported effect on luteinizing hormone and follicle-stimulating hormone pathways was also observed. PMID:24056979

  1. Synaptic generation of an intracellular retrograde signal requires activation of the tyrosine kinase and mitogen-activated protein kinase signaling cascades in Aplysia.

    PubMed

    Stough, Shara; Kopec, Ashley M; Carew, Thomas J

    2015-11-01

    Cellular changes underlying memory formation can be generated in an activity-dependent manner at specific synapses. Thus an important question concerns the mechanisms by which synaptic signals communicate with the cell body to mediate these cellular changes. A monosynaptic circuit that is enhanced by sensitization in Aplysia is well-suited to study this question because three different subcellular compartments: (i) the sensorimotor SN-MN synapses, (ii) the SN projections to MNs via axonal connections, (iii) the SN cell bodies, can all be manipulated and studied independently. Here, we report that activity-dependent (AD) training in either the entire SN-MN circuit or in only the synaptic compartment, activates MAPK in a temporally and spatially specific pattern. Specifically, we find (i) MAPK activation is first transiently generated at SN-MN synapses during training, (ii) immediately after training MAPK is transiently activated in SN-MN axonal connections and persistently activated in SN cell bodies, and finally, (iii) MAPK is activated in SN cell bodies and SN-MN synapses 1h after training. These data suggest that there is an intracellularly transported retrograde signal generated at the synapse which is later responsible for delayed MAPK activation at SN somata. Finally, we find that this retrograde signal requires activation of tyrosine kinase (TK) and MEK signaling cascades at the synapses.

  2. Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-04-01

    Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.

  3. Genetic Algorithm Calibration of Probabilistic Cellular Automata for Modeling Mining Permit Activity

    USGS Publications Warehouse

    Louis, S.J.; Raines, G.L.

    2003-01-01

    We use a genetic algorithm to calibrate a spatially and temporally resolved cellular automata to model mining activity on public land in Idaho and western Montana. The genetic algorithm searches through a space of transition rule parameters of a two dimensional cellular automata model to find rule parameters that fit observed mining activity data. Previous work by one of the authors in calibrating the cellular automaton took weeks - the genetic algorithm takes a day and produces rules leading to about the same (or better) fit to observed data. These preliminary results indicate that genetic algorithms are a viable tool in calibrating cellular automata for this application. Experience gained during the calibration of this cellular automata suggests that mineral resource information is a critical factor in the quality of the results. With automated calibration, further refinements of how the mineral-resource information is provided to the cellular automaton will probably improve our model.

  4. Statistics of cellular signal transduction as a race to the nucleus by multiple random walkers in compartment/phosphorylation space.

    PubMed

    Lu, Ting; Shen, Tongye; Zong, Chenghang; Hasty, Jeff; Wolynes, Peter G

    2006-11-07

    Cellular signal transduction often involves a reaction network of phosphorylation and transport events arranged with a ladder topology. If we keep track of the location of the phosphate groups describing an abstract state space, a simple model of signal transduction involving enzymes can be mapped on to a problem of how multiple biased random walkers compete to reach their target in the nucleus yielding a signal. Here, the first passage time probability and the survival probability for multiple walkers can be used to characterize the response of the network. The statistics of the first passage through the network has an asymmetric distribution with a long tail arising from the hierarchical structure of the network. This distribution implies a significant difference between the mean and the most probable signal transduction time. The response patterns for various external inputs generated by our model agree with recent experiments. In addition, the model predicts that there is an optimal phosphorylation enzyme concentration for rapid signal transduction.

  5. Metal oxide nanoparticles interact with immune cells and activate different cellular responses

    PubMed Central

    Simón-Vázquez, Rosana; Lozano-Fernández, Tamara; Dávila-Grana, Angela; González-Fernández, Africa

    2016-01-01

    Besides cell death, nanoparticles (Nps) can induce other cellular responses such as inflammation. The potential immune response mediated by the exposure of human lymphoid cells to metal oxide Nps (moNps) was characterized using four different moNps (CeO2, TiO2, Al2O3, and ZnO) to study the three most relevant mitogen-activated protein kinase subfamilies and the nuclear factor kappa-light-chain-enhancer of the activated B-cell inhibitor, IκBα, as well as the expression of several genes by immune cells incubated with these Nps. The moNps activated different signaling pathways and altered the gene expression in human lymphocyte cells. The ZnO Nps were the most active and the release of Zn2+ ions was the main mechanism of toxicity. CeO2 Nps induced the smallest changes in gene expression and in the IκBα protein. The effects of the particles were strongly dependent on the type and concentration of the Nps and on the cell activation status prior to Np exposure. PMID:27695324

  6. Fenofibrate suppresses cellular metabolic memory of high glucose in diabetic retinopathy via a sirtuin 1-dependent signalling pathway.

    PubMed

    Zhao, Shuzhi; Li, Jun; Wang, Na; Zheng, Bingqing; Li, Tao; Gu, Qing; Xu, Xun; Zheng, Zhi

    2015-10-01

    Inflammation is a major contributing factor in the development of diabetic microvascular complications, regardless of whether improved glycaemic control is achieved. Studies have increasingly indicated that fenofibrate, a lipid‑lowering therapeutic agent in clinical use, exerts a potential anti‑inflammatory effect, which is mediated by sirtuin 1 (SIRT1; an NAD+‑dependent deacetylase) in endothelial cells. The aim of the present study was to investigate the inhibitory effect of fenofibrate on metabolic memory (via the regulation of SIRT1), and inflammatory responses in cell and animal models of diabetic retinopathy (DR). The data demonstrated that high glucose treatment in human retinal endothelial cells (HRECs) inhibited the expression and deacetylase activity of SIRT1. The reduction of SIRT1 expression and deacetylase activity persisted following a return to normal glucose levels. Furthermore, nuclear factor‑κB expression was observed to be negatively correlated with SIRT1 expression and activity in HRECs under high glucose levels and the subsequent return to normal glucose levels. Fenofibrate treatment abrogated these changes. Knockdown of SIRT1 attenuated the effect of fenofibrate on high glucose‑induced NF‑κB expression. In addition, fenofibrate upregulated SIRT1 expression through peroxisome proliferator‑activated receptor α in high glucose‑induced metabolic memory. These findings indicate that fenofibrate is important in anti‑inflammatory processes and suppresses the cellular metabolic memory of high glucose‑induced stress via the SIRT1‑dependent signalling pathway. Thus, treatment with fenofibrate may offer a promising therapeutic strategy for halting the development of DR and other complications of diabetes.

  7. The journey of antiphospholipid antibodies from cellular activation to antiphospholipid syndrome.

    PubMed

    Willis, Rohan; Gonzalez, E B; Brasier, A R

    2015-03-01

    Pathogenic antiphospholipid antibodies (aPL) are the driving factors of recurrent pregnancy loss and thrombosis that characterize antiphospholipid syndrome (APS). Current evidence indicates that aPL induce a procoagulant phenotype in the vasculature and abnormal cellular proliferation and differentiation in placental tissues to cause the typical clinical features; however, the molecular mechanisms underlying these processes remain incompletely understood. Inflammation serves as a necessary link between the observed procoagulant phenotype and actual thrombus development and is an important mediator of the placental injury in APS patients. However, the underlying mechanisms for these events have also not been fully elucidated. In this review, we will outline the available data that give us our current understanding of the pathophysiology of APS, especially as it relates to the development of thromboembolic and obstetric pathological phenomena in these patients. We will also describe the intracellular signaling pathways activated by aPL in various cellular subtypes and outline the current evidence linking these pathways to clinical phenotypes. Finally, we will discuss the implications of distinct molecular patterns defining clinical phenotypes of APS patients.

  8. Meta-analysis of retrograde signaling in Arabidopsis thaliana reveals a core module of genes embedded in complex cellular signaling networks.

    PubMed

    Gläßer, Christine; Haberer, Georg; Finkemeier, Iris; Pfannschmidt, Thomas; Kleine, Tatjana; Leister, Dario; Dietz, Karl-Josef; Häusler, Rainer Erich; Grimm, Bernhard; Mayer, Klaus Franz Xaver

    2014-07-01

    Plastid-to-nucleus signaling is essential for the coordination and adjustment of cellular metabolism in response to environmental and developmental cues of plant cells. A variety of operational retrograde signaling pathways have been described that are thought to be triggered by reactive oxygen species, photosynthesis redox imbalance, tetrapyrrole intermediates, and other metabolic traits. Here we report a meta-analysis based on transcriptome and protein interaction data. Comparing the output of these pathways reveals the commonalities and peculiarities stimulated by six different sources impinging on operational retrograde signaling. Our study provides novel insights into the interplay of these pathways, supporting the existence of an as-yet unknown core response module of genes being regulated under all conditions tested. Our analysis further highlights affiliated regulatory cis-elements and classifies abscisic acid and auxin-based signaling as secondary components involved in the response cascades following a plastidial signal. Our study provides a global analysis of structure and interfaces of different pathways involved in plastid-to-nucleus signaling and a new view on this complex cellular communication network.

  9. Cross-linked bromelain inhibits lipopolysaccharide-induced cytokine production involving cellular signaling suppression in rats.

    PubMed

    Hou, Rolis Chien-Wei; Chen, Yuh-Shuen; Huang, Jing-Rong; Jeng, Kee-Ching G

    2006-03-22

    Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. It has been cross-linked with organic acids and polysaccharides by gamma irradiation. The cross-linked (CL)-bromelain preparation resisted an acidic environment of pH 3 for 2 h and preserved 80% of its enzyme activity. Pretreatment of rats with CL-bromelain intragastrically for 7 days significantly reduced serum cytokine production induced by injected i.p. with 2.5 mg/kg of lipopolysaccharide (LPS). Bromelain significantly reduced serum glutamate-oxalacetate transaminase induced by LPS. The anti-inflammatory effect of CL-bromelain was correlated with reduced LPS-induced NF-kappaB activity and cyclooxygenase 2 (COX-2) mRNA expression in rat livers. In addition, CL-bromelain dose-dependently inhibited LPS-induced COX-2 mRNA and prostaglandin E2 (PGE2) in BV-2 microglial cells. CL-Bromelain also suppressed the LPS-activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). In conclusion, the anti-inflammatory effects of the CL-bromelain preparation in vivo and in vitro suggest its therapeutic potentials.

  10. Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency

    PubMed Central

    Mologni, Luca; Poggio, Teresa; Varesio, Lydia M.; Menotti, Matteo; Bombelli, Silvia; Rigolio, Roberta; Manazza, Andrea D.; Di Giacomo, Filomena; Ambrogio, Chiara; Giudici, Giovanni; Casati, Cesare; Mastini, Cristina; Compagno, Mara; Turner, Suzanne D.; Gambacorti-Passerini, Carlo; Chiarle, Roberto; Voena, Claudia

    2016-01-01

    Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK. NPM-ALK deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive due to heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or re-localization of NPM-ALK to the cytoplasm by NPM genetic knock-out or knock-down caused ERK1/2 increased phosphorylation and cell death through the engagement of an ATM/Chk2 and γH2AX mediated DNA damage response. Remarkably, human NPM-ALK amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A “drug holiday” where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification. PMID:26657151

  11. Cellular Stress Response and Immune Signaling in Retinal Ischemia–Reperfusion Injury

    PubMed Central

    Minhas, Gillipsie; Sharma, Jyoti; Khan, Nooruddin

    2016-01-01

    Ischemia–reperfusion injury is a well-known pathological hallmark associated with diabetic retinopathy, glaucoma, and other related retinopathies that ultimately can lead to visual impairment and vision loss. Retinal ischemia pathogenesis involves a cascade of detrimental events that include energy failure, excitotoxic damage, calcium imbalance, oxidative stress, and eventually cell death. Retina for a long time has been known to be an immune privileged site; however, recent investigations reveal that retina, as well as the central nervous system, elicits immunological responses during various stress cues. Stress condition, such as reperfusion of blood supply post-ischemia results in the sequestration of different immune cells, inflammatory mediators including cytokines, chemokines, etc., to the ischemic region, which in turn facilitates induction of inflammatory conditions in these tissues. The immunological activation during injury or stress per se is beneficial for repair and maintenance of cellular homeostasis, but whether the associated inflammation is good or bad, during ischemia–reperfusion injury, hitherto remains to be explored. Keeping all these notions in mind, the current review tries to address the immune response and host stress response mechanisms involved in ischemia–reperfusion injury with the focus on the retina. PMID:27822213

  12. Angiotensin-converting enzyme (ACE) inhibitors modulate cellular retinol-binding protein 1 and adiponectin expression in adipocytes via the ACE-dependent signaling cascade.

    PubMed

    Kohlstedt, Karin; Gershome, Cynthia; Trouvain, Caroline; Hofmann, Wolf-Karsten; Fichtlscherer, Stephan; Fleming, Ingrid

    2009-03-01

    Inhibitors of the angiotensin-converting enzyme (ACE) decrease angiotensin II production and activate an intracellular signaling cascade that affects gene expression in endothelial cells. Because ACE inhibitors have been reported to delay the onset of type 2 diabetes, we determined ACE signaling-modulated gene expression in endothelial cells and adipocytes. Using differential gene expression analysis, several genes were identified that were 3-fold up- or down-regulated by ramiprilat in cells expressing wild-type ACE versus cells expressing a signaling-dead ACE mutant. One up-regulated gene was the cellular retinol-binding protein 1 (CRBP1). In adipocytes, the overexpression of CRBP1 enhanced (4- to 5-fold) the activity of promoters containing response elements for retinol-dependent nuclear receptors [retinoic acid receptor (RAR) and retinoid X receptor (RXR)] or peroxisome proliferator-activated receptors (PPAR). CRBP1 overexpression also enhanced the promoter activity (by 470 +/- 40%) and expression/release of the anti-inflammatory and antiatherogenic adipokine adiponectin (cellular adiponectin by 196 +/- 24%, soluble adiponectin by 228 +/- 74%). Significantly increased adiponectin secretion was also observed after ACE inhibitor treatment of human preadipocytes, an effect prevented by small interfering RNA against CRBP1. Furthermore, in ob/ob mice, ramipril markedly potentiated both the basal (approximately 2-fold) and rosiglitazonestimulated circulating levels of adiponectin. In patients with coronary artery disease or type 2 diabetes, ACE inhibition also significantly increased plasma adiponectin levels (1.6- or 2.1-fold, respectively). In summary, ACE inhibitors affect adipocyte homeostasis via CRBP1 through the activation of RAR/RXR-PPAR signaling and up-regulation of adiponectin. The latter may contribute to the beneficial effects of ACE inhibitors on the development of type 2 diabetes in patients with an activated renin-angiotensin system.

  13. N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

    PubMed

    Yoshida, Hiroyuki; Nagaoka, Aya; Nakamura, Sachiko; Tobiishi, Megumi; Sugiyama, Yoshinori; Inoue, Shintaro

    2014-01-03

    Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.

  14. Heparin activates Wnt signaling for neuronal morphogenesis.

    PubMed

    Colombres, Marcela; Henríquez, Juan Pablo; Reig, Germán F; Scheu, Jessica; Calderón, Rosario; Alvarez, Alejandra; Brandan, Enrique; Inestrosa, Nibaldo C

    2008-09-01

    Wnt factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Wnt signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3beta (GSK-3beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Wnt signaling for neuronal morphogenesis.

  15. A conformational change within the WAVE2 complex regulates its degradation following cellular activation

    PubMed Central

    Joseph, Noah; Biber, Guy; Fried, Sophia; Reicher, Barak; Levy, Omer; Sabag, Batel; Noy, Elad; Barda-Saad, Mira

    2017-01-01

    WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation. PMID:28332566

  16. Effects of PPARγ Agonist Pioglitazone on Redox-Sensitive Cellular Signaling in Young Spontaneously Hypertensive Rats

    PubMed Central

    Dovinová, Ima; Barancik, Miroslav; Zorad, Stefan; Gajdosechová, Lucia; Gresová, Linda; Cacanyiova, Sona; Kristek, Frantisek; Balis, Peter; Chan, Julie Y. H.

    2013-01-01

    PPARγ receptor plays an important role in oxidative stress response. Its agonists can influence vascular contractility in experimental hypertension. Our study was focused on the effects of a PPARγ agonist pioglitazone (PIO) on blood pressure regulation, vasoactivity of vessels, and redox-sensitive signaling at the central (brainstem, BS) and peripheral (left ventricle, LV) levels in young prehypertensive rats. 5-week-old SHR were treated either with PIO (10 mg/kg/day, 2 weeks) or with saline using gastric gavage. Administration of PIO significantly slowed down blood pressure increase and improved lipid profile and aortic relaxation after insulin stimulation. A significant increase in PPARγ expression was found only in BS, not in LV. PIO treatment did not influence NOS changes, but had tissue-dependent effect on SOD regulation and increased SOD activity, observed in LV. The treatment with PIO differentially affected also the levels of other intracellular signaling components: Akt kinase increased in the the BS, while β-catenin level was down-regulated in the BS and up-regulated in the LV. We found that the lowering of blood pressure in young SHR can be connected with insulin sensitivity of vessels and that β-catenin and SOD levels are important agents mediating PIO effects in the BS and LV. PMID:24454335

  17. Roles of vitamin D in amyotrophic lateral sclerosis: possible genetic and cellular signaling mechanisms

    PubMed Central

    2013-01-01

    Evidence suggests that there are aberrations in the vitamin D-endocrine system in subjects with amyotrophic lateral sclerosis (ALS). Here, we review the relationship between vitamin D and ALS. Vitamin D deficiency was reported in patients with ALS. Dietary vitamin D3 supplementation improves functional capacity in the G93A transgenic mouse model of ALS. Genetic studies have provided an opportunity to identify the proteins that link vitamin D to ALS pathology, including major histocompatibility complex (MHC) class II molecules, toll-like receptors, poly(ADP-ribose) polymerase-1, heme oxygenase-1, and calcium-binding proteins, as well as the reduced form of nicotinamide adenine dinucleotide phosphate. Vitamin D also exerts its effect on ALS through cell-signaling mechanisms, including glutamate, matrix metalloproteinases, mitogen-activated protein kinase pathways, the Wnt/β-catenin signaling pathway, prostaglandins, reactive oxygen species, and nitric oxide synthase. In conclusion, vitamin D may have a role in ALS. Further investigation of vitamin D in ALS patients is needed. PMID:23570271

  18. Pyrazinoic acid decreases the proton motive force, respiratory ATP synthesis activity, and cellular ATP levels.

    PubMed

    Lu, Ping; Haagsma, Anna C; Pham, Hoang; Maaskant, Janneke J; Mol, Selena; Lill, Holger; Bald, Dirk

    2011-11-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  19. Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells

    PubMed Central

    Morgan, J. Brian; Liu, Yang; Coothankandaswamy, Veena; Mahdi, Fakhri; Jekabsons, Mika B.; Gerwick, William H.; Valeriote, Frederick A.; Zhou, Yu-Dong; Nagle, Dale G.

    2015-01-01

    The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells. PMID:25803180

  20. Intrinsic cellular signaling mechanisms determine the sensitivity of cancer cells to virus-induced apoptosis

    PubMed Central

    Wang, Yunfei; Li, Dawei; Luo, Jian; Tian, Guimei; Zhao, Lisa Y.; Liao, Daiqing

    2016-01-01

    Cancer cells of epithelial and mesenchymal phenotypes exhibit different sensitivities to apoptosis stimuli, but the mechanisms underlying this phenomenon remain partly understood. We constructed a novel recombinant adenovirus expressing Ad12 E1A (Ad-E1A12) that can strongly induce apoptosis. Ad-E1A12 infection of epithelial cancer cells displayed dramatic detachment and apoptosis, whereas cancer cells of mesenchymal phenotypes with metastatic propensity were markedly more resistant to this virus. Notably, forced detachment of epithelial cells did not further sensitize them to Ad-E1A12-induced apoptosis, suggesting that cell detachment is a consequence rather than the cause of Ad-E1A12-induced apoptosis. Ad-E1A12 increased phosphorylation of AKT1 and ribosomal protein S6 through independent mechanisms in different cell types. Ad-E1A12–induced AKT1 phosphorylation was PI3K-dependent in epithelial cancer cells, and mTOR-dependent in mesenchymal cancer cells. Epithelial cancer cells upon Ad-E1A12-induced detachment could not sustain AKT activation due to AKT1 degradation, but AKT1 activation was maintained in mesenchymal cancer cells. Expression of epithelial cell-restricted miR-200 family in mesenchymal cells limited mTOR signaling and sensitized them to Ad-E1A12-induced cell killing. Thus, epithelial cancer cells rely on the canonical PI3K-AKT signaling pathway for survival, while mesenchymal cancer cells deploy the PI3K-independent mTORC2-AKT axis in response to strong death stimuli. PMID:27849011

  1. Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells.

    PubMed

    Wittig, Anja; Gehrke, Helge; Del Favero, Giorgia; Fritz, Eva-Maria; Al-Rawi, Marco; Diabaté, Silvia; Weiss, Carsten; Sami, Haider; Ogris, Manfred; Marko, Doris

    2017-01-13

    Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways-both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration

  2. Amorphous Silica Particles Relevant in Food Industry Influence Cellular Growth and Associated Signaling Pathways in Human Gastric Carcinoma Cells

    PubMed Central

    Wittig, Anja; Gehrke, Helge; Del Favero, Giorgia; Fritz, Eva-Maria; Al-Rawi, Marco; Diabaté, Silvia; Weiss, Carsten; Sami, Haider; Ogris, Manfred; Marko, Doris

    2017-01-01

    Nanostructured silica particles are commonly used in biomedical and biotechnical fields, as well as, in cosmetics and food industry. Thus, their environmental and health impacts are of great interest and effects after oral uptake are only rarely investigated. In the present study, the toxicological effects of commercially available nano-scaled silica with a nominal primary diameter of 12 nm were investigated on the human gastric carcinoma cell line GXF251L. Besides the analysis of cytotoxic and proliferative effects and the comparison with effects of particles with a nominal primary diameter of 200 nm, emphasis was also given to their influence on the cellular epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPK) signaling pathways—both of them deeply involved in the regulation of cellular processes like cell cycle progression, differentiation or proliferation. The investigated silica nanoparticles (NPs) were found to stimulate cell proliferation as measured by microscopy and the sulforhodamine B assay. In accordance, the nuclear level of the proliferation marker Ki-67 was enhanced in a concentration-dependent manner. At high particle concentrations also necrosis was induced. Finally, silica NPs affected the EGFR and MAPK pathways at various levels dependent on concentration and time. However, classical activation of the EGFR, to be reflected by enhanced levels of phosphorylation, could be excluded as major trigger of the proliferative stimulus. After 45 min of incubation the level of phosphorylated EGFR did not increase, whereas enhanced levels of total EGFR protein were observed. These results indicate interference with the complex homeostasis of the EGFR protein, whereby up to 24 h no impact on the transcription level was detected. In addition, downstream on the level of the MAP kinases ERK1/2 short term incubation appeared to affect total protein levels without clear increase in phosphorylation. Depending on the concentration

  3. Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

    PubMed Central

    Afriat, Reuven; Horowitz, Shulamith; Priel, Esther

    2013-01-01

    To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists. PMID:24013388

  4. Spatio-temporal analysis of brain electrical activity in epilepsy based on cellular nonlinear networks

    NASA Astrophysics Data System (ADS)

    Gollas, Frank; Tetzlaff, Ronald

    2009-05-01

    Epilepsy is the most common chronic disorder of the nervous system. Generally, epileptic seizures appear without foregoing sign or warning. The problem of detecting a possible pre-seizure state in epilepsy from EEG signals has been addressed by many authors over the past decades. Different approaches of time series analysis of brain electrical activity already are providing valuable insights into the underlying complex dynamics. But the main goal the identification of an impending epileptic seizure with a sufficient specificity and reliability, has not been achieved up to now. An algorithm for a reliable, automated prediction of epileptic seizures would enable the realization of implantable seizure warning devices, which could provide valuable information to the patient and time/event specific drug delivery or possibly a direct electrical nerve stimulation. Cellular Nonlinear Networks (CNN) are promising candidates for future seizure warning devices. CNN are characterized by local couplings of comparatively simple dynamical systems. With this property these networks are well suited to be realized as highly parallel, analog computer chips. Today available CNN hardware realizations exhibit a processing speed in the range of TeraOps combined with low power consumption. In this contribution new algorithms based on the spatio-temporal dynamics of CNN are considered in order to analyze intracranial EEG signals and thus taking into account mutual dependencies between neighboring regions of the brain. In an identification procedure Reaction-Diffusion CNN (RD-CNN) are determined for short segments of brain electrical activity, by means of a supervised parameter optimization. RD-CNN are deduced from Reaction-Diffusion Systems, which usually are applied to investigate complex phenomena like nonlinear wave propagation or pattern formation. The Local Activity Theory provides a necessary condition for emergent behavior in RD-CNN. In comparison linear spatio

  5. The deiodinases and the control of intracellular thyroid hormone signaling during cellular differentiation☆

    PubMed Central

    Dentice, Monica; Marsili, Alessandro; Zavacki, AnnMarie; Larsen, P. Reed; Salvatore, Domenico

    2013-01-01

    Background Thyroid hormone influences gene expression in virtually all vertebrates. Its action is initiated by the activation of T4 to T3, an outer ring deiodination reaction that is catalyzed by the type 1 or the type 2 iodothyronine selenodeiodinases (D1 or D2). Inactivation of T4 and T3 occurs via inner ring deiodination catalyzed by the type 3 iodothyronine selenodeiodinases (D3). The T4 concentration is generally quite stable in human plasma, with T3 levels also remaining constant. Deiodinase actions are tightly regulated in both pre- and post-natal life when they are required to make local adjustments of intracellular T3 concentrations in a precise spatio- and temporal manner. Although all the signals governing the dynamic expression of deiodinases in specific cell types are not known, many important regulatory factors have been deciphered. Scope of review This review provides striking examples from the recent literature illustrating how the expression of D2 and D3 is finely tuned during maturation of different organs, and how their action play a critical role in different settings to control intracellular T3 availability. Major conclusions Emerging evidence indicates that in various cell contexts, D2 and D3 are expressed in a dynamic balance, in which the expression of one enzyme is coordinately regulated with that of the other to tightly control intracellular T3 levels commensurate with cell requirements at that time. General significance Deiodinases control TH action in a precise spatio-temporal fashion thereby providing a novel mechanism for the local paracrine and autocrine regulation of TH action. This remarkable tissue-specific regulation of intracellular thyroid status remains hidden due to the maintenance of constant circulating TH concentrations by the hypothalamic–pituitary–thyroid axis. This article is part of a Special Issue entitled Thyroid hormone signalling. PMID:22634734

  6. Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

    PubMed Central

    Exell, Jack C.; Thompson, Mark J.; Finger, L. David; Shaw, Steven J.; Debreczeni, Judit; Ward, Thomas A.; McWhirter, Claire; Siöberg, Catrine L. B.; Martinez Molina, Daniel; Mark Abbott, W.; Jones, Clifford D.; Nissink, J. Willem M.; Durant, Stephen T.; Grasby, Jane A.

    2016-01-01

    The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed. PMID:27526030

  7. Learning about Cellular Respiration: An Active Approach Illustrating the Process of Scientific Inquiry.

    ERIC Educational Resources Information Center

    Johnson, Margaret (Peg)

    1998-01-01

    Details the active-learning approach to teaching cellular respiration in an introductory, one-semester course for nonmajors. Focuses on a laboratory exercise designed to answer the question of what happens to food when eaten. Contains 19 references. (DDR)

  8. Technology Learning Activities: Columbus Sailed the Ocean Blue, the Cellular Connection, Emergency Shelter.

    ERIC Educational Resources Information Center

    Etchison, Cindy; Deal, Walter F., III

    1992-01-01

    Presents learning activities such as planning and building a sailboat, manufacturing cellular phone cases, and designing and building emergency shelters. Includes the context, the challenge, resources used, objectives, materials needed, and an evaluation. (JOW)

  9. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    PubMed Central

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited. PMID:28074895

  10. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    NASA Astrophysics Data System (ADS)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  11. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    NASA Astrophysics Data System (ADS)

    Song, Zhen

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

  12. Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation.

    PubMed

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Im, Sanghun; Fukuda, Tomohiko; Gu, Jianguo

    2015-12-04

    Integrin α5β1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5β1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5β1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5β1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3-5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3-5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors.

  13. Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation*

    PubMed Central

    Hang, Qinglei; Isaji, Tomoya; Hou, Sicong; Im, Sanghun; Fukuda, Tomohiko; Gu, Jianguo

    2015-01-01

    Integrin α5β1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5β1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5β1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5β1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3–5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3–5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors. PMID:26483551

  14. Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

    PubMed Central

    Andrusiak, Matthew G.; Jin, Yishi

    2016-01-01

    Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

  15. Cellular targets of estrogen signaling in regeneration of inner ear sensory epithelia

    PubMed Central

    McCullar, Jennifer S.; Oesterle, Elizabeth C.

    2010-01-01

    Estrogen signaling in auditory and vestibular sensory epithelia is a newly emerging focus propelled by the role of estrogen signaling in many other proliferative systems. Understanding the pathways with which estrogen interacts can provide a means to identify how estrogen may modulate proliferative signaling in inner ear sensory epithelia. Reviewed herein are two signaling families, EGF and TGFβ. Both pathways are involved in regulating proliferation of supporting cells in mature vestibular sensory epithelia and have well characterized interactions with estrogen signaling in other systems. It is becoming increasingly clear that elucidating the complexity of signaling in regeneration will be necessary for development of therapeutics that can initiate regeneration and prevent progression to a pathogenic state. PMID:19450430

  16. The effect of neutral-surface iron oxide nanoparticles on cellular uptake and signaling pathways

    PubMed Central

    Kim, Eunjoo; Kim, Joon Mee; Kim, Lucia; Choi, Suk Jin; Park, In Suh; Han, Jee Young; Chu, Young Chae; Choi, Eun Sook; Na, Kun; Hong, Soon-Sun

    2016-01-01

    In recent years, iron oxide nanoparticles (IONPs) have been applied widely to biomedical fields. However, the relationship between the physicochemical properties of IONPs and their biological behavior is not fully understood yet. We prepared 3-methacryloxypropyltrimethoxysilane (MPS)-coated IONPs, which have a neutral hydrophobic surface, and compared their biological behavior to that of Resovist (ferucarbotran), a commercialized IONP formulation modified with carboxymethyl dextran. The rate of MPS-IONP uptake by human aortic endothelial cells (HAoECs) was higher than ferucarbotran uptake, indicating that the neutral hydrophobic nature of MPS-IONPs allowed them to be absorbed more readily through the plasma membrane. However, the signaling pathways activated by MPS-IONPs and ferucarbotran were comparable, suggesting that surface charge is not a key factor for inducing changes in HAoECs. In vivo fate analysis showed that MPS-IONPs accumulated for longer periods in tissues than hydrophilic ferucarbotran. These findings could enlarge our understanding of NP behavior for advanced applications in the biomedical field. PMID:27695320

  17. Cellular aspartyl proteases promote the unconventional secretion of biologically active HIV-1 matrix protein p17

    PubMed Central

    Caccuri, Francesca; Iaria, Maria Luisa; Campilongo, Federica; Varney, Kristen; Rossi, Alessandro; Mitola, Stefania; Schiarea, Silvia; Bugatti, Antonella; Mazzuca, Pietro; Giagulli, Cinzia; Fiorentini, Simona; Lu, Wuyuan; Salmona, Mario; Caruso, Arnaldo

    2016-01-01

    The human immune deficiency virus type 1 (HIV-1) matrix protein p17 (p17), although devoid of a signal sequence, is released by infected cells and detected in blood and in different organs and tissues even in HIV-1-infected patients undergoing successful combined antiretroviral therapy (cART). Extracellularly, p17 deregulates the function of different cells involved in AIDS pathogenesis. The mechanism of p17 secretion, particularly during HIV-1 latency, still remains to be elucidated. A recent study showed that HIV-1-infected cells can produce Gag without spreading infection in a model of viral latency. Here we show that in Gag-expressing cells, secretion of biologically active p17 takes place at the plasma membrane and occurs following its interaction with phosphatidylinositol-(4,5)-bisphosphate and its subsequent cleavage from the precursor Gag (Pr55Gag) operated by cellular aspartyl proteases. These enzymes operate a more complex Gag polypeptide proteolysis than the HIV-1 protease, thus hypothetically generating slightly truncated or elongated p17s in their C-terminus. A 17 C-terminal residues excised p17 was found to be structurally and functionally identical to the full-length p17 demonstrating that the final C-terminal region of p17 is irrelevant for the protein’s biological activity. These findings offer new opportunities to identify treatment strategies for inhibiting p17 release in the extracellular microenvironment. PMID:27905556

  18. Activation of human natural killer cells by the soluble form of cellular prion protein

    SciTech Connect

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  19. Evaluation of Cellular Antioxidant and Antiproliferative Activities of Five Main Phyllanthus Emblica L. Cultivars in China.

    PubMed

    Li, Y; Sun, H Y; Yu, X Y; Liu, D; Wan, H X

    2015-01-01

    The cell-based antioxidant activity assay as more biological relevant assay was considered to be more accurate to predict antioxidant activity in vivo than chemical activity assays. In the present study, the five main Phyllanthus emblica L. cultivars in China were subjected for cellular antioxidant activity based on HepG2 cells as well as antiproliferative activity. Total phenolics, total flavonoids and oxygen radical absorbance capacity were also measured. The results showed that Qingyougan, Binggan and Boligan (832±100, 774±52 and 704±28 μmol of quercetin equivalents/100 g) had higher cellular antioxidant activity than Tianyougan and Yougan (553±50 and 457±24 μmol of quercetin equivalents/100 g) in phosphate buffered saline wash protocol whereas, Boligan (3735±217 μmol of quercetin equivalents/100 g) had the highest cellular antioxidant activity and Tianyougan (2025±171 μmol of quercetin equivalents/100 g) had the lowest cellular antioxidant activity in no phosphate buffered saline wash protocol. The highest and lowest antiproliferative activities were observed in Binggan and Tianyougan (median effective dose: 6.95±0.11 and 14.03±0.10 mg/ml), respectively. The significant correlation was only observed between total flavonoids and cellular antioxidant activity from no phosphate buffered saline wash protocol (R(2) =0.908, P<0.05), and total flavonoids and antiproliferative activity (R(2) =0.887, P<0.05), suggesting the major contribution of flavonoids to the bioactivities of emblica. Overall, the data obtained revealed that different Phyllanthus emblica L. cultivars had strong cellular antioxidant and antiproliferative activities, thus should be recommended to increase consumption for health.

  20. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

  1. Spontaneous Motion in Hierarchically Assembled Active Cellular Materials

    NASA Astrophysics Data System (ADS)

    Chen, Daniel

    2013-03-01

    With exquisite precision and reproducibility, cells orchestrate the cooperative action of thousands of nanometer-sized molecular motors to carry out mechanical tasks at much larger length scales, such as cell motility, division and replication. Besides their biological importance, such inherently far-from-equilibrium processes are an inspiration for the development of soft materials with highly sought after biomimetic properties such as autonomous motility and self-healing. I will describe our exploration of such a class of biologically inspired soft active materials. Starting from extensile bundles comprised of microtubules and kinesin, we hierarchically assemble active analogs of polymeric gels, liquid crystals and emulsions. At high enough concentration, microtubule bundles form an active gel network capable of generating internally driven chaotic flows that enhance transport and fluid mixing. When confined to emulsion droplets, these 3D networks buckle onto the water-oil interface forming a dense thin film of bundles exhibiting cascades of collective buckling, fracture, and self-healing driven by internally generated stresses from the kinesin clusters. When compressed against surfaces, this active nematic cortex exerts traction stresses that propel the locomotion of the droplet. Taken together, these observations exemplify how assemblies of animate microscopic objects exhibit collective biomimetic properties that are fundamentally distinct from those found in materials assembled from inanimate building blocks. These assemblies, in turn, enable the generation of a new class of materials that exhibit macroscale flow phenomena emerging from nanoscale components.

  2. Cellular imaging at 1.5 T: detecting cells in neuroinflammation using active labeling with superparamagnetic iron oxide.

    PubMed

    Oweida, Ayman J; Dunn, Elizabeth A; Foster, Paula J

    2004-04-01

    The ability to visualize cell infiltration in experimental auto-immune encephalomyelitis (EAE), a well-known animal model for multiple sclerosis in humans, was investigated using a clinical 1.5-T magnetic resonance imaging (MRI) scanner, a custom-built, high-strength gradient coil insert, a 3-D fast imaging employing steady-state acquisition (FIESTA) imaging sequence and a superparamagnetic iron oxide (SPIO) contrast agent. An "active labeling" approach was used with SPIO administered intravenously during inflammation in EAE. Our results show that small, discrete regions of signal void corresponding to iron accumulation in EAE brain can be detected using FIESTA at 1.5 T. This work provides early evidence that cellular abnormalities that are the basis of diseases can be probed using cellular MRI and supports our earlier work which indicates that tracking of iron-labeled cells will be possible using clinical MR scanners.

  3. Neurons in the barrel cortex turn into processing whisker and odor signals: a cellular mechanism for the storage and retrieval of associative signals

    PubMed Central

    Wang, Dangui; Zhao, Jun; Gao, Zilong; Chen, Na; Wen, Bo; Lu, Wei; Lei, Zhuofan; Chen, Changfeng; Liu, Yahui; Feng, Jing; Wang, Jin-Hui

    2015-01-01

    Associative learning and memory are essential to logical thinking and cognition. How the neurons are recruited as associative memory cells to encode multiple input signals for their associated storage and distinguishable retrieval remains unclear. We studied this issue in the barrel cortex by in vivo two-photon calcium imaging, electrophysiology, and neural tracing in our mouse model that the simultaneous whisker and olfaction stimulations led to odorant-induced whisker motion. After this cross-modal reflex arose, the barrel and piriform cortices connected. More than 40% of barrel cortical neurons became to encode odor signal alongside whisker signal. Some of these neurons expressed distinct activity patterns in response to acquired odor signal and innate whisker signal, and others encoded similar pattern in response to these signals. In the meantime, certain barrel cortical astrocytes encoded odorant and whisker signals. After associative learning, the neurons and astrocytes in the sensory cortices are able to store the newly learnt signal (cross-modal memory) besides the innate signal (native-modal memory). Such associative memory cells distinguish the differences of these signals by programming different codes and signify the historical associations of these signals by similar codes in information retrievals. PMID:26347609

  4. Antioxidant activity of puha (Sonchus oleraceus L.) as assessed by the cellular antioxidant activity (CAA) assay.

    PubMed

    McDowell, Arlene; Thompson, Scott; Stark, Mirjam; Ou, Zong-Quan; Gould, Kevin S

    2011-12-01

    There is considerable interest in antioxidant dietary components that can be protective against degenerative diseases in humans. Puha (Sonchus oleraceus L.) is a rich source of polyphenols, and exhibits strong antioxidant activity as measured by the 2,2-diphenylpicrylhydrazyl (DPPH) assay. However, the potential of puha to protect against degenerative diseases requires that low molecular weight antioxidants (LMWA) are absorbed by, and active in, human cells. The cellular antioxidant activity (CAA) assay was used to investigate the antioxidant activity of puha leaf extracts. Preparation methods of freezing and freeze-drying reduced the total polyphenolic content compared with fresh puha, but did not affect the LMWA potential as determined by the DPPH assay. The IC(50) values were 0.012 ± 0.003 mg/mL and 0.010 ± 0.005 mg/mL for freeze-dried and fresh puha leaves, respectively. Using the CAA assay, it was shown that LMWAs from foliar extracts of puha were effectively absorbed into HepG2 cells, and exerted antioxidant activity at levels comparable to those of extracts from blueberry fruits, the much-touted antioxidant superfood. Methylene blue staining of HepG2 cells indicated that puha extracts were not cytotoxic at concentrations below 100 mg DW/mL. The data indicate the potential of puha as a nutraceutical supplement for human health.

  5. Heat dissipation guides activation in signaling proteins

    PubMed Central

    Weber, Jeffrey K.; Shukla, Diwakar; Pande, Vijay S.

    2015-01-01

    Life is fundamentally a nonequilibrium phenomenon. At the expense of dissipated energy, living things perform irreversible processes that allow them to propagate and reproduce. Within cells, evolution has designed nanoscale machines to do meaningful work with energy harnessed from a continuous flux of heat and particles. As dictated by the Second Law of Thermodynamics and its fluctuation theorem corollaries, irreversibility in nonequilibrium processes can be quantified in terms of how much entropy such dynamics produce. In this work, we seek to address a fundamental question linking biology and nonequilibrium physics: can the evolved dissipative pathways that facilitate biomolecular function be identified by their extent of entropy production in general relaxation processes? We here synthesize massive molecular dynamics simulations, Markov state models (MSMs), and nonequilibrium statistical mechanical theory to probe dissipation in two key classes of signaling proteins: kinases and G-protein–coupled receptors (GPCRs). Applying machinery from large deviation theory, we use MSMs constructed from protein simulations to generate dynamics conforming to positive levels of entropy production. We note the emergence of an array of peaks in the dynamical response (transient analogs of phase transitions) that draw the proteins between distinct levels of dissipation, and we see that the binding of ATP and agonist molecules modifies the observed dissipative landscapes. Overall, we find that dissipation is tightly coupled to activation in these signaling systems: dominant entropy-producing trajectories become localized near important barriers along known biological activation pathways. We go on to classify an array of equilibrium and nonequilibrium molecular switches that harmonize to promote functional dynamics. PMID:26240354

  6. Molecular and Cellular Mechanisms for Trapping and Activating Emotional Memories

    PubMed Central

    Cai, Denise J.; Sano, Yoshitake; Lee, Yong-Seok; Zhou, Yu; Bekal, Pallavi; Deisseroth, Karl; Silva, Alcino J.

    2016-01-01

    Recent findings suggest that memory allocation to specific neurons (i.e., neuronal allocation) in the amygdala is not random, but rather the transcription factor cAMP-response element binding protein (CREB) modulates this process, perhaps by regulating the transcription of channels that control neuronal excitability. Here, optogenetic studies in the mouse lateral amygdala (LA) were used to demonstrate that CREB and neuronal excitability regulate which neurons encode an emotional memory. To test the role of CREB in memory allocation, we overexpressed CREB in the lateral amygdala to recruit the encoding of an auditory-fear conditioning (AFC) memory to a subset of neurons. Then, post-training activation of these neurons with Channelrhodopsin-2 was sufficient to trigger recall of the memory for AFC, suggesting that CREB regulates memory allocation. To test the role of neuronal excitability in memory allocation, we used a step function opsin (SFO) to transiently increase neuronal excitability in a subset of LA neurons during AFC. Post-training activation of these neurons with Volvox Channelrhodopsin-1 was able to trigger recall of that memory. Importantly, our studies show that activation of the SFO did not affect AFC by either increasing anxiety or by strengthening the unconditioned stimulus. Our findings strongly support the hypothesis that CREB regulates memory allocation by modulating neuronal excitability. PMID:27579481

  7. Desmosomes: Regulators of Cellular Signaling and Adhesion in Epidermal Health and Disease

    PubMed Central

    Johnson, Jodi L.; Najor, Nicole A.; Green, Kathleen J.

    2014-01-01

    Desmosomes are intercellular junctions that mediate cell–cell adhesion and anchor the intermediate filament network to the plasma membrane, providing mechanical resilience to tissues such as the epidermis and heart. In addition to their critical roles in adhesion, desmosomal proteins are emerging as mediators of cell signaling important for proper cell and tissue functions. In this review we highlight what is known about desmosomal proteins regulating adhesion and signaling in healthy skin—in morphogenesis, differentiation and homeostasis, wound healing, and protection against environmental damage. We also discuss how human diseases that target desmosome molecules directly or interfere indirectly with these mechanical and signaling functions to contribute to pathogenesis. PMID:25368015

  8. Desmosomes: regulators of cellular signaling and adhesion in epidermal health and disease.

    PubMed

    Johnson, Jodi L; Najor, Nicole A; Green, Kathleen J

    2014-11-03

    Desmosomes are intercellular junctions that mediate cell-cell adhesion and anchor the intermediate filament network to the plasma membrane, providing mechanical resilience to tissues such as the epidermis and heart. In addition to their critical roles in adhesion, desmosomal proteins are emerging as mediators of cell signaling important for proper cell and tissue functions. In this review we highlight what is known about desmosomal proteins regulating adhesion and signaling in healthy skin-in morphogenesis, differentiation and homeostasis, wound healing, and protection against environmental damage. We also discuss how human diseases that target desmosome molecules directly or interfere indirectly with these mechanical and signaling functions to contribute to pathogenesis.

  9. Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light

    NASA Astrophysics Data System (ADS)

    Lin, Liang; Liu, Ling; Zhao, Bing; Xie, Ran; Lin, Wei; Li, He; Li, Yaya; Shi, Minlong; Chen, Ye-Guang; Springer, Timothy A.; Chen, Xing

    2015-05-01

    Receptor-mediated signal transduction modulates complex cellular behaviours such as cell growth, migration and differentiation. Although photoactivatable proteins have emerged as a powerful tool for controlling molecular interactions and signalling cascades at precise times and spaces using light, many of these light-sensitive proteins are activated by ultraviolent or visible light, which has limited tissue penetration. Here, we report a single-walled carbon nanotube (SWCNT)-assisted approach that enables near-infrared light-triggered activation of transforming growth factor β (TGF-β) signal transduction, an important signalling pathway in embryonic development and cancer progression. The protein complex of TGF-β and its latency-associated peptide is conjugated onto SWCNTs, where TGF-β is inactive. Upon near-infrared irradiation, TGF-β is released through the photothermal effect of SWCNTs and becomes active. The released TGF-β activates downstream signal transduction in live cells and modulates cellular behaviours. Furthermore, preliminary studies show that the method can be used to mediate TGF-β signalling in living mice.

  10. Active voltammetric microsensors with neural signal processing.

    SciTech Connect

    Vogt, M. C.

    1998-12-11

    Many industrial and environmental processes, including bioremediation, would benefit from the feedback and control information provided by a local multi-analyte chemical sensor. For most processes, such a sensor would need to be rugged enough to be placed in situ for long-term remote monitoring, and inexpensive enough to be fielded in useful numbers. The multi-analyte capability is difficult to obtain from common passive sensors, but can be provided by an active device that produces a spectrum-type response. Such new active gas microsensor technology has been developed at Argonne National Laboratory. The technology couples an electrocatalytic ceramic-metallic (cermet) microsensor with a voltammetric measurement technique and advanced neural signal processing. It has been demonstrated to be flexible, rugged, and very economical to produce and deploy. Both narrow interest detectors and wide spectrum instruments have been developed around this technology. Much of this technology's strength lies in the active measurement technique employed. The technique involves applying voltammetry to a miniature electrocatalytic cell to produce unique chemical ''signatures'' from the analytes. These signatures are processed with neural pattern recognition algorithms to identify and quantify the components in the analyte. The neural signal processing allows for innovative sampling and analysis strategies to be employed with the microsensor. In most situations, the whole response signature from the voltammogram can be used to identify, classify, and quantify an analyte, without dissecting it into component parts. This allows an instrument to be calibrated once for a specific gas or mixture of gases by simple exposure to a multi-component standard rather than by a series of individual gases. The sampled unknown analytes can vary in composition or in concentration, the calibration, sensing, and processing methods of these active voltammetric microsensors can detect, recognize, and

  11. Optimization of the Design of Pre-Signal System Using Improved Cellular Automaton

    PubMed Central

    Li, Yan; Li, Ke; Tao, Siran; Chen, Kuanmin

    2014-01-01

    The pre-signal system can improve the efficiency of intersection approach under rational design. One of the main obstacles in optimizing the design of pre-signal system is that driving behaviors in the sorting area cannot be well evaluated. The NaSch model was modified by considering slow probability, turning-deceleration rules, and lane changing rules. It was calibrated with field observed data to explore the interactions among design parameters. The simulation results of the proposed model indicate that the length of sorting area, traffic demand, signal timing, and lane allocation are the most important influence factors. The recommendations of these design parameters are demonstrated. The findings of this paper can be foundations for the design of pre-signal system and show promising improvement in traffic mobility. PMID:25435871

  12. Optimization of the design of pre-signal system using improved cellular automaton.

    PubMed

    Li, Yan; Li, Ke; Tao, Siran; Wan, Xia; Chen, Kuanmin

    2014-01-01

    The pre-signal system can improve the efficiency of intersection approach under rational design. One of the main obstacles in optimizing the design of pre-signal system is that driving behaviors in the sorting area cannot be well evaluated. The NaSch model was modified by considering slow probability, turning-deceleration rules, and lane changing rules. It was calibrated with field observed data to explore the interactions among design parameters. The simulation results of the proposed model indicate that the length of sorting area, traffic demand, signal timing, and lane allocation are the most important influence factors. The recommendations of these design parameters are demonstrated. The findings of this paper can be foundations for the design of pre-signal system and show promising improvement in traffic mobility.

  13. Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

    PubMed

    Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

    2013-05-17

    Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

  14. Oma1 Links Mitochondrial Protein Quality Control and TOR Signaling To Modulate Physiological Plasticity and Cellular Stress Responses.

    PubMed

    Bohovych, Iryna; Kastora, Stavroula; Christianson, Sara; Topil, Danelle; Kim, Heejeong; Fangman, Teresa; Zhou, You J; Barrientos, Antoni; Lee, Jaekwon; Brown, Alistair J P; Khalimonchuk, Oleh

    2016-09-01

    A network of conserved proteases known as the intramitochondrial quality control (IMQC) system is central to mitochondrial protein homeostasis and cellular health. IMQC proteases also appear to participate in establishment of signaling cues for mitochondrion-to-nucleus communication. However, little is known about this process. Here, we show that in Saccharomyces cerevisiae, inactivation of the membrane-bound IMQC protease Oma1 interferes with oxidative-stress responses through enhanced production of reactive oxygen species (ROS) during logarithmic growth and reduced stress signaling via the TORC1-Rim15-Msn2/Msn4 axis. Pharmacological or genetic prevention of ROS accumulation in Oma1-deficient cells restores this defective TOR signaling. Additionally, inactivation of the Oma1 ortholog in the human fungal pathogen Candida albicans also alters TOR signaling and, unexpectedly, leads to increased resistance to neutrophil killing and virulence in the invertebrate animal model Galleria mellonella Our findings reveal a novel and evolutionarily conserved link between IMQC and TOR-mediated signaling that regulates physiological plasticity and pancellular oxidative-stress responses.

  15. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

    PubMed

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-11-15

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

  16. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  17. Activation of human natural killer cells by the soluble form of cellular prion protein.

    PubMed

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP(C)) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP(C) in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP(C) protein on human natural killer (NK) cells. Recombinant soluble PrP(C) protein was generated by fusion of human PrP(C) with the Fc portion of human IgG1 (PrP(C)-Fc). PrP(C)-Fc binds to the surface of human NK cells, particularly to CD56(dim) NK cells. PrP(C)-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP(C)-Fc facilitated the IL-15-induced proliferation of NK cells. PrP(C)-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP(C)-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP(C)-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  18. Cellular uptake and anticancer activity of carboxylated gallium corroles.

    PubMed

    Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H; Gray, Harry B; Termini, John; Lim, Punnajit

    2016-04-19

    We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax= 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 > 3 > 2 > 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging.

  19. Cellular uptake and anticancer activity of carboxylated gallium corroles

    PubMed Central

    Pribisko, Melanie; Palmer, Joshua; Grubbs, Robert H.; Gray, Harry B.; Termini, John; Lim, Punnajit

    2016-01-01

    We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50 values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50 values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax = 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 >> 3 > 2 >> 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging. PMID:27044076

  20. Associations of Unilateral Whisker and Olfactory Signals Induce Synapse Formation and Memory Cell Recruitment in Bilateral Barrel Cortices: Cellular Mechanism for Unilateral Training Toward Bilateral Memory

    PubMed Central

    Gao, Zilong; Chen, Lei; Fan, Ruicheng; Lu, Wei; Wang, Dangui; Cui, Shan; Huang, Li; Zhao, Shidi; Guan, Sudong; Zhu, Yan; Wang, Jin-Hui

    2016-01-01

    Somatosensory signals and operative skills learned by unilateral limbs can be retrieved bilaterally. In terms of cellular mechanism underlying this unilateral learning toward bilateral memory, we hypothesized that associative memory cells in bilateral cortices and synapse innervations between them were produced. In the examination of this hypothesis, we have observed that paired unilateral whisker and odor stimulations led to odorant-induced whisker motions in bilateral sides, which were attenuated by inhibiting the activity of barrel cortices. In the mice that showed bilateral cross-modal responses, the neurons in both sides of barrel cortices became to encode this new odor signal alongside the innate whisker signal. Axon projections and synapse formations from the barrel cortex, which was co-activated with the piriform cortex, toward its contralateral barrel cortex (CBC) were upregulated. Glutamatergic synaptic transmission in bilateral barrel cortices was upregulated and GABAergic synaptic transmission was downregulated. The associative activations of the sensory cortices facilitate new axon projection, glutamatergic synapse formation and GABAergic synapse downregulation, which drive the neurons to be recruited as associative memory cells in the bilateral cortices. Our data reveal the productions of associative memory cells and synapse innervations in bilateral sensory cortices for unilateral training toward bilateral memory. PMID:28018178

  1. Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

    PubMed

    Fick, Laura J; Belsham, Denise D

    2010-08-15

    Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

  2. Inside-out Signaling Promotes Dynamic Changes in the Carcinoembryonic Antigen-related Cellular Adhesion Molecule 1 (CEACAM1) Oligomeric State to Control Its Cell Adhesion Properties*

    PubMed Central

    Patel, Prerna C.; Lee, Hannah S. W.; Ming, Aaron Y. K.; Rath, Arianna; Deber, Charles M.; Yip, Christopher M.; Rocheleau, Jonathan V.; Gray-Owen, Scott D.

    2013-01-01

    Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded 432GXXXG436 motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the 432GXXXG436 motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the 432GXXXG436 mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling. PMID:24005674

  3. Separating Fluid Shear Stress from Acceleration during Vibrations in Vitro: Identification of Mechanical Signals Modulating the Cellular Response

    PubMed Central

    Uzer, Gunes; Manske, Sarah L; Chan, M Ete; Chiang, Fu-Pen; Rubin, Clinton T; Frame, Mary D; Judex, Stefan

    2012-01-01

    The identification of the physical mechanism(s) by which cells can sense vibrations requires the determination of the cellular mechanical environment. Here, we quantified vibration-induced fluid shear stresses in vitro and tested whether this system allows for the separation of two mechanical parameters previously proposed to drive the cellular response to vibration – fluid shear and peak accelerations. When peak accelerations of the oscillatory horizontal motions were set at 1g and 60Hz, peak fluid shear stresses acting on the cell layer reached 0.5Pa. A 3.5-fold increase in fluid viscosity increased peak fluid shear stresses 2.6-fold while doubling fluid volume in the well caused a 2-fold decrease in fluid shear. Fluid shear was positively related to peak acceleration magnitude and inversely related to vibration frequency. These data demonstrated that peak shear stress can be effectively separated from peak acceleration by controlling specific levels of vibration frequency, acceleration, and/or fluid viscosity. As an example for exploiting these relations, we tested the relevance of shear stress in promoting COX-2 expression in osteoblast like cells. Across different vibration frequencies and fluid viscosities, neither the level of generated fluid shear nor the frequency of the signal were able to consistently account for differences in the relative increase in COX-2 expression between groups, emphasizing that the eventual identification of the physical mechanism(s) requires a detailed quantification of the cellular mechanical environment. PMID:23074384

  4. Bluetooth telemedicine processor for multichannel biomedical signal transmission via mobile cellular networks.

    PubMed

    Rasid, Mohd Fadlee A; Woodward, Bryan

    2005-03-01

    One of the emerging issues in m-Health is how best to exploit the mobile communications technologies that are now almost globally available. The challenge is to produce a system to transmit a patient's biomedical signals directly to a hospital for monitoring or diagnosis, using an unmodified mobile telephone. The paper focuses on the design of a processor, which samples signals from sensors on the patient. It then transmits digital data over a Bluetooth link to a mobile telephone that uses the General Packet Radio Service. The modular design adopted is intended to provide a "future-proofed" system, whose functionality may be upgraded by modifying the software.

  5. PATHLOGIC-S: A Scalable Boolean Framework for Modelling Cellular Signalling

    PubMed Central

    Fearnley, Liam G.; Nielsen, Lars K.

    2012-01-01

    Curated databases of signal transduction have grown to describe several thousand reactions, and efficient use of these data requires the development of modelling tools to elucidate and explore system properties. We present PATHLOGIC-S, a Boolean specification for a signalling model, with its associated GPL-licensed implementation using integer programming techniques. The PATHLOGIC-S specification has been designed to function on current desktop workstations, and is capable of providing analyses on some of the largest currently available datasets through use of Boolean modelling techniques to generate predictions of stable and semi-stable network states from data in community file formats. PATHLOGIC-S also addresses major problems associated with the presence and modelling of inhibition in Boolean systems, and reduces logical incoherence due to common inhibitory mechanisms in signalling systems. We apply this approach to signal transduction networks including Reactome and two pathways from the Panther Pathways database, and present the results of computations on each along with a discussion of execution time. A software implementation of the framework and model is freely available under a GPL license. PMID:22879903

  6. Transient Inhibition of FGFR2b-ligands signaling leads to irreversible loss of cellular β-catenin organization and signaling in AER during mouse limb development.

    PubMed

    Danopoulos, Soula; Parsa, Sara; Al Alam, Denise; Tabatabai, Reza; Baptista, Sheryl; Tiozzo, Caterina; Carraro, Gianni; Wheeler, Matthew; Barreto, Guillermo; Braun, Thomas; Li, Xiaokun; Hajihosseini, Mohammad K; Bellusci, Saverio

    2013-01-01

    The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤ 24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.

  7. Re-evaluation of the Role of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) in Cellular Calcium Signaling*

    PubMed Central

    Lin-Moshier, Yaping; Sebastian, Peter J.; Higgins, LeeAnn; Sampson, Natalie D.; Hewitt, Jane E.; Marchant, Jonathan S.

    2013-01-01

    Changes in cytoplasmic Ca2+ concentration, resulting from activation of intracellular Ca2+ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca2+ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca2+ channels, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca2+ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP3Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest. PMID:23148228

  8. Fluctuation analysis of activity biosensor images for the study of information flow in signaling pathways.

    PubMed

    Vilela, Marco; Halidi, Nadia; Besson, Sebastien; Elliott, Hunter; Hahn, Klaus; Tytell, Jessica; Danuser, Gaudenz

    2013-01-01

    Comprehensive understanding of cellular signal transduction requires accurate measurement of the information flow in molecular pathways. In the past, information flow has been inferred primarily from genetic or protein-protein interactions. Although useful for overall signaling, these approaches are limited in that they typically average over populations of cells. Single-cell data of signaling states are emerging, but these data are usually snapshots of a particular time point or limited to averaging over a whole cell. However, many signaling pathways are activated only transiently in specific subcellular regions. Protein activity biosensors allow measurement of the spatiotemporal activation of signaling molecules in living cells. These data contain highly complex, dynamic information that can be parsed out in time and space and compared with other signaling events as well as changes in cell structure and morphology. We describe in this chapter the use of computational tools to correct, extract, and process information from time-lapse images of biosensors. These computational tools allow one to explore the biosensor signals in a multiplexed approach in order to reconstruct the sequence of signaling events and consequently the topology of the underlying pathway. The extraction of this information, dynamics and topology, provides insight into how the inputs of a signaling network are translated into its biochemical or mechanical outputs.

  9. Glycitein activates extracellular signal-regulated kinase via vascular endothelial growth factor receptor signaling in nontumorigenic (RWPE-1) prostate epithelial cells.

    PubMed

    Clubbs, Elizabeth A; Bomser, Joshua A

    2007-08-01

    Increased consumption of soy is associated with a decreased risk for prostate cancer; however, the specific cellular mechanisms responsible for this anticancer activity are unknown. Dietary modulation of signaling cascades controlling cellular growth, proliferation and differentiation has emerged as a potential chemopreventive mechanism. The present study examined the effects of four soy isoflavones (genistein, daidzein, glycitein and equol) on extracellularsignal-regulated kinase (ERK1/2) activity in a nontumorigenic prostate epithelial cell line (RWPE-1). All four isoflavones (10 micromol/L) significantly increased ERK1/2 activity in RWPE-1 cells, as determined by immunoblotting. Isoflavone-induced ERK1/2 activation was rapid and sustained for approximately 2 h posttreatment. Glycitein, the most potent activator of ERK1/2, decreased RWPE-1 cell proliferation by 40% (P<.01). Glycitein-induced ERK1/2 activation was dependent, in part, on tyrosine kinase activity associated with vascular endothelial growth factor receptor (VEGFR). The presence of both VEGFR1 and VEGFR2 in the RWPE-1 cell line was confirmed by immunocytochemistry. Treatment of RWPE-1 cells with VEGF(165) resulted in transient ERK1/2 activation and increased cellular proliferation. The ability of isoflavones to modulate ERK1/2 signaling cascade via VEGFR signaling in the prostate may be responsible, in part, for the anticancer activity of soy.

  10. Evidence of cellular stress and caspase-3 resulting from a combined two-frequency signal in the cerebrum and cerebellum of Sprague-dawley rats

    PubMed Central

    López-Furelos, Alberto; Leiro-Vidal, José Manuel; Salas-Sánchez, Aarón Ángel; Ares-Pena, Francisco José; López-Martín, María Elena

    2016-01-01

    Multiple simultaneous exposures to electromagnetic signals induced adjustments in mammal nervous systems. In this study, we investigated the non-thermal SAR (Specific Absorption Rate) in the cerebral or cerebellar hemispheres of rats exposed in vivo to combined electromagnetic field (EMF) signals at 900 and 2450 MHz. Forty rats divided into four groups of 10 were individually exposed or not exposed to radiation in a GTEM chamber for one or two hours. After radiation, we used the Chemiluminescent Enzyme-Linked Immunosorbent Assay (ChELISA) technique to measure cellular stress levels, indicated by the presence of heat shock proteins (HSP) 90 and 70, as well as caspase-3-dependent pre-apoptotic activity in left and right cerebral and cerebellar hemispheres of Sprague Dawley rats. Twenty-four hours after exposure to combined or single radiation, significant differences were evident in HSP 90 and 70 but not in caspase 3 levels between the hemispheres of the cerebral cortex at high SAR levels. In the cerebellar hemispheres, groups exposed to a single radiofrequency (RF) and high SAR showed significant differences in HSP 90, 70 and caspase-3 levels compared to control animals. The absorbed energy and/or biological effects of combined signals were not additive, suggesting that multiple signals act on nervous tissue by a different mechanism. PMID:27589837

  11. Cellular Factor XIIIA Transglutaminase Localizes in Caveolae and Regulates Caveolin-1 Phosphorylation, Homo-oligomerization and c-Src Signaling in Osteoblasts

    PubMed Central

    Wang, Shuai; Kaartinen, Mari T.

    2015-01-01

    Transglutaminases (TGs) are a family of widely distributed enzymes that catalyze protein crosslinking by forming a covalent isopeptide bond between the substrate proteins. We have shown that MC3T3-E1 osteoblasts express Factor XIII-A (FXIII-A), and that the extracellular crosslinking activity of FXIII-A is involved in regulating matrix secretion and deposition. In this study, we have investigated the localization and potential role of intracellular FXIII-A. Conventional immunofluorescence microscopy and TIRF microscopy analyses showed that FXIII-A co-localizes with caveolin-1 in specialized membrane structures, caveolae, in differentiating osteoblasts. The caveolae-disrupting agent methyl-β-cyclodextrin abolished FXIII-A staining and co-localization with caveolin-1 from the osteoblast plasma membrane. The presence of FXIII-A in caveolae was confirmed by preparing caveolae-enriched cellular fractions using sucrose density gradient ultracentrifugation followed by western blotting. Despite this association of FXIII-A with caveolae, there was no detectable transglutaminase activity in caveolae, as measured by monodansylcadaverine incorporation. TG inhibitor NC9—which can alter TG enzyme conformation—localized to caveolae and displaced FXIII-A from these structures when added to the osteoblast cultures. The decreased FXIII-A levels in caveolae after NC9 treatment increased c-Src activation, which resulted in caveolin-1 phosphorylation, homo-oligomerization and Akt phosphorylation, suggesting cellular FXIII-A has a role in regulating c-Src signaling in osteoblasts. PMID:26231113

  12. Detection of silent cells, synchronization and modulatory activity in developing cellular networks.

    PubMed

    Hjorth, Johannes J J; Dawitz, Julia; Kroon, Tim; Pires, Johny; Dassen, Valerie J; Berkhout, Janna A; Emperador Melero, Javier; Nadadhur, Aish G; Alevra, Mihai; Toonen, Ruud F; Heine, Vivi M; Mansvelder, Huibert D; Meredith, Rhiannon M

    2016-04-01

    Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers.

  13. Hierarchical random cellular neural networks for system-level brain-like signal processing.

    PubMed

    Kozma, Robert; Puljic, Marko

    2013-09-01

    Sensory information processing and cognition in brains are modeled using dynamic systems theory. The brain's dynamic state is described by a trajectory evolving in a high-dimensional state space. We introduce a hierarchy of random cellular automata as the mathematical tools to describe the spatio-temporal dynamics of the cortex. The corresponding brain model is called neuropercolation which has distinct advantages compared to traditional models using differential equations, especially in describing spatio-temporal discontinuities in the form of phase transitions. Phase transitions demarcate singularities in brain operations at critical conditions, which are viewed as hallmarks of higher cognition and awareness experience. The introduced Monte-Carlo simulations obtained by parallel computing point to the importance of computer implementations using very large-scale integration (VLSI) and analog platforms.

  14. Traffic states and fundamental diagram in cellular automaton model of vehicular traffic controlled by signals

    NASA Astrophysics Data System (ADS)

    Nagatani, Takashi

    2009-04-01

    We present a cellular automaton (CA) model for vehicular traffic controlled by traffic lights. The CA model is not described by a set of rules, but is given by a simple difference equation. The vehicular motion varies highly with both signals’ characteristics and vehicular density. The dependence of tour time on both cycle time and vehicular density is clarified. In the dilute limit of vehicles, the vehicular motion is compared with that by the nonlinear-map model. The fundamental diagrams are derived numerically. It is shown that the fundamental diagram depends highly on the signals’ characteristics. The traffic states are shown for various values of cycle time in the fundamental diagram. We also study the effect of a slow vehicle on the traffic flow.

  15. The Cellular Prion Protein Controls Notch Signaling in Neural Stem/Progenitor Cells.

    PubMed

    Martin-Lannerée, Séverine; Halliez, Sophie; Hirsch, Théo Z; Hernandez-Rapp, Julia; Passet, Bruno; Tomkiewicz, Céline; Villa-Diaz, Ana; Torres, Juan-Maria; Launay, Jean-Marie; Béringue, Vincent; Vilotte, Jean-Luc; Mouillet-Richard, Sophie

    2017-03-01

    The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrP(C) , the discovery of its participation to the self-renewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrP(C) depletion, we establish that PrP(C) deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrP(C) in a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp(-/-) embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrP(C) results in altered expression of Nestin and Olig2 as well as N-cadherin distribution. We further provide evidence that PrP(C) controls the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrP(C) . As a whole, our study delineates a molecular scenario through which PrP(C) takes part to the self-renewal of neural stem and progenitor cells. Stem Cells 2017;35:754-765.

  16. Sex-biased cellular signaling: molecular basis for sex differences in neuropsychiatric diseases.

    PubMed

    Valentino, Rita J; Bangasser, Debra A

    2016-12-01

    The recognition that there are fundamental biological sex differences that extend beyond those that define sexual behavior and reproductive function has inspired the drive toward inclusion of both sexes in research design. This is supported by an underlying clinical rationale that studying both sexes is necessary to elucidate pathophysiology and develop treatments for the entire population. However, at a more basic level, sex differences, like genetic differences, can be exploited to better understand biology. Here, we discuss how sex differences at the molecular level of cell signaling and protein trafficking are amplified to create a state of vulnerability that under the right conditions can result in symptoms of neuropsychiatry disease. Although this dialogue focuses on the specific example of corticotropin-releasing factor, the potential for analogous sex differences in signaling and/or trafficking of receptors for other neuromodulators has broad biological and therapeutic implications.

  17. Sex-biased cellular signaling: molecular basis for sex differences in neuropsychiatric diseases

    PubMed Central

    Valentino, Rita J.; Bangasser, Debra A.

    2016-01-01

    The recognition that there are fundamental biological sex differences that extend beyond those that define sexual behavior and reproductive function has inspired the drive toward inclusion of both sexes in research design. This is supported by an underlying clinical rationale that studying both sexes is necessary to elucidate pathophysiology and develop treatments for the entire population. However, at a more basic level, sex differences, like genetic differences, can be exploited to better understand biology. Here, we discuss how sex differences at the molecular level of cell signaling and protein trafficking are amplified to create a state of vulnerability that under the right conditions can result in symptoms of neuropsychiatry disease. Although this dialogue focuses on the specific example of corticotropin-releasing factor, the potential for analogous sex differences in signaling and/or trafficking of receptors for other neuromodulators has broad biological and therapeutic implications. PMID:28179810

  18. The primary cilium is a self-adaptable, integrating nexus for mechanical stimuli and cellular signaling.

    PubMed

    Nguyen, An M; Young, Y-N; Jacobs, Christopher R

    2015-11-24

    Mechanosensation is crucial for cells to sense and respond to mechanical signals within their local environment. While adaptation allows a sensor to be conditioned by stimuli within the environment and enables its operation in a wide range of stimuli intensities, the mechanisms behind adaptation remain controversial in even the most extensively studied mechanosensor, bacterial mechanosensitive channels. Primary cilia are ubiquitous sensory organelles. They have emerged as mechanosensors across diverse tissues, including kidney, liver and the embryonic node, and deflect with mechanical stimuli. Here, we show that both mechanical and chemical stimuli can alter cilium stiffness. We found that exposure to flow stiffens the cilium, which deflects less in response to subsequent exposures to flow. We also found that through a process involving acetylation, the cell can biochemically regulate cilium stiffness. Finally, we show that this altered stiffness directly affects the responsiveness of the cell to mechanical signals. These results demonstrate a potential mechanism through which the cell can regulate its mechanosensing apparatus.

  19. Cellular signaling protective against noise-induced hearing loss – A role for novel intrinsic cochlear signaling involving corticotropin-releasing factor?

    PubMed

    Vetter, Douglas E

    2015-09-01

    Hearing loss afflicts approximately 15% of the world's population, and crosses all socioeconomic boundaries. While great strides have been made in understanding the genetic components of syndromic and non-syndromic hearing loss, understanding of the mechanisms underlying noise-induced hearing loss (NIHL) have come much more slowly. NIHL is not simply a mechanism by which older individuals loose their hearing. Significantly, the incidence of NIHL is increasing, and is now involving ever younger populations. This may predict future increased occurrences of hearing loss. Current research has shown that even short-term exposures to loud sounds generating what was previously considered temporary hearing loss, actually produces an almost immediate and permanent loss of specific populations of auditory nerve fibers. Additionally, recurrent exposures to intense sound may hasten age-related hearing loss. While NIHL is a significant medical concern, to date, few compounds have delivered significant protection, arguing that new targets need to be identified. In this commentary, we will explore cellular signaling processes taking place in the cochlea believed to be involved in protection against hearing loss, and highlight new data suggestive of novel signaling not previously recognized as occurring in the cochlea, that is perhaps protective of hearing. This includes a recently described local hypothalamic-pituitary-adrenal axis (HPA)-like signaling system fully contained in the cochlea. This system may represent a local cellular stress-response system based on stress hormone release similar to the systemic HPA axis. Its discovery may hold hope for new drug therapies that can be delivered directly to the cochlea, circumventing systemic side effects.

  20. From Cellular Attractor Selection to Adaptive Signal Control for Traffic Networks

    PubMed Central

    Tian, Daxin; Zhou, Jianshan; Sheng, Zhengguo; Wang, Yunpeng; Ma, Jianming

    2016-01-01

    The management of varying traffic flows essentially depends on signal controls at intersections. However, design an optimal control that considers the dynamic nature of a traffic network and coordinates all intersections simultaneously in a centralized manner is computationally challenging. Inspired by the stable gene expressions of Escherichia coli in response to environmental changes, we explore the robustness and adaptability performance of signalized intersections by incorporating a biological mechanism in their control policies, specifically, the evolution of each intersection is induced by the dynamics governing an adaptive attractor selection in cells. We employ a mathematical model to capture such biological attractor selection and derive a generic, adaptive and distributed control algorithm which is capable of dynamically adapting signal operations for the entire dynamical traffic network. We show that the proposed scheme based on attractor selection can not only promote the balance of traffic loads on each link of the network but also allows the global network to accommodate dynamical traffic demands. Our work demonstrates the potential of bio-inspired intelligence emerging from cells and provides a deep understanding of adaptive attractor selection-based control formation that is useful to support the designs of adaptive optimization and control in other domains. PMID:26972968

  1. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology

    NASA Technical Reports Server (NTRS)

    Ingber, Donald E.

    2002-01-01

    Great advances have been made in the identification of the soluble angiogenic factors, insoluble extracellular matrix (ECM) molecules, and receptor signaling pathways that mediate control of angiogenesis--the growth of blood capillaries. This review focuses on work that explores how endothelial cells integrate these chemical signals with mechanical cues from their local tissue microenvironment so as to produce functional capillary networks that exhibit specialized form as well as function. These studies have revealed that ECM governs whether an endothelial cell will switch between growth, differentiation, motility, or apoptosis programs in response to a soluble stimulus based on its ability to mechanically resist cell tractional forces and thereby produce cell and cytoskeletal distortion. Transmembrane integrin receptors play a key role in this mechanochemical transduction process because they both organize a cytoskeletal signaling complex within the focal adhesion and preferentially focus mechanical forces on this site. Molecular filaments within the internal cytoskeleton--microfilaments, microtubules, and intermediate filaments--also contribute to the cell's structural and functional response to mechanical stress through their role as discrete support elements within a tensegrity-stabilized cytoskeletal array. Importantly, a similar form of mechanical control also has been shown to be involved in the regulation of contractility in vascular smooth muscle cells and cardiac myocytes. Thus, the mechanism by which cells perform mechanochemical transduction and the implications of these findings for morphogenetic control are discussed in the wider context of vascular development and cardiovascular physiology.

  2. Reconstruction of cellular signal transduction networks using perturbation assays and linear programming.

    PubMed

    Knapp, Bettina; Kaderali, Lars

    2013-01-01

    Perturbation experiments for example using RNA interference (RNAi) offer an attractive way to elucidate gene function in a high throughput fashion. The placement of hit genes in their functional context and the inference of underlying networks from such data, however, are challenging tasks. One of the problems in network inference is the exponential number of possible network topologies for a given number of genes. Here, we introduce a novel mathematical approach to address this question. We formulate network inference as a linear optimization problem, which can be solved efficiently even for large-scale systems. We use simulated data to evaluate our approach, and show improved performance in particular on larger networks over state-of-the art methods. We achieve increased sensitivity and specificity, as well as a significant reduction in computing time. Furthermore, we show superior performance on noisy data. We then apply our approach to study the intracellular signaling of human primary nave CD4(+) T-cells, as well as ErbB signaling in trastuzumab resistant breast cancer cells. In both cases, our approach recovers known interactions and points to additional relevant processes. In ErbB signaling, our results predict an important role of negative and positive feedback in controlling the cell cycle progression.

  3. From Cellular Attractor Selection to Adaptive Signal Control for Traffic Networks

    NASA Astrophysics Data System (ADS)

    Tian, Daxin; Zhou, Jianshan; Sheng, Zhengguo; Wang, Yunpeng; Ma, Jianming

    2016-03-01

    The management of varying traffic flows essentially depends on signal controls at intersections. However, design an optimal control that considers the dynamic nature of a traffic network and coordinates all intersections simultaneously in a centralized manner is computationally challenging. Inspired by the stable gene expressions of Escherichia coli in response to environmental changes, we explore the robustness and adaptability performance of signalized intersections by incorporating a biological mechanism in their control policies, specifically, the evolution of each intersection is induced by the dynamics governing an adaptive attractor selection in cells. We employ a mathematical model to capture such biological attractor selection and derive a generic, adaptive and distributed control algorithm which is capable of dynamically adapting signal operations for the entire dynamical traffic network. We show that the proposed scheme based on attractor selection can not only promote the balance of traffic loads on each link of the network but also allows the global network to accommodate dynamical traffic demands. Our work demonstrates the potential of bio-inspired intelligence emerging from cells and provides a deep understanding of adaptive attractor selection-based control formation that is useful to support the designs of adaptive optimization and control in other domains.

  4. BDMC33, A Curcumin Derivative Suppresses Inflammatory Responses in Macrophage-Like Cellular System: Role of Inhibition in NF-κB and MAPK Signaling Pathways

    PubMed Central

    Lee, Ka-Heng; Chow, Yuh-Lit; Sharmili, Vidyadaran; Abas, Faridah; Alitheen, Noorjahan Banu Mohamed; Shaari, Khozirah; Israf, Daud Ahmad; Lajis, Nordin Haji; Syahida, Ahmad

    2012-01-01

    Our preliminary screening has shown that curcumin derivative BDMC33 [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] exerted promising nitric oxide inhibitory activity in activated macrophages. However, the molecular basis and mechanism for its pharmacological action is yet to be elucidated. The aim of this study was to investigate the anti-inflammatory properties of BDMC33 and elucidate its underlying mechanism action in macrophage cells. Our current study demonstrated that BDMC33 inhibits the secretion of major pro-inflammatory mediators in stimulated macrophages, and includes NO, TNF-α and IL-1β through interference in both nuclear factor kappaB (NF-κB) and mitogen activator protein kinase (MAPK) signaling cascade in IFN-γ/LPS-stimulated macrophages. Moreover, BDMC33 also interrupted LPS signaling through inhibiting the surface expression of CD-14 accessory molecules. In addition, the inhibitory action of BDMC33 not only restricted the macrophages cell (RAW264.7), but also inhibited the secretion of NO and TNF-α in IFN-γ/LPS-challenged microglial cells (BV-2). The experimental data suggests the inflammatory action of BDMC33 on activated macrophage-like cellular systems, which could be used as a future therapeutic agent in the management of chronic inflammatory diseases. PMID:22489138

  5. Supramolecular organizing centers (SMOCs) as signaling machines in innate immune activation.

    PubMed

    Qiao, Qi; Wu, Hao

    2015-11-01

    Innate immunity offers the first line of defense against infections and other types of danger such as tumorigenesis. Its discovery provides tremendous therapeutic opportunities for numerous human diseases. Delving into the structural basis of signal transduction by innate immune receptors, our lab has recently helped to establish the new paradigm in which innate immune receptors transduce ligand-binding signals through formation of higher-order assemblies containing intracellular adapters, signaling enzymes and their substrates. These large signalosome assemblies may be visible under light microscopy as punctate structures in the µm scale, connecting to the underlying molecular structures in the nm scale. They drive proximity-induced enzyme activation, and provide a mechanism for signaling amplification by nucleated polymerization. These supramolecular signaling complexes also open new questions on their cellular organization and mode of regulation, pose challenges to our methodology, and afford valuable implications in drug discovery against these medically important pathways.

  6. The Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway as a Discovery Target in Stroke.

    PubMed

    Sun, Jing; Nan, Guangxian

    2016-05-01

    Protein kinases are critical modulators of a variety of intracellular and extracellular signal transduction pathways, and abnormal phosphorylation events can contribute to disease progression in a variety of diseases. As a result, protein kinases have emerged as important new drug targets for small molecule therapeutics. The mitogen-activated protein kinase (MAPK) signaling pathway transmits signals from the cell membrane to the nucleus in response to a variety of different stimuli. Because this pathway controls a broad spectrum of cellular processes, including growth, inflammation, and stress responses, it is accepted as a therapeutic target for cancer and peripheral inflammatory disorders. There is also increasing evidence that MAPK is an important regulator of ischemic and hemorrhagic cerebral vascular disease, raising the possibility that it might be a drug discovery target for stroke. In this review, we discuss the MAPK signaling pathway in association with its activation in stroke-induced brain injury.

  7. Cellular Telephones Measure Activity and Lifespace in Community-Dwelling Adults: Proof of Principle

    PubMed Central

    Schenk, Ana Katrin; Witbrodt, Bradley C.; Hoarty, Carrie A.; Carlson, Richard H.; Goulding, Evan H.; Potter, Jane F.; Bonasera, Stephen J.

    2011-01-01

    OBJECTIVES To describe a system that uses off-the-shelf sensor and telecommunication technologies to continuously measure individual lifespace and activity levels in a novel way. DESIGN Proof of concept involving three field trials of 30, 30, and 21 days. SETTING Omaha, Nebraska, metropolitan and surrounding rural region. PARTICIPANTS Three participants (48-year-old man, 33-year-old woman, and 27-year-old male), none with any functional limitations. MEASUREMENTS Cellular telephones were used to detect in-home position and in-community location and to measure physical activity. Within the home, cellular telephones and Bluetooth transmitters (beacons) were used to locate participants at room-level resolution. Outside the home, the same cellular telephones and global positioning system (GPS) technology were used to locate participants at a community-level resolution. Physical activity was simultaneously measured using the cellular telephone accelerometer. RESULTS This approach had face validity to measure activity and lifespace. More importantly, this system could measure the spatial and temporal organization of these metrics. For example, an individual’s lifespace was automatically calculated across multiple time intervals. Behavioral time budgets showing how people allocate time to specific regions within the home were also automatically generated. CONCLUSION Mobile monitoring shows much promise as an easily deployed system to quantify activity and lifespace, important indicators of function, in community-dwelling adults. PMID:21288235

  8. Regulation of TGF-β signaling, exit from the cell cycle, and cellular migration through cullin cross-regulation

    PubMed Central

    Abbas, Tarek; Keaton, Mignon; Dutta, Anindya

    2013-01-01

    Deregulation of the cell cycle and genome instability are common features of cancer cells and various mechanisms exist to preserve the integrity of the genome and guard against cancer. The cullin 4-RING ubiquitin ligase (CRL4) with the substrate receptor Cdt2 (CRL4Cdt2) promotes cell cycle progression and prevents genome instability through ubiquitylation and degradation of Cdt1, p21, and Set8 during S phase of the cell cycle and following DNA damage. Two recently published studies report the ubiquitin-dependent degradation of Cdt2 via the cullin 1-RING ubiquitin ligase (CRL1) in association with the substrate specificity factor and tumor suppressor FBXO11 (CRL1FBXO11). The newly identified pathway restrains the activity of CRL4Cdt2 on p21 and Set8 and regulates cellular response to TGF-β, exit from the cell cycle and cellular migration. Here, we show that the CRL1FBXO11 also promotes the degradation of Cdt2 during an unperturbed cell cycle to promote efficient progression through S and G2/M phases of the cell cycle. We discuss how this new method of regulating the abundance of Cdt2 participates in various cellular activities. PMID:23892434

  9. Thermal stress and cellular signaling processes in hemocytes of native (Mytilus californianus) and invasive (M. galloprovincialis) mussels: cell cycle regulation and DNA repair.

    PubMed

    Yao, Cui-Luan; Somero, George N

    2013-06-01

    In a previous study using hemocytes from native and invasive congeners of Mytilus (Mytilus californianus and Mytilus galloprovincialis, respectively) we showed that DNA damage and cell signaling transduction processes related to the cellular stress response and apoptosis were induced by acute temperature stress. The present study extends this work by examining effects of acute heat- and cold stress on total hemocyte counts (THCs) and expression of key regulatory molecules involved in responding to stress: tumor suppressor factor (p53), cell cycle arrest activator (p21), and a DNA base excision repair enzyme (apurinic/apyrimidinic endonuclease (APE)). Hyperthermia (28 °C, 32 °C) led to significant decreases of THCs in both species. The extent of decrease in THC was temperature-, time-, and species-dependent; lower THC values were found in M. californianus, the more cold-adapted species. Western blot analyses of hemocyte extracts with antibodies specific for p53 protein, several site-specific phosphorylation states of p53, p21 protein, and APE indicated that heat- and cold (2 °C) stress induced a time-dependent activation of stress-related proteins in response to DNA damage; these stress-induced changes could govern cell cycle arrest or DNA damage repair. Our results show that the downstream regulatory response to temperature-induced cell damage may play an important role in deciding cellular fate following heat- and cold stress. Compared to M. californianus, the more warm-adapted M. galloprovincialis appears to have a higher temperature tolerance due to a lesser reduction in THC, faster signaling activation and transduction, and stronger DNA repair ability following heat stress.

  10. Cytochrome P450 17A1 inhibitor abiraterone attenuates cellular growth of prostate cancer cells independently from androgen receptor signaling by modulation of oncogenic and apoptotic pathways.

    PubMed

    Grossebrummel, Hannah; Peter, Tilmann; Mandelkow, Robert; Weiss, Martin; Muzzio, Damian; Zimmermann, Uwe; Walther, Reinhard; Jensen, Federico; Knabbe, Cornelius; Zygmunt, Marek; Burchardt, Martin; Stope, Matthias B

    2016-02-01

    Abiraterone provides significant survival advantages in prostate cancer (PC), however, the current understanding of the molecular mechanisms of abiraterone is still limited. Therefore, the abiraterone impact on androgen receptor (AR)-positive LNCaP and AR-negative PC-3 cells was assessed by cellular and molecular analyses. The present study demonstrated, that abiraterone treatment significantly decreased cell growth, AR expression, and AR activity of AR-positive LNCaP cells. Notably, AR-negative PC-3 cells exhibited comparable reductions in cellular proliferation, associated with DNA fragmentation and pro-apoptotic modulation of p21, caspase-3, survivin, and transforming growth factor β (TGFβ). Our observations suggest that the attenuation of AR signaling is not the only rationale to explain the abiraterone anticancer activity. Abiraterone efficacy may play a more global role in PC progression control than originally hypothesized. In this regard, abiraterone is not only a promising drug for treatment of AR-negative PC stages, even more, abiraterone may represent an alternative for treatment of other malignancies besides prostate cancer.

  11. Micro-simulation of vehicle conflicts involving right-turn vehicles at signalized intersections based on cellular automata.

    PubMed

    Chai, C; Wong, Y D

    2014-02-01

    At intersection, vehicles coming from different directions conflict with each other. Improper geometric design and signal settings at signalized intersection will increase occurrence of conflicts between road users and results in a reduction of the safety level. This study established a cellular automata (CA) model to simulate vehicular interactions involving right-turn vehicles (as similar to left-turn vehicles in US). Through various simulation scenarios for four case cross-intersections, the relationships between conflict occurrences involving right-turn vehicles with traffic volume and right-turn movement control strategies are analyzed. Impacts of traffic volume, permissive right-turn compared to red-amber-green (RAG) arrow, shared straight-through and right-turn lane as well as signal setting are estimated from simulation results. The simulation model is found to be able to provide reasonable assessment of conflicts through comparison of existed simulation approach and observed accidents. Through the proposed approach, prediction models for occurrences and severity of vehicle conflicts can be developed for various geometric layouts and traffic control strategies.

  12. Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa.

    PubMed

    Moradali, M Fata; Ghods, Shirin; Rehm, Bernd H A

    2017-03-03

    The exopolysaccharide, alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa represents a survival advantage by contributing to formation of characteristic biofilms during infection. Membrane anchored proteins Alg8 (catalytic subunit) and Alg44 (co-polymerase) constitute the alginate polymerase which is being activated by the second messenger molecule c-di-GMP, but the mechanism of activation remains elusive. To shed light on the c-di-GMP mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops including H323 (loop A), T457 and E460 (loop B) surrounding the catalytic site in the predicted model. Activity of respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 co-polymerase constituted a stable dimer, with its periplasmic domains required for protein localization, alginate polymerization and modification. Superfolder GFP fusions of Alg8 and Alg44 showed a non-uniform, punctuate and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44 including their subcellular localization and distribution.IMPORTANCE The exopolysaccharide, alginate, is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principle

  13. Secretory proteins characteristic of environmental changes in cellular signal transduction: Expression in oral fluid

    NASA Astrophysics Data System (ADS)

    Mednieks, M. I.; Burke, J. C.; Sivakumar, T. P.; Hand, A. R.; Grindeland, R. E.

    2000-01-01

    Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to those in gingival crevicular fluid (GCF), which reflects the protein composition of serum. The ratio of cARP in saliva to that in GCF can be used as a measure of basal compared to hyper-or hypo-gravity values. An ultimate goal is to test hyper and zero G responses in human saliva to determine if cARP is a suitable index of acute and chronic stress. A miniaturized test kit for saliva collection has been designed. Samples can be collected and stored till analyses are carried out that will distinguish the effects of increased gravity from those of one and zero G. Such tests can serve as an individualized monitoring system for physiologic responses either in space or on earth. .

  14. The Impact of Silica Nanoparticle Design on Cellular Toxicity and Hemolytic Activity

    PubMed Central

    Yu, Tian; Malugin, Alexander; Ghandehari, Hamidreza

    2011-01-01

    Understanding the toxicity of silica nanoparticles (SiO2) on the cellular level is crucial for rational design of these nanomaterials for biomedical applications. Herein, we explore the impacts of geometry, porosity and surface charge of SiO2 on cellular toxicity and hemolytic activity. Nonporous Stöber silica nanospheres (115 nm diameter), mesoporous silica nanospheres (120 nm diameter, aspect ratio 1), mesoporous silica nanorods with aspect ratio of 2, 4 and 8 (width by length 80 × 200 nm, 150 × 600 nm, 130 × 1000 nm) as well as their cationic counterparts were evaluated on macrophages, lung carcinoma cells, and human erythrocytes. It was shown that the toxicity of SiO2 is cell-type dependent and that surface charge and pore size govern cellular toxicity. Using inductively coupled plasma mass spectrometry, the cellular association of SiO2 was quantitated with the association amount increasing in the following order: mesoporous SiO2 (aspect ratio 1, 2, 4, 8) < amine-modified mesoporous SiO2 (aspect ratio 1, 2, 4, 8) < amine-modified nonporous Stöber SiO2 < nonporous Stöber SiO2. Geometry did not seem to influence the extent of SiO2 association at early or extended time points. The level of cellular association of the nanoparticles was directly linked to the extent of plasma membrane damage, suggesting a biological cause-and-effect relationship. Hemolysis assay showed that the hemolytic activity was porosity- and geometry- dependent for bare SiO2 and surface charge-dependent for amine-modified SiO2. A good correlation between hemolytic activity and cellular association was found on a similar dosage basis. These results can provide useful guidelines for the rational design of SiO2 in nanomedicine. PMID:21630682

  15. The Effect of Gap Junctional Coupling on the Spatiotemporal Patterns of Ca2+ Signals and the Harmonization of Ca2+-Related Cellular Responses

    PubMed Central

    Dougoud, Michaël; Mazza, Christian; Schwaller, Beat; Pecze, László

    2016-01-01

    Calcium ions (Ca2+) are important mediators of a great variety of cellular activities e.g. in response to an agonist activation of a receptor. The magnitude of a cellular response is often encoded by frequency modulation of Ca2+ oscillations and correlated with the stimulation intensity. The stimulation intensity highly depends on the sensitivity of a cell to a certain agonist. In some cases, it is essential that neighboring cells produce a similar and synchronized response to an agonist despite their different sensitivity. In order to decipher the presumed function of Ca2+ waves spreading among connecting cells, a mathematical model was developed. This model allows to numerically modifying the connectivity probability between neighboring cells, the permeability of gap junctions and the individual sensitivity of cells to an agonist. Here, we show numerically that strong gap junctional coupling between neighbors ensures an equilibrated response to agonist stimulation via formation of Ca2+ phase waves, i.e. a less sensitive neighbor will produce the same or similar Ca2+ signal as its highly sensitive neighbor. The most sensitive cells within an ensemble are the wave initiator cells. The Ca2+ wave in the cytoplasm is driven by a sensitization wave front in the endoplasmic reticulum. The wave velocity is proportional to the cellular sensitivity and to the strength of the coupling. The waves can form different patterns including circular rings and spirals. The observed pattern depends on the strength of noise, gap junctional permeability and the connectivity probability between neighboring cells. Our simulations reveal that one highly sensitive region gradually takes the lead within the entire noisy system by generating directed circular phase waves originating from this region. PMID:28027293

  16. Regulation of HTLV-1 tax stability, cellular trafficking and NF-κB activation by the ubiquitin-proteasome pathway.

    PubMed

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-10-23

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.

  17. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

    PubMed

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

  18. The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways

    PubMed Central

    Youns, Mаhmoud; Abdel Halim Hegazy, Wael

    2017-01-01

    Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes. PMID:28052097

  19. Exponential stability of delayed and impulsive cellular neural networks with partially Lipschitz continuous activation functions.

    PubMed

    Song, Xueli; Xin, Xing; Huang, Wenpo

    2012-05-01

    The paper discusses exponential stability of distributed delayed and impulsive cellular neural networks with partially Lipschitz continuous activation functions. By relative nonlinear measure method, some novel criteria are obtained for the uniqueness and exponential stability of the equilibrium point. Our method abandons usual assumptions on global Lipschitz continuity, boundedness and monotonicity of activation functions. Our results are generalization and improvement of some existing ones. Finally, two examples and their simulations are presented to illustrate the correctness of our analysis.

  20. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    SciTech Connect

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  1. A smart fluorescence nanoprobe for the detection of cellular alkaline phosphatase activity and early osteogenic differentiation.

    PubMed

    Cao, Feng-Yi; Fan, Jin-Xuan; Long, Yue; Zeng, Xuan; Zhang, Xian-Zheng

    2016-07-01

    In the past decades, biomaterials were designed to induce stem cell toward osteogenic differentiation. However, conventional methods for evaluation osteogenic differentiation all required a process of cell fixation or lysis, which induce waste of a large number of cells. In this study, a fluorescence nanoprobe was synthesized by combining phosphorylated fluoresceinamine isomer I (FLA) on the surface of mesoporous silica-coated superparamagnetic iron oxide (Fe3O4@mSiO2) nanoparticles. In the presence of alkaline phosphatase (ALP), the phosphorylated FLA on the nanoprobe would be hydrolyzed, resulting in a fluorescence recovery of FLA. During early osteogenic differentiation, a high-level expression of cellular ALP was induced, which accelerated the hydrolysis of phosphorylated FLA, resulting in an enhancement of cellular fluorescence intensity. This fluorescence nanoprobe provides us a rapid and non-toxic method for the detection of cellular ALP activity and early osteogenic differentiation.

  2. The inactive-active phase transition in the noisy additive (exclusive-or) probabilistic cellular automaton

    NASA Astrophysics Data System (ADS)

    Mendonça, J. Ricardo G.

    2016-07-01

    We investigate the inactive-active phase transition in an array of additive (exclusive-or) cellular automata (CA) under noise. The model is closely related with the Domany-Kinzel (DK) probabilistic cellular automaton (PCA), for which there are rigorous as well as numerical estimates on the transition probabilities. Here, we characterize the critical behavior of the noisy additive cellular automaton by mean field analysis and finite-size scaling and show that its phase transition belongs to the directed percolation universality class of critical behavior. As a by-product of our analysis, we argue that the critical behavior of the noisy elementary CA 90 and 102 (in Wolfram’s enumeration scheme) must be the same. We also perform an empirical investigation of the mean field equations to assess their quality and find that away from the critical point (but not necessarily very far away) the mean field approximations provide a reasonably good description of the dynamics of the PCA.

  3. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway.

  4. Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

    PubMed Central

    Ng, Mei Ying; Wang, Mei; Casey, Patrick J.; Gan, Yunn-Hwen; Hagen, Thilo

    2017-01-01

    Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways. PMID:28166272

  5. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    PubMed Central

    Andrews, Joel F.; Sykora, Landon J.; Barik-Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-01-01

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington’s, Parkinson’s diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S). PMID:22504047

  6. At the interface of antioxidant signalling and cellular function: Key polyphenol effects

    PubMed Central

    Kerimi, Asimina

    2016-01-01

    The hypothesis that dietary (poly)phenols promote well‐being by improving chronic disease‐risk biomarkers, such as endothelial dysfunction, chronic inflammation and plasma uric acid, is the subject of intense current research, involving human interventions studies, animal models and in vitro mechanistic work. The original claim that benefits were due to the direct antioxidant properties of (poly)phenols has been mostly superseded by detailed mechanistic studies on specific molecular targets. Nevertheless, many proposed mechanisms in vivo and in vitro are due to modulation of oxidative processes, often involving binding to specific proteins and effects on cell signalling. We review the molecular mechanisms for 3 actions of (poly)phenols on oxidative processes where there is evidence in vivo from human intervention or animal studies. (1) Effects of (poly) phenols on pathways of chronic inflammation leading to prevention of some of the damaging effects associated with the metabolic syndrome. (2) Interaction of (poly)phenols with endothelial cells and smooth muscle cells, leading to effects on blood pressure and endothelial dysfunction, and consequent reduction in cardiovascular disease risk. (3) The inhibition of xanthine oxidoreductase leading to modulation of intracellular superoxide and plasma uric acid, a risk factor for developing type 2 diabetes. PMID:26887821

  7. Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling.

    PubMed

    Luu, Tony C; Bhattacharya, Pompeya; Chan, William K

    2008-09-22

    Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.

  8. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    SciTech Connect

    Andrews, Joel F.; Sykora, Landon J.; Barik Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna; Barik, Sailen; Shevde, Lalita A.; Samant, Rajeev S.

    2012-06-10

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

  9. Malignant peripheral nerve sheath tumour (MPNST): the clinical implications of cellular signalling pathways.

    PubMed

    Katz, Daniela; Lazar, Alexander; Lev, Dina

    2009-10-19

    Malignant peripheral nerve sheath tumour (MPNST) is a rare malignancy accounting for 3-10% of all soft tissue sarcomas. Most MPNSTs arise in association with peripheral nerves or deep neurofibromas and may originate from neural crest cells, although the specific cell of origin is uncertain. Approximately half of MPNSTs occur in the setting of neurofibromatosis type 1 (NF1), an autosomal dominant disorder with an incidence of approximately one in 3500 persons; the remainder of MPNSTs develop sporadically. In addition to a variety of clinical manifestations, approximately 8-13% of NF1 patients develop MPNSTs, which are the leading cause of NF1-related mortality. Surgical resection is the mainstay of MPNST clinical management. However, because of invasive growth, propensity to metastasise, and limited sensitivity to chemotherapy and radiation, MPNST has a guarded to poor prognosis. Five-year survival rates of only 20-50% indicate an urgent need for improved therapeutic approaches. Recent work in this field has identified several altered intracellular signal transduction cascades and deregulated tyrosine kinase receptors, posing the possibility of personalised, targeted therapeutics. However, expanded knowledge of MPNST molecular pathobiology will be needed to meaningfully apply such approaches for the benefit of afflicted patients.

  10. Human ECG signal parameters estimation during controlled physical activity

    NASA Astrophysics Data System (ADS)

    Maciejewski, Marcin; Surtel, Wojciech; Dzida, Grzegorz

    2015-09-01

    ECG signal parameters are commonly used indicators of human health condition. In most cases the patient should remain stationary during the examination to decrease the influence of muscle artifacts. During physical activity, the noise level increases significantly. The ECG signals were acquired during controlled physical activity on a stationary bicycle and during rest. Afterwards, the signals were processed using a method based on Pan-Tompkins algorithms to estimate their parameters and to test the method.

  11. Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

    PubMed

    Zhang, Tianyi; Liao, Yang; Hsu, Fu-Ning; Zhang, Robin; Searle, Jennifer S; Pei, Xun; Li, Xuan; Ryoo, Hyung Don; Ji, Jun-Yuan; Du, Wei

    2014-05-01

    Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

  12. MECHANISTIC PATHWAYS AND BIOLOGICAL ROLES FOR RECEPTOR-INDEPENDENT ACTIVATORS OF G-PROTEIN SIGNALING

    PubMed Central

    Blumer, Joe B.; Smrcka, Alan V.; Lanier, S.M.

    2007-01-01

    Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents, plays an important role in adaptive processes of organs, and aberrant processing of signals through these transducing systems is a component of various disease states. In addition to GPCR-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Gαβγ heterotrimer or Gα and Gαβγ subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Gα and Gαβγ) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Gαβγ. Such regulatory accessory proteins include the family of RGS proteins that accelerate the GTPase activity of Gα and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor independent activators of G-protein signaling or AGS proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways and provide a platform for diverse functions of both the heterotrimeric Gαβγ and the individual Gα and Gαβγ subunits. PMID:17240454

  13. AMP-activated protein kinase regulates L-arginine mediated cellular responses

    PubMed Central

    2013-01-01

    Background Our prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown. Method Human umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye. Results Short-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased

  14. Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

    PubMed

    Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

    2015-06-01

    Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

  15. Low probability of intercept-based adaptive radar waveform optimization in signal-dependent clutter for joint radar and cellular communication systems

    NASA Astrophysics Data System (ADS)

    Shi, Chenguang; Salous, Sana; Wang, Fei; Zhou, Jianjiang

    2016-12-01

    In this paper, we investigate the problem of low probability of intercept (LPI)-based adaptive radar waveform optimization in signal-dependent clutter for joint radar and cellular communication systems, where the radar system optimizes the transmitted waveform such that the interference caused to the cellular communication systems is strictly controlled. Assuming that the precise knowledge of the target spectra, the power spectral densities (PSDs) of signal-dependent clutters, the propagation losses of corresponding channels and the communication signals is known by the radar, three different LPI based criteria for radar waveform optimization are proposed to minimize the total transmitted power of the radar system by optimizing the multicarrier radar waveform with a predefined signal-to-interference-plus-noise ratio (SINR) constraint and a minimum required capacity for the cellular communication systems. These criteria differ in the way the communication signals scattered off the target are considered in the radar waveform design: (1) as useful energy, (2) as interference or (3) ignored altogether. The resulting problems are solved analytically and their solutions represent the optimum power allocation for each subcarrier in the multicarrier radar waveform. We show with numerical results that the LPI performance of the radar system can be significantly improved by exploiting the scattered echoes off the target due to cellular communication signals received at the radar receiver.

  16. Low probability of intercept-based adaptive radar waveform optimization in signal-dependent clutter for joint radar and cellular communication systems.

    PubMed

    Shi, Chenguang; Salous, Sana; Wang, Fei; Zhou, Jianjiang

    2016-01-01

    In this paper, we investigate the problem of low probability of intercept (LPI)-based adaptive radar waveform optimization in signal-dependent clutter for joint radar and cellular communication systems, where the radar system optimizes the transmitted waveform such that the interference caused to the cellular communication systems is strictly controlled. Assuming that the precise knowledge of the target spectra, the power spectral densities (PSDs) of signal-dependent clutters, the propagation losses of corresponding channels and the communication signals is known by the radar, three different LPI based criteria for radar waveform optimization are proposed to minimize the total transmitted power of the radar system by optimizing the multicarrier radar waveform with a predefined signal-to-interference-plus-noise ratio (SINR) constraint and a minimum required capacity for the cellular communication systems. These criteria differ in the way the communication signals scattered off the target are considered in the radar waveform design: (1) as useful energy, (2) as interference or (3) ignored altogether. The resulting problems are solved analytically and their solutions represent the optimum power allocation for each subcarrier in the multicarrier radar waveform. We show with numerical results that the LPI performance of the radar system can be significantly improved by exploiting the scattered echoes off the target due to cellular communication signals received at the radar receiver.

  17. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga Volvox carteri and their potential role in cellular differentiation.

    PubMed

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in multicellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation.

  18. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga volvox carteri and their potential role in cellular differentiation

    PubMed Central

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in muticellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation. PMID:25874475

  19. Activation of DNA damage response signaling by condensed chromatin.

    PubMed

    Burgess, Rebecca C; Burman, Bharat; Kruhlak, Michael J; Misteli, Tom

    2014-12-11

    The DNA damage response (DDR) occurs in the context of chromatin, and architectural features of chromatin have been implicated in DNA damage signaling and repair. Whereas a role of chromatin decondensation in the DDR is well established, we show here that chromatin condensation is integral to DDR signaling. We find that, in response to DNA damage chromatin regions transiently expand before undergoing extensive compaction. Using a protein-chromatin-tethering system to create defined chromatin domains, we show that interference with chromatin condensation results in failure to fully activate DDR. Conversely, forced induction of local chromatin condensation promotes ataxia telangiectasia mutated (ATM)- and ATR-dependent activation of upstream DDR signaling in a break-independent manner. Whereas persistent chromatin compaction enhanced upstream DDR signaling from irradiation-induced breaks, it reduced recovery and survival after damage. Our results demonstrate that chromatin condensation is sufficient for activation of DDR signaling and is an integral part of physiological DDR signaling.

  20. Activity Dependent Signal Transduction in Skeletal Muscle

    NASA Technical Reports Server (NTRS)

    Hamilton, Susan L.

    1999-01-01

    The overall goals of this project are: 1) to define the initial signal transduction events whereby the removal of gravitational load from antigravity muscles, such as the soleus, triggers muscle atrophy, and 2) to develop countermeasures to prevent this from happening. Our rationale for this approach is that, if countermeasures can be developed to regulate these early events, we could avoid having to deal with the multiple cascades of events that occur downstream from the initial event. One of our major findings is that hind limb suspension causes an early and sustained increase in intracellular Ca(2+) concentration ([Ca (2+)](sub i)). In most cells the consequences of changes in ([Ca (2+)](sub i))depend on the amplitude, frequency and duration of the Ca(2+) signal and on other factors in the intracellular environment. We propose that muscle remodeling in microgravity represents a change in the balance among several CA(2+) regulated signal transduction pathways, in particular those involving the transcription factors NFAT and NFkB and the pro-apoptotic protein BAD. Other Ca(2+) sensitive pathways involving PKC, ras, rac, and CaM kinase II may also contribute to muscle remodeling.

  1. Crosstalk and Signaling Switches in Mitogen-Activated Protein Kinase Cascades

    PubMed Central

    Fey, Dirk; Croucher, David R.; Kolch, Walter; Kholodenko, Boris N.

    2012-01-01

    Mitogen-activated protein kinase (MAPK) cascades control cell fate decisions, such as proliferation, differentiation, and apoptosis by integrating and processing intra- and extracellular cues. However, similar MAPK kinetic profiles can be associated with opposing cellular decisions depending on cell type, signal strength, and dynamics. This implies that signaling by each individual MAPK cascade has to be considered in the context of the entire MAPK network. Here, we develop a dynamic model of feedback and crosstalk for the three major MAPK cascades; extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), c-Jun N-terminal kinase (JNK), and also include input from protein kinase B (AKT) signaling. Focusing on the bistable activation characteristics of the JNK pathway, this model explains how pathway crosstalk harmonizes different MAPK responses resulting in pivotal cell fate decisions. We show that JNK can switch from a transient to sustained activity due to multiple positive feedback loops. Once activated, positive feedback locks JNK in a highly active state and promotes cell death. The switch is modulated by the ERK, p38, and AKT pathways. ERK activation enhances the dual specificity phosphatase (DUSP) mediated dephosphorylation of JNK and shifts the threshold of the apoptotic switch to higher inputs. Activation of p38 restores the threshold by inhibiting ERK activity via the PP1 or PP2A phosphatases. Finally, AKT activation inhibits the JNK positive feedback, thus abrogating the apoptotic switch and allowing only proliferative signaling. Our model facilitates understanding of how cancerous deregulations disturb MAPK signal processing and provides explanations for certain drug resistances. We highlight a critical role of DUSP1 and DUSP2 expression patterns in facilitating the switching of JNK activity and show how oncogene induced ERK hyperactivity prevents the normal apoptotic switch explaining the failure of certain drugs to

  2. K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

    PubMed Central

    Katsnelson, Michael A.; Rucker, L. Graham; Russo, Hana M.; Dubyak, George R.

    2015-01-01

    Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal. PMID:25762778

  3. Fractionation, enzyme inhibitory and cellular antioxidant activity of bioactives from purple sweet potato (Ipomoea batatas).

    PubMed

    Esatbeyoglu, Tuba; Rodríguez-Werner, Miriam; Schlösser, Anke; Winterhalter, Peter; Rimbach, Gerald

    2017-04-15

    Sweet potato (Ipomoea batatas L.) is mainly cultivated in Asia. The deep purple color of purple sweet potato (PSP) is due to the high content of acylated anthocyanins. In the present study, PSP-derived polyphenols were identified using HPLC-PDA and HPLC-ESI-MS(n) analyses. After concentration of the polyphenols from PSP, preparative separation into two fractions, designated anthocyanins (AF) and copigments (CF), was carried out using adsorptive membrane chromatography. In enzyme inhibitory assays, all PSP samples inhibited the enzymes α-amylase, α-glucosidase and xanthine oxidase. Additionally, the cell signaling cellular antioxidant properties of the PSP extracts were investigated in cultured cells. PSP induced the transcription factor Nrf2, which regulates the expression of genes encoding heme oxygenase 1 (Hmox1), glutamate-cysteine ligase catalytic subunit (Gclc) and paraoxonase 1 (PON1). Furthermore, PSP enhanced cellular glutathione concentrations and decreased lipid peroxidation in cultured hepatocytes. Overall, these results suggest that PSP extracts exhibit enzyme inhibitory and cellular antioxidant properties, especially PSP CF.

  4. LED-activated pheophorbide a induces cellular destruction of colon cancer cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.; Liu, L.; Xia, X. S.

    2010-07-01

    Pheophorbide a (Pa) from Chinese herbal medicine Scutellaria Barbata and Silkworm Excreta shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that LED-activated Pa induced the cellular destruction of colon cancer HT-29 cells. The results showed that Pa resulted in a drug-dose dependent photocytotoxicity in the HT-29 cells, meaning the photocytotoxicity of Pa depends on the drug concentration (0 - 2 μM). We further investigated the apoptosis of the HT-29 cells 18 hours after photosensitization of Pa using a confocal laser scanning microscopy with Hoechst 33258 staining. These data demonstrated that LED-activated Pa could significantly induce the cellular destruction of the HT-29 cells.

  5. Light-activated hypericin induces cellular destruction of nasopharyngeal carcinoma cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.

    2010-01-01

    Hypericin from Hypericum perforatum plants shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that light-activated hypericin induced the cellular destruction of nasopharyngeal carcinoma cells. The result showed that hypericin resulted in a drug- and light-dose dependent cytotoxicity in the CNE-2 cells, meaning the photocytotoxicity of hypericin depends on both of the drug concentration (0 - 2.5 μM) and light-doses (1 - 8 J/cm2). We further investigated the apoptosis of the CNE-2 cells 8 hours after photosensitization of hypericin using fluorescence microscopy with Hoechst 33258 staining. Flow cytometry with annexin V-FITC and PI staining was used to analyze early and late apoptosis. These data demonstrated that light-activated hypericin could significantly lead to the cellular destruction of the CNE-2 cells and induce early apoptosis as a prominent mode of cell death.

  6. Embryo as an active granular fluid: stress-coordinated cellular constriction chains

    NASA Astrophysics Data System (ADS)

    Holcomb, Michael; Gao, Guo-Jie; Thomas, Jeffrey; Blawzdziewicz, Jerzy

    2016-11-01

    Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity.

  7. Preliminary cellular-automata forecast of permit activity from 1998 to 2010, Idaho and Western Montana

    USGS Publications Warehouse

    Raines, G.L.; Zientek, M.L.; Causey, J.D.; Boleneus, D.E.

    2002-01-01

    For public land management in Idaho and western Montana, the U.S. Forest Service (USFS) has requested that the U.S. Geological Survey (USGS) predict where mineral-related activity will occur in the next decade. Cellular automata provide an approach to simulation of this human activity. Cellular automata (CA) are defined by an array of cells, which evolve by a simple transition rule, the automaton. Based on exploration trends, we assume that future exploration will focus in areas of past exploration. Spatial-temporal information about mineral-related activity, that is permits issued by USFS and Bureau of Land Management (BLM) in the last decade, and spatial information about undiscovered resources, provide a basis to calibrate a CA. The CA implemented is a modified annealed voting rule that simulates mineral-related activity with spatial and temporal resolution of 1 mi2 and 1 year based on activity from 1989 to 1998. For this CA, the state of the economy and exploration technology is assumed constant for the next decade. The calibrated CA reproduces the 1989-1998-permit activity with an agreement of 94%, which increases to 98% within one year. Analysis of the confusion matrix and kappa correlation statistics indicates that the CA underestimates high activity and overestimates low activity. Spatially, the major differences between the actual and calculated activity are that the calculated activity occurs in a slightly larger number of small patches and is slightly more uneven than the actual activity. Using the calibrated CA in a Monte Carlo simulation projecting from 1998 to 2010, an estimate of the probability of mineral activity shows high levels of activity in Boise, Caribou, Elmore, Lincoln, and western Valley counties in Idaho and Beaverhead, Madison, and Stillwater counties in Montana, and generally low activity elsewhere. ?? 2002 International Association for Mathematical Geology.

  8. Aldosterone regulates cellular turnover and mitogen-activated protein kinase family expression in the neonatal rat kidney.

    PubMed

    Yim, Hyung Eun; Yoo, Kee Hwan; Bae, In Sun; Jang, Gi Young; Hong, Young Sook; Lee, Joo Won

    2009-06-01

    Growing evidence indicates that aldosterone is a potent mitogenic signal regulating genes involved in antiapoptosis, cell proliferation and growth. We investigated the role of endogenous aldosterone in renal development, cell proliferation and apoptosis, and mitogen-activated protein kinase (MAPK) family expression. Newborn rats were treated with either spironolactone (200 mg/kg/d) in olive oil or only olive oil for 7 days. TUNEL assay and proliferating cell nuclear antigen (PCNA) stain were performed on kidney sections. Immunoblots, immunohistochemical (IHC) stain, and reverse transcriptase-PCR for MAPKs were performed. PCNA-positive proliferating cells decreased and apoptotic cells increased significantly with spironolactone (P < 0.05). In the spironolactone-treated group, c-jun N-terminal kinase (JNK)-2 expression increased, whereas extracellular signal regulated kinase (ERK)-2 and p38 expressions decreased in immunoblots (P < 0.05) and IHC stain. ERK-2 and p38 mRNA expressions increased in the spironolactone-treated group (P < 0.05). This study demonstrates that aldosterone blockade in the developing kidney decreases cellular proliferation, increases apoptosis, and modulates the expressions of JNK-2, ERK-2, and p38. Aldosterone possibly participates in renal development and MAPK family may serve as, in part, the signaling intermediate through the mineralocorticoid receptor (MR) in the developing kidney. J. Cell. Physiol. 219: 724-733, 2009. (c) 2009 Wiley-Liss, Inc.

  9. Potential Function of Exogenous Vimentin on the Activation of Wnt Signaling Pathway in Cancer Cells

    PubMed Central

    Satelli, Arun; Hu, Jiemiao; Xia, Xueqing; Li, Shulin

    2016-01-01

    Cancer cell signaling, growth, morphology, proliferation and tumorigenic potential are largely depending on the signaling molecules present naturally in the tumor microenvironment and the identification of key molecules that drive the tumor progression is critical for the development of new modalities for the prevention of tumor progression. High concentrations of vimentin in the blood of cancer patients have been reported, however the function of blood circulating vimentin remains unknown. Here, we investigated the functional role of exogenously supplemented vimentin on colon cancer cells and examined the Wnt Signaling activation and cancer cell invasion. Vimentin when supplemented to the cancer cells remained bound to the surface of the cancer cells. Furthermore, bound vimentin activates Wnt signaling pathway as detectable by increased β-catenin accumulation in the nucleus with concomitant activation of β-catenin-dependent transcription of Wnt signaling downstream targets. Functionally, there was an increase in the rate of cellular invasion in these cancer cells upon binding with vimentin. Our results thus suggest that free vimentin in the tumor microenvironment acts as a positive regulator of the β-catenin signaling pathway, thus providing a basis for cancer invasive properties. PMID:27698922

  10. Embryo as an active granular fluid: stress-coordinated cellular constriction chains

    NASA Astrophysics Data System (ADS)

    Gao, Guo-Jie Jason; Holcomb, Michael C.; Thomas, Jeffrey H.; Blawzdziewicz, Jerzy

    2016-10-01

    Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. However, systematic methods of studying how mechanical stress and feedback help to harmonize cellular activities within a tissue have yet to be developed. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity. Our particle-based AGF model will be useful in analyzing mechanical feedback effects in a wide variety of morphogenesis and organogenesis phenomena.

  11. SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity.

    PubMed

    Wei, Wei-Yen; Li, Hui-Chun; Chen, Chiung-Yao; Yang, Chee-Hing; Lee, Shen-Kao; Wang, Chia-Wen; Ma, Hsin-Chieh; Juang, Yue-Li; Lo, Shih-Yen

    2012-04-01

    The pathogenesis of SARS-CoV remains largely unknown. To study the function of the SARS-CoV nucleocapsid protein, we have conducted a yeast two-hybrid screening experiment to identify cellular proteins that may interact with the SARS-CoV nucleocapsid protein. Pyruvate kinase (liver) was found to interact with SARS-CoV nucleocapsid protein in this experiment. The binding domains of these two proteins were also determined using the yeast two-hybrid system. The physical interaction between the SARS-CoV nucleocapsid and cellular pyruvate kinase (liver) proteins was further confirmed by GST pull-down assay, co-immunoprecipitation assay and confocal microscopy. Cellular pyruvate kinase activity in hepatoma cells was repressed by SARS-CoV nucleocapsid protein in either transiently transfected or stably transfected cells. PK deficiency in red blood cells is known to result in human hereditary non-spherocytic hemolytic anemia. It is reasonable to assume that an inhibition of PKL activity due to interaction with SARS-CoV N protein is likely to cause the death of the hepatocytes, which results in the elevation of serum alanine aminotransferase and liver dysfunction noted in most SARS patients. Thus, our results suggest that SARS-CoV could reduce pyruvate kinase activity via its nucleocapsid protein, and this may in turn cause disease.

  12. Obatoclax, saliphenylhalamide and gemcitabine inhibit Zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism.

    PubMed

    Kuivanen, Suvi; Bespalov, Maxim M; Nandania, Jatin; Ianevski, Aleksandr; Velagapudi, Vidya; De Brabander, Jef K; Kainov, Denis E; Vapalahti, Olli

    2017-03-01

    An epidemic of Zika virus (ZIKV) infection associated with congenital abnormalities such as microcephaly, is ongoing in the Americas and the Pacific. Currently there are no approved therapies to treat this emerging viral disease. Here, we tested three cell-directed broad-spectrum antiviral compounds against ZIKV replication using human retinal pigment epithelial (RPE) cells and a low-passage ZIKV strain isolated from fetal brain. We found that obatoclax, SaliPhe, and gemcitabine inhibited ZIKV infections at noncytotoxic concentrations. Moreover, all three compounds prevented production of viral RNA and proteins as well as activation of cellular caspase 8, 3 and 7. However, these compounds differentially affected ZIKV-mediated transcription, translation and posttranslational modifications of cellular factors as well as metabolic pathways indicating that these agents possess different mechanisms of action. Interestingly, combination of obatoclax and SaliPhe at nanomolar concentrations had a synergistic effect against ZIKV infection. Thus, our results provided the foundation for development of broad-spectrum cell-directed antivirals or their combinations for treatment of ZIKV and other emerging viral diseases.

  13. Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

    PubMed

    Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A

    2016-12-24

    Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α2 -Macroglobulin (α2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α2 M (α2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 9999: 1-9, 2017. © 2016 Wiley Periodicals, Inc.

  14. Hyaluronan and dextran modified tubes resist cellular activation with blood contact.

    PubMed

    Eckmann, David M; Tsai, Irene Y; Tomczyk, Nancy; Weisel, John W; Composto, Russell J

    2013-08-01

    This study was undertaken to evaluate the effects of thin film hyaluronic acid and dextran surface coatings to blunt cellular activation in a laboratory model of extracorporeal blood circulation. The inner lumen surface of polyurethane (PU) and poly(vinyl) chloride (PVC) tubing was grafted with hyaluronic acid and dextran. Surfaces were characterized for the presence of the grafted layer using ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Persistence of the surface layer was maintained for up to 5 days of continuous exposure to shear flow using a Chandler loop apparatus. The Chandler loop method was used to study human whole blood activation activity. Whole blood aggregometry and flow cytometry measures of CD18, CD62L, CD62P, Annexin V and myeloperoxidase performed on blood samples exposed to the tubing for up to three hours were complemented by scanning electron microscopy (SEM) analysis of adherent cells and state of activation. In these studies commercial hospital products and uncoated PVC and PU tubes were used as controls. We found that hyaluranized PU and PVC conferred the greatest resistance to blood activation and that dextranization of the PU and PU tubing also provided significant diminution of the bioresponses measured. Based on our findings, we suggest that surface coating with hyaluronic acid or dextran acts as a potent shield from blood cellular activation during forms of extracorporeal circulation.

  15. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  16. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    PubMed Central

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  17. Safeners recruit multiple signalling pathways for the orchestrated induction of the cellular xenobiotic detoxification machinery in Arabidopsis.

    PubMed

    Behringer, Carina; Bartsch, Klaus; Schaller, Andreas

    2011-11-01

    Safeners enhance herbicide tolerance in crop plants but not in target weeds, thus improving herbicide selectivity. The safeners isoxadifen-ethyl and mefenpyr-diethyl protect cereal crops from sulfonyl urea herbicides in postemergence application. The two safeners were shown here to induce the cellular xenobiotic detoxification machinery in Arabidopsis thaliana when applied to leaves in a way mimicking field application. Gene expression profiling revealed the induction of 446 genes potentially involved in the detoxification process. Transgenic Arabidopsis plants expressing a reporter gene under control of a safener-responsive maize promoter were used as a model system to study the safener signalling pathway. Reporter gene analysis in the tga2/3/5/6, sid2-2 and npr1 mutants as compared with the wild-type background showed that safener inducibility required TGA transcription factors and salicylic acid (SA) in a NON-EXPRESSOR of PR-1 (NPR1)-independent pathway converging on two as-1 promoter elements. For the majority of the safener-responsive Arabidopsis genes, a similar dependence on TGA transcription factors and/or SA was shown by gene expression profiling in wild-type plants as compared with the tga2/3/5/6 and sid2-2 mutants. Thirty-eight percent of the genes, however, were induced by safeners in a TGA/SA-independent manner. These genes are likely to be controlled by WRKY transcription factors and cognate W-boxes in their promoters.

  18. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation

    PubMed Central

    Covarrubias, Anthony J; Aksoylar, Halil Ibrahim; Yu, Jiujiu; Snyder, Nathaniel W; Worth, Andrew J; Iyer, Shankar S; Wang, Jiawei; Ben-Sahra, Issam; Byles, Vanessa; Polynne-Stapornkul, Tiffany; Espinosa, Erika C; Lamming, Dudley; Manning, Brendan D; Zhang, Yijing; Blair, Ian A; Horng, Tiffany

    2016-01-01

    Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation. DOI: http://dx.doi.org/10.7554/eLife.11612.001 PMID:26894960

  19. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.

    PubMed

    Covarrubias, Anthony J; Aksoylar, Halil Ibrahim; Yu, Jiujiu; Snyder, Nathaniel W; Worth, Andrew J; Iyer, Shankar S; Wang, Jiawei; Ben-Sahra, Issam; Byles, Vanessa; Polynne-Stapornkul, Tiffany; Espinosa, Erika C; Lamming, Dudley; Manning, Brendan D; Zhang, Yijing; Blair, Ian A; Horng, Tiffany

    2016-02-19

    Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation.

  20. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

    NASA Technical Reports Server (NTRS)

    Baldwin, Kenneth M.; Haddad, Fadia

    2002-01-01

    The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

  1. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms.

    PubMed

    Baldwin, Kenneth M; Haddad, Fadia

    2002-11-01

    The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

  2. The collagen triple helix repeat containing 1 facilitates hepatitis B virus-associated hepatocellular carcinoma progression by regulating multiple cellular factors and signal cascades.

    PubMed

    Zhang, Rui; Cao, Yanhua; Bai, Lan; Zhu, Chengliang; Li, Rui; He, Hui; Liu, Yingle; Wu, Kailang; Liu, Fang; Wu, Jianguo

    2015-12-01

    Hepatitis B virus (HBV) infection is one of the major causes of acute and chronic liver diseases, fulminant hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HCC accounts for more than 85% of primary liver cancers and is the seventh most common cancer and the third leading cause of cancer-related deaths. However, the mechanism by which HBV induces HCC is largely unknown. Collagen triple helixes repeat containing 1 (CTHRC1) is a secreted protein and has characteristics of a circulating hormone with potentially broad implications for cell metabolism and physiology. CTHRC1 is associated with human cancers, but its effect on HCC is unknown. Here, we revealed that CTHRC1 expression is highly correlated with HCC progression in HBV-infected patients, and demonstrated that HBV stimulates CTHRC1 expression by activating nuclear factor-kappa B (NF-κB) and cAMP response element binding protein (CREB), through extracellular signal-regulated kinase/c-Jun N-terminal kinase (ERK/c-JNK) pathway. In addition, CTHRC1 activates hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) through regulating phosphoinosmde-3-kinase/protein kinase B/mammalian target of rapamycin (PI-3K/AKT/mTOR) pathway. More interestingly, CTHRC1 enhances colony formation, migration, and invasion of hepatoma cells by regulating p53 and stimulating matrix metalloproteinase-9 (MMP-9) expression. In addition, knock-down of CTHRC1 results in the repression of HBV-associated carcinogenesis in nude mice. Thus, we revealed a novel mechanism by which HBV facilitates HCC development through activating the oncoprotein CTHRC1, which in turn enhances HBV-related HCC progression by stimulates colony formation, migration, and invasion of hepatoma cells through regulating multiple cellular factors and signal cascades.

  3. Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides

    SciTech Connect

    Brodie, A.E.; Reed, D.J. )

    1990-01-01

    The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.

  4. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models

    PubMed Central

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q.

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  5. Activating Cell Death Ligand Signaling Through Proteasome Inhibition

    DTIC Science & Technology

    2009-05-01

    Activating Cell Death Ligand Signaling Through Proteasome Inhibition PRINCIPAL INVESTIGATOR: Steven R Schwarze...SUBTITLE Activating Cell Death Ligand Signaling Through 5a. CONTRACT NUMBER Proteasome Inhibition 5b. GRANT NUMBER W81XWH-08-1-0392 5c...proteasome inhibition can act as an anti-neoplastic agent in vivo by sensitizing cancer cells to cell death ligands in the tumor microenvironment

  6. SIGNALING TO THE P53 TUMOR SUPPRESSOR THROUGH PATHWAYS ACTIVATED BY GENOTOXIC AND NON-GENOTOXIC STRESSES.

    SciTech Connect

    ANDERSON,C.W.APPELLA,E.

    2002-07-01

    The p53 tumor suppressor is a tetrameric transcription factor that is post-translational modified at {approx}18 different sites by phosphorylation, acetylation, or sumoylation in response to various cellular stress conditions. Specific posttranslational modifications, or groups of modifications, that result from the activation of different stress-induced signaling pathways are thought to modulate p53 activity to regulate cell fate by inducing cell cycle arrest, apoptosis, or cellular senescence. Here we review the posttranslational modifications to p53 and the pathways that produce them in response to both genotoxic and non-genotoxic stresses.

  7. Hub-activated signal transmission in complex networks

    NASA Astrophysics Data System (ADS)

    Jahnke, Sven; Memmesheimer, Raoul-Martin; Timme, Marc

    2014-03-01

    A wide range of networked systems exhibit highly connected nodes (hubs) as prominent structural elements. The functional roles of hubs in the collective nonlinear dynamics of many such networks, however, are not well understood. Here, we propose that hubs in neural circuits may activate local signal transmission along sequences of specific subnetworks. Intriguingly, in contrast to previous suggestions of the functional roles of hubs, here, not the hubs themselves, but nonhub subnetworks transfer the signals. The core mechanism relies on hubs and nonhubs providing activating feedback to each other. It may, thus, induce the propagation of specific pulse and rate signals in neuronal and other communication networks.

  8. Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

    PubMed Central

    Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

    2013-01-01

    The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

  9. Signal peptides are allosteric activators of the protein translocase

    PubMed Central

    Gouridis, Giorgos; Karamanou, Spyridoula; Gelis, Ioannis; Kalodimos, Charalampos G.; Economou, Anastassios

    2010-01-01

    Extra-cytoplasmic polypeptides are usually synthesized as “preproteins” carrying aminoterminal, cleavable signal peptides1 and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA2,3. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA2,3. Preprotein targeting to SecA is thought to involve signal peptides4 and chaperones like SecB5,6. Here we reveal that signal peptides have a novel role beyond targeting: they are essential allosteric activators of the translocase. Upon docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, “triggering” that drives the translocase to a lower activation energy state; then “trapping” that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus and, finally, “secretion” during which trapped mature domains undergo multiple turnovers of translocation in segments7. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases. PMID:19924216

  10. PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes

    PubMed Central

    Anta, B; Pérez-Rodríguez, A; Castro, J; García- Domínguez, C A; Ibiza, S; Martínez, N; Durá, L M; Hernández, S; Gragera, T; Peña-Jiménez, D; Yunta, M; Zarich, N; Crespo, P; Serrador, J M; Santos, E; Muñoz, A; Oliva, J L; Rojas-Cabañeros, J M

    2016-01-01

    The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment. PMID:27468687

  11. PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes.

    PubMed

    Anta, B; Pérez-Rodríguez, A; Castro, J; García-Domínguez, C A; Ibiza, S; Martínez, N; Durá, L M; Hernández, S; Gragera, T; Peña-Jiménez, D; Yunta, M; Zarich, N; Crespo, P; Serrador, J M; Santos, E; Muñoz, A; Oliva, J L; Rojas-Cabañeros, J M

    2016-07-28

    The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.

  12. Comparison of phytochemical profiles, antioxidant and cellular antioxidant activities of different varieties of blueberry (Vaccinium spp.).

    PubMed

    Wang, Huailing; Guo, Xinbo; Hu, Xiaodan; Li, Tong; Fu, Xiong; Liu, Rui Hai

    2017-02-15

    Numerous reports have demonstrated that the consumption of fruits and vegetables is beneficial for the human health. Blueberries, in particular, are rich in phytochemicals including free and bound forming. Phytochemical profiles of 14 varieties of blueberry were compared in this study. 12 compounds were analyzed and had significant changes in blueberry fruits. Total antioxidant activities in different blueberry varieties varied about 2.6times by oxygen radical absorbance capacity (ORAC) assay, and 2times by peroxyl radical scavenging capacity (PSC) assay. The cellular antioxidant activities (CAA) in different varieties varied about 3.9times without phosphate buffer saline (PBS) wash, and 4.7times with PBS wash by CAA assay. Blueberry extracts had potent antiproliferative activities against HepG2 human liver cancer cells, indicating the potential protective benefits associated with their use as functional foods. The anti-proliferative activity was observed to be dose-dependent in blueberry extracts.

  13. Celebrating Soft Matter's 10th Anniversary: Cell division: a source of active stress in cellular monolayers.

    PubMed

    Doostmohammadi, Amin; Thampi, Sumesh P; Saw, Thuan B; Lim, Chwee T; Ladoux, Benoit; Yeomans, Julia M

    2015-10-07

    We introduce the notion of cell division-induced activity and show that the cell division generates extensile forces and drives dynamical patterns in cell assemblies. Extending the hydrodynamic models of lyotropic active nematics we describe turbulent-like velocity fields that are generated by the cell division in a confluent monolayer of cells. We show that the experimentally measured flow field of dividing Madin-Darby Canine Kidney (MDCK) cells is reproduced by our modeling approach. Division-induced activity acts together with intrinsic activity of the cells in extensile and contractile cell assemblies to change the flow and director patterns and the density of topological defects. Finally we model the evolution of the boundary of a cellular colony and compare the fingering instabilities induced by cell division to experimental observations on the expansion of MDCK cell cultures.

  14. Imaging large-scale cellular activity in spinal cord of freely behaving mice

    PubMed Central

    Sekiguchi, Kohei J.; Shekhtmeyster, Pavel; Merten, Katharina; Arena, Alexander; Cook, Daniela; Hoffman, Elizabeth; Ngo, Alexander; Nimmerjahn, Axel

    2016-01-01

    Sensory information from mechanoreceptors and nociceptors in the skin plays key roles in adaptive and protective motor behaviours. To date, very little is known about how this information is encoded by spinal cord cell types and their activity patterns, particularly under freely behaving conditions. To enable stable measurement of neuronal and glial cell activity in behaving mice, we have developed fluorescence imaging approaches based on two- and miniaturized one-photon microscopy. We show that distinct cutaneous stimuli activate overlapping ensembles of dorsal horn neurons, and that stimulus type and intensity is encoded at the single-cell level. In contrast, astrocytes show large-scale coordinated calcium responses to intense but not weak sensory inputs. Sensory-evoked activity is potently suppressed by anaesthesia. By revealing the cellular and computational logic of spinal cord networks under behaving conditions, our approach holds promise for better understanding of healthy and aberrant spinal cord processes. PMID:27121084

  15. Photochemopreventive effect of pomegranate fruit extract on UVA-mediated activation of cellular pathways in normal human epidermal keratinocytes.

    PubMed

    Syed, Deeba N; Malik, Arshi; Hadi, Naghma; Sarfaraz, Sami; Afaq, Farrukh; Mukhtar, Hasan

    2006-01-01

    UVA is the major portion (90-99%) of solar radiation reaching the surface of the earth and has been described to lead to formation of benign and malignant tumors. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species and is responsible for immunosuppression, photodermatoses, photoaging and photocarcinogenesis. Pomegranate fruit extract (PFE) possesses strong antioxidant and anti-inflammatory properties. Our recent studies have shown that PFE treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated activation of MAPK and NF-kappaB pathways. Signal transducers and activators of transcription 3 (STAT3), Protein Kinase B/AKT and Map Kinases (MAPKs), which are activated by a variety of factors, modulate cell proliferation, apoptosis and other biological activities. The goal of this study was to determine whether PFE affords protection against UVA-mediated activation of STAT3, AKT and extracellular signal-regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/cm2 of UVA exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. Pretreatment of NHEK with PFE (60-100 microg/mL) for 24 h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. mTOR, structurally related to PI3K, is involved in the regulation of p70S6K, which in turn phosphorylates the S6 protein of the 40S ribosomal subunit. We found that UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. PFE pretreatment resulted in a dose-dependent inhibition in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. Our data further demonstrate that PFE pretreatment of NHEK resulted in significant inhibition of UVA exposure-mediated increases in Ki-67 and PCNA. PFE pretreatment of NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of

  16. Danger signals activating the immune response after trauma.

    PubMed

    Hirsiger, Stefanie; Simmen, Hans-Peter; Werner, Clément M L; Wanner, Guido A; Rittirsch, Daniel

    2012-01-01

    Sterile injury can cause a systemic inflammatory response syndrome (SIRS) that resembles the host response during sepsis. The inflammatory response following trauma comprises various systems of the human body which are cross-linked with each other within a highly complex network of inflammation. Endogenous danger signals (danger-associated molecular patterns; DAMPs; alarmins) as well as exogenous pathogen-associated molecular patterns (PAMPs) play a crucial role in the initiation of the immune response. With popularization of the "danger theory," numerous DAMPs and PAMPs and their corresponding pathogen-recognition receptors have been identified. In this paper, we highlight the role of the DAMPs high-mobility group box protein 1 (HMGB1), interleukin-1α (IL-1α), and interleukin-33 (IL-33) as unique dual-function mediators as well as mitochondrial danger signals released upon cellular trauma and necrosis.

  17. Photomodulation of cellular and subcellular activities by He-Ne laser

    NASA Astrophysics Data System (ADS)

    Moro, Loredana; Greco, Margherita; Marra, Ersilia; Passarella, Salvatore

    2003-12-01

    In hepatocytes, proliferation of tetraploid and binuclear cells and an increase in cytosolic and mitochondrial protein synthesis are caused by He-Ne laser irradiation. To gain some insight into the mechanism of photomodulation of cellular and subcellular activities in isolated hepatocytes, intracellular mediators of cell photostimulation were investigated in intact cells and isolated mitochondria. Irradiation of isolated hepatocytes and isolated rat liver mitochondria was carried out with He-Ne laser (wavelength: 632.8 nm; fluence: 0.24 J cm-2; fluence rate: 12 mW cm-2). Changes in cytosolic [(Ca2+)c] and mitochondrial [(Ca2+)m] calcium concentration, and in mitochondrial (Δψm) and plasma (Δψc) membrane potential were, monitored using either colorimetric or fluorescent probes. C-fos expression was studied by Northern and immunoblotting analysis. As a result of irradiation, an increase in (Ca2+)c and a calcium-dependent increase in Δψc were found. The increase in (Ca2+)c, in turn, caused an increase in c-fos expression. Finally, an increase in (Ca2+)m, probably owing to the increase in Δψm, was found. Increase in (Ca2+)c, leading to activation of gene expression, and a general activation of mitochondrial metabolism, could play a major role in the mechanism of photostimulation of cellular activities by He-Ne laser.

  18. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements.

    PubMed

    Wolfe, Kelly L; Liu, Rui Hai

    2007-10-31

    A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.

  19. Signaling Components of Redox Active Endosomes: The Redoxosomes

    PubMed Central

    Oakley, Fredrick D.; Abbott, Duane; Li, Qiang

    2009-01-01

    Abstract Subcellular compartmentalization of reactive oxygen species (ROS) plays a critical role in transmitting cell signals in response to environmental stimuli. In this regard, signals at the plasma membrane have been shown to trigger NADPH oxidase-dependent ROS production within the endosomal compartment and this step can be required for redox-dependent signal transduction. Unique features of redox-active signaling endosomes can include NADPH oxidase complex components (Nox1, Noxo1, Noxa1, Nox2, p47phox, p67phox, and/or Rac1), ROS processing enzymes (SOD1 and/or peroxiredoxins), chloride channels capable of mediating superoxide transport and/or membrane gradients required for Nox activity, and novel redox-dependent sensors that control Nox activity. This review will discuss the cytokine and growth factor receptors that likely mediate signaling through redox-active endosomes, and the common mechanisms whereby they act. Additionally, the review will cover ligand-independent environmental injuries, such as hypoxia/reoxygenation injury, that also appear to facilitate cell signaling through NADPH oxidase at the level of the endosome. We suggest that redox-active endosomes encompass a subset of signaling endosomes that we have termed redoxosomes. Redoxosomes are uniquely equipped with redox-processing proteins capable of transmitting ROS signals from the endosome interior to redox-sensitive effectors on the endosomal surface. In this manner, redoxosomes can control redox-dependent effector functions through the spatial and temporal regulation of ROS as second messengers. Antioxid. Redox Signal. 11, 1313–1333. PMID:19072143

  20. Disruption of the Membrane Nuclease Gene (MBOVPG45_0215) of Mycoplasma bovis Greatly Reduces Cellular Nuclease Activity

    PubMed Central

    Sharma, Shukriti; Tivendale, Kelly A.; Markham, Philip F.

    2015-01-01

    ABSTRACT Although the complete genome sequences of three strains of Mycoplasma bovis are available, few studies have examined gene function in this important pathogen. Mycoplasmas lack the biosynthetic machinery for the de novo synthesis of nucleic acid precursors, so nucleases are likely to be essential for them to acquire nucleotide precursors. Three putative membrane nucleases have been annotated in the genome of M. bovis strain PG45, MBOVPG45_0089 and MBOVPG45_0310, both of which have the thermonuclease (TNASE_3) functional domain, and MBOVPG45_0215 (mnuA), which has an exonuclease/endonuclease/phosphatase domain. While previous studies have demonstrated the function of TNASE_3 domain nucleases in several mycoplasmas, quantitative comparisons of the contributions of different nucleases to cellular nuclease activity have been lacking. Mapping of a library of 319 transposon mutants of M. bovis PG45 by direct genome sequencing identified mutants with insertions in MBOVPG45_0310 (the Δ0310 mutant) and MBOVPG45_0215 (the Δ0215 mutant). In this study, the detection of the product of MBOVPG45_0215 in the Triton X-114 fraction of M. bovis cell lysates, its cell surface exposure, and its predicted signal peptide suggested that it is a surface-exposed lipoprotein nuclease. Comparison of a ΔmnuA mutant with wild-type M. bovis on native and denatured DNA gels and in digestion assays using double-stranded phage λ DNA and closed circular plasmid DNA demonstrated that inactivation of this gene abolishes most of the cellular exonuclease and endonuclease activity of M. bovis. This activity could be fully restored by complementation with the wild-type mnuA gene, demonstrating that MnuA is the major cellular nuclease of M. bovis. IMPORTANCE Nucleases are thought to be important contributors to virulence and crucial for the maintenance of a nutritional supply of nucleotides in mycoplasmas that are pathogenic in animals. This study demonstrates for the first time that of the

  1. Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

    PubMed Central

    Allen, Erin M. G.; Florang, Virginia R.; Davenport, Laurie L.; Jinsmaa, Yunden; Doorn, Jonathan A.

    2015-01-01

    The incidence of Parkinson’s disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure–activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity. PMID:23763672

  2. Sipuleucel-T (Provenge): active cellular immunotherapy for advanced prostate cancer.

    PubMed

    McKarney, I

    2007-09-01

    (1) Sipuleucel-T (Provenge) is an active cellular immunotherapy (therapeutic vaccine) that is designed to stimulate the patient's T-cells to recognize and attack prostate cancer cells that express prostatic acid phosphatase (PAP) antigen. (2) Sipuleucel-T demonstrated a survival benefit in men with advanced androgen-independent prostate cancer (AIPC), although this preliminary finding requires confirmation in larger trials. (3) Mild to moderate myalgia, chills, fever, and tremor are the most commonly reported adverse events for patients receiving sipuleucel-T. These events generally resolve quickly. (4) More studies are needed to evaluate sipuleucel-T in the earlier stages of prostate cancer and in combination with conventional therapies.

  3. Chemical and cellular antioxidant activity of phytochemicals purified from olive mill waste waters.

    PubMed

    Angelino, Donato; Gennari, Lorenzo; Blasa, Manuela; Selvaggini, Roberto; Urbani, Stefania; Esposto, Sonia; Servili, Maurizio; Ninfali, Paolino

    2011-03-09

    The isolation and identification of a phytocomplex from olive mill waste waters (OMWW) was achieved. The isolated phytocomplex is made up of the following three phenolic compounds: hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and the dialdehydic form of decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA). The purification of this phytocomplex was reached by partial dehydration of the OMWW, followed by liquid-liquid extraction with ethyl acetate and middle pressure liquid chromatography (MPLC) on a Sephadex LH-20 column. The phytocomplex accounted for 6% of the total phenolic content of the OMWW. The phytocomplex and individual compounds were tested for antioxidant capacity by the oxygen radical absorbance capacity (ORAC) method. The ORAC phytocomplex produced 10,000 ORAC units/g dry weight, whereas the cellular antioxidant activity, measured by the cellular antioxidant activity in red blood cell (CAA-RBC) method, demonstrated that the phytocomplex and all of the components are able to permeate the cell membrane thus exhibiting antioxidant activity inside the red blood cells. Our phytocomplex could be employed in the formulation of fortified foods and nutraceuticals, with the goal to obtain substantial health protective effects due to the suitable combination of the component molecules.

  4. How the Venus flytrap actively snaps: hydrodynamic measurements at the cellular level

    NASA Astrophysics Data System (ADS)

    Colombani, Mathieu; Forterre, Yoel; GEP Team

    2012-11-01

    Although they lack muscle, plants have evolved a remarkable range of mechanisms to create rapid motion, from the rapid folding of sensitive plants to seed dispersal. Of these spectacular examples that have long fascinated scientists, the carnivorous plant Venus flytrap, whose leaves snap together in a fraction of second to capture insects, has long been a paradigm for study. Recently, we have shown that this motion involves a snap-buckling instability due to the shell-like geometry of the leaves of the trap. However, the origin of the movement that allows the plant to cross the instability threshold and actively bend remains largely unknown. In this study, we investigate this active motion using a micro-fluidic pressure probe that gives direct hydraulic and mechanical measurements at the cellular level (osmotic pressure, cell membrane permeability, cell wall elasticity). Our results challenge the role of osmotically-driven water flows usually put forward to explain Venus flytrap's active closure.

  5. Cellular antioxidant activity of feijoada whole meal coupled with an in vitro digestion.

    PubMed

    Faller, Ana Luisa Kremer; Fialho, Eliane; Liu, Rui Hai

    2012-05-16

    Consumption of plant food rich meals, such as feijoada, a traditional meal in Brazil, is associated with the reduction of chronic disease. The objectives of this study were to determine phytochemical content and antioxidant activity by chemical and cellular antioxidant assays (CAA) of feijoada with or without in vitro digestion. Feijoada showed no difference in phenolics and flavonoids after digestion. Bound and residue contributions to total phenolics were 20.9% and 32.2%, respectively, suggesting that phenolics reach the colon after intake. Flavonoids in residue and bound fractions represented 50% of total flavonoids. Antioxidant activity of feijoada without digestion was higher than that with digestion; however, it showed lower antiproliferative activity and CAA. Feijoada with in vitro digestion also yielded phenolics with higher CAA. Analyses of whole meals should be used to evaluate phytochemicals present in food mixtures consumed, especially with digestion models coupled with CAA resulting in information similar to those in physiological conditions.

  6. P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.

    PubMed

    Hernández-Negrete, Ivette; Carretero-Ortega, Jorge; Rosenfeldt, Hans; Hernández-García, Ricardo; Calderón-Salinas, J Victor; Reyes-Cruz, Guadalupe; Gutkind, J Silvio; Vázquez-Prado, José

    2007-08-10

    Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

  7. Effect of Cellular Location of Human Carboxylesterase 2 on CPT-11 Hydrolysis and Anticancer Activity

    PubMed Central

    Hsieh, Yuan-Ting; Lin, Hsuan-Pei; Chen, Bing-Mae; Huang, Ping-Ting; Roffler, Steve R.

    2015-01-01

    CPT-11 is an anticancer prodrug that is clinically used for the treatment of metastatic colorectal cancer. Hydrolysis of CPT-11 by human carboxylesterase 2 (CE2) generates SN-38, a topoisomerase I inhibitor that is the active anti-tumor agent. Expression of CE2 in cancer cells is under investigation for the tumor-localized activation of CPT-11. CE2 is normally expressed in the endoplasmic reticulum of cells but can be engineered to direct expression of active enzyme on the plasma membrane or as a secreted form. Although previous studies have investigated different locations of CE2 expression in cancer cells, it remains unclear if CE2 cellular location affects CPT-11 anticancer activity. In the present study, we directly compared the influence of CE2 cellular location on substrate hydrolysis and CPT-11 cytotoxicity. We linked expression of CE2 and enhanced green fluorescence protein (eGFP) via a foot-and-mouth disease virus 2A (F2A) peptide to facilitate fluorescence-activated cell sorting to achieve similar expression levels of ER-located, secreted or membrane-anchored CE2. Soluble CE2 was detected in the medium of cells that expressed secreted and membrane-anchored CE2, but not in cells that expressed ER-retained CE2. Cancer cells that expressed all three forms of CE2 were more sensitive to CPT-11 as compared to unmodified cancer cells, but the membrane-anchored and ER-retained forms of CE2 were consistently more effective than secreted CE2. We conclude that expression of CE2 in the ER or on the membrane of cancer cells is suitable for enhancing CPT-11 anticancer activity. PMID:26509550

  8. Coco is a dual activity modulator of TGFβ signaling

    PubMed Central

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.

    2015-01-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling. PMID:26116664

  9. Coco is a dual activity modulator of TGFβ signaling.

    PubMed

    Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H

    2015-08-01

    The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling.

  10. CFTR impairment upregulates c-Src activity through IL-1β autocrine signaling.

    PubMed

    Massip-Copiz, María Macarena; Clauzure, Mariángeles; Valdivieso, Ángel Gabriel; Santa-Coloma, Tomás Antonio

    2017-02-15

    Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1β receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1β→c-Src and IL-1β→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1β constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

  11. Reverse signaling initiated from GITRL induces NF-kappaB activation through ERK in the inflammatory activation of macrophages.

    PubMed

    Bae, Eun Mi; Kim, Won-Jung; Suk, Kyoungho; Kang, Young-Mo; Park, Jeong-Euy; Kim, Won Young; Choi, Eun Mi; Choi, Beom Kyu; Kwon, Byoung S; Lee, Won-Ha

    2008-01-01

    Glucocorticoid-induced TNF receptor family related protein ligand (GITRL) is known to interact with its cognate receptor GITR. In order to investigate the potential role of GITRL in the pro-inflammatory activation of macrophages and the signaling pathway induced by GITRL, we stimulated the macrophage cell line, THP-1, and primary macrophages with an anti-GITRL monoclonal antibody or a GITR:Fc fusion protein and analyzed the cellular responses. The stimulation of GITRL induced the expression of pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9 and up-regulated ICAM-1 expression levels, which was responsible for enhanced cellular aggregation and adhesion to extracellular matrix proteins. The activation of these pro-inflammatory mediators required the activation of ERK1/2 mitogen-activated protein kinase (MAPK) and negatively regulated by p38 MAPK and JNK. Immunofluorescence analysis detected nuclear translocation of the NF-kappaB p50 subunit and this was blocked by ERK inhibitor, indicating that GITRL stimulation induced ERK1/2 phosphorylation and subsequent activation of NF-kappaB. Furthermore, the expression of GITRL and GITR was detected in macrophages in inflammatory disease specimens such as atherosclerotic plaques and synovial tissues of rheumatoid arthritis. These observations raise the possibility that the GITRL-mediated inflammatory activation of macrophages is involved in the pathogenesis of inflammatory diseases.

  12. Sunlight UV-induced skin cancer relies upon activation of the p38α signaling pathway.

    PubMed

    Liu, Kangdong; Yu, Donghoon; Cho, Yong-Yeon; Bode, Ann M; Ma, Weiya; Yao, Ke; Li, Shengqing; Li, Jixia; Bowden, G Tim; Dong, Ziming; Dong, Zigang

    2013-04-01

    The activation of cellular signal transduction pathways by solar ultraviolet (SUV) irradiation plays a vital role in skin tumorigenesis. Although many pathways have been studied using pure ultraviolet A (UVA) or ultraviolet B (UVB) irradiation, the signaling pathways induced by SUV (i.e., sunlight) are not understood well enough to permit improvements for prevention, prognosis, and treatment. Here, we report parallel protein kinase array studies aimed at determining the dominant signaling pathway involved in SUV irradiation. Our results indicated that the p38-related signal transduction pathway was dramatically affected by SUV irradiation. SUV (60 kJ UVA/m(2)/3.6 kJ UVB/m(2)) irradiation stimulates phosphorylation of p38α (MAPK14) by 5.78-fold, MSK2 (RPS6KA4) by 6.38-fold, and HSP27 (HSPB1) by 34.56-fold compared with untreated controls. By investigating the tumorigenic role of SUV-induced signal transduction in wild-type and p38 dominant-negative (p38 DN) mice, we found that p38 blockade yielded fewer and smaller tumors. These results establish that p38 signaling is critical for SUV-induced skin carcinogenesis.

  13. Antioxidant activity of rosmarinic acid and its principal metabolites in chemical and cellular systems: Importance of physico-chemical characteristics.

    PubMed

    Adomako-Bonsu, Amma G; Chan, Sue Lf; Pratten, Margaret; Fry, Jeffrey R

    2017-04-01

    Persistent accumulation of reactive oxygen species causes cellular oxidative stress which contributes strongly towards the induction and progression of various diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. In the current study, rosmarinic acid (RA) and its key metabolites were evaluated in non-cellular and cellular antioxidant assays, using quercetin (Q) as a positive control. The non-cellular assay was performed as scavenging of DPPH radical, whilst the cellular assay was performed as protection from an oxidant stress. Radical-scavenging activity of RA and two of its primary metabolites, CA and DHPLA, were comparable to that of Q, whilst FA was of lower potency and m-CoA was inactive. In the cellular assay, RA and CA were markedly less potent than Q, with DHPLA, FA and m-CoA being inactive, this being true in short-term (5-h) or long-term (20-h) exposure conditions. However, antioxidant potency of Q and methyl rosmarinate, a non-ionisable ester of RA, was similar in the non-cellular and short-term cellular assays. It is proposed that marked ionisation of organic acids such as RA and its metabolites at physiological pH greatly limits their intracellular accumulation, and so attenuates intrinsic antioxidant ability demonstrated in the non-cellular assay. This study demonstrates some of the factors that prevent well-known phytochemicals from progressing further along the drug discovery chain.

  14. Tac-beta1 inhibits FAK activation and Src signaling.

    PubMed

    Berrier, Allison L; Jones, Christopher W; LaFlamme, Susan E

    2008-03-28

    The binding of integrins to extracellular matrix triggers signals that promote cell spreading. We previously demonstrated that expression of the integrin beta1 cytoplasmic domain in the context of a chimeric transmembrane receptor with the Tac subunit of the interleukin-2 receptor (Tac-beta1) inhibits cell spreading. To study the mechanism whereby Tac-beta1 inhibits cell spreading, we examined the effect of Tac-beta1 on early signaling events following integrin engagement namely FAK and Src signaling. We infected primary fibroblasts with adenoviruses expressing Tac or Tac-beta1 and found that Tac-beta1 prevented FAK activation by inhibiting the phosphorylation of FAK at Tyr-397. In contrast, Src activation was maintained, as phosphorylation of Src at Tyr-419 and Tyr-530 were not responsive to expression of Tac-beta1. Importantly, adhesion-induced tyrosine phosphorylation of the Src substrates p130Cas and paxillin was inhibited, indicating that Src signaling was blocked by Tac-beta1. These Src-dependent signaling events were found to require FAK signaling. Our results suggest that Tac-beta1 inhibits cell spreading, at least in part, by preventing the phosphorylation of FAK at Tyr-397 and the assembly of signaling complexes necessary for phosphorylation of p130Cas and other downstream effectors.

  15. Dermal quercetin smartCrystals®: Formulation development, antioxidant activity and cellular safety.

    PubMed

    Hatahet, T; Morille, M; Hommoss, A; Dorandeu, C; Müller, R H; Bégu, S

    2016-05-01

    Flavonoids are natural plant pigments, which possess high antioxidative and antiradical activities. However, their poor water solubility led to a limited bioavailability. To overcome this major hurdle, quercetin nanocrystals were produced implementing smartCrystals® technology. This process combines bead milling and subsequent high-pressure homogenization at relatively low pressure (300bar). To test the possibility to develop a dermal formulation from quercetin smartCrystals®, quercetin nanosuspensions were admixed to Lutrol® F127 and hydroxythylcellulose nonionic gels. The physicochemical properties (morphology, size and charge), saturation solubility, dissolution velocity and the antioxidant properties (DPPH assay) as well as the cellular interaction of the produced quercetin smartCrystals® were studied and compared to crude quercetin powder. Quercetin smartCrystals® showed a strong increase in the saturation solubility and the dissolution velocity (7.6 fold). SmartCrystals® loaded or not into gels proved to be physically stable over a period of three months at 25°C. Interestingly, in vitro DPPH assay confirmed the preservation of quercetin antioxidative properties after nanonization. In parallel, the nanocrystalline form did not display cellular toxicity, even at high concentration (50μg/ml), as assayed on an epithelial cell line (VERO cells). In addition, the nanocrystalline form confirmed a protective activity for VERO cells against hydrogen peroxide induced toxicity in vitro. This new formulation presents a promising approach to deliver quercetin efficiently to skin in well-tolerated formulations.

  16. The influence of active components of Eleutherococcus senticosus on cellular defence and physical fitness in man.

    PubMed

    Szołomicki, J; Samochowiec, L; Wójcicki, J; Droździk, M; Szołomicki, S

    2000-02-01

    The influence of active components of Eleutherococcus senticosus, contained in Taiga Wurzel preparation, were studied on cellular defence and physical fitness in man. 50 healthy volunteers of both sexes were selected, and basic clinical examination and laboratory tests were performed in all subjects. All were randomly subdivided into two study groups: group A with 35 subjects receiving Taiga Wurzel and group B with 15 subjects receiving Echinacea. 20 healthy males were randomly selected from both groups and underwent an ergospirometric study. The preparations were administered for 30 days as follows: Taiga Wurzel 25 drops three times daily, Echinacea 40 drops three times daily. After 1 month blood was drawn for control tests. Changes in the following blood parameters were observed in comparison to initial values in group A: total and LDL cholesterol, triglycerides and glucose. No alterations were seen in group B. The ergospirometric test revealed a higher oxygen plateau in group A (Taiga Wurzel). On the basis of the present study the following conclusions were drawn: active components in Eleutherococcus senticosus contained in Taiga Wurzel preparation affect cellular defence and physical fitness, as well as lipid metabolism.

  17. Dependence of cellular activity at protein adsorbed biointerfaces with nano- to microscale dimensionality.

    PubMed

    Nune, C; Misra, R D K; Somani, M C; Karjalainen, L P

    2014-06-01

    Protein adsorption is one of the first-few events that occur when a biomedical device comes in contact with the physiological system. The adsorption process is subsequently followed by communication with cells and formation of tissue. Given the strong interest in nanostructured surfaces, we describe here the impact of grain structure from nanograined (NG) regime to coarse-grained (CG) regime on the self-assembly of proteins (bovine serum albumin) and consequent functional response of osteoblasts. The objective is accomplished using the innovative concept of "phase reversion" that enables a wide range of grain size (from NG to CG regime) to be obtained using an identical set of parameters, besides additional attributes of high strength/weight ratio and wear resistance. Depending on the grain structure a consistent and significant change in the adsorption characteristics of protein was observed at biointerface, such that the cell density was statistically different. The high surface coverage and leaf-like conformation of adsorbed protein on NG surface as compared to bare branch-like structure with low surface coverage on the CG surface, was beneficial in favorably modulating cellular activity (osteoblast functions: cell attachment, proliferation, actin, vinculin, and fibronectin expression). This is the first report that elucidates the impact of grain structure from NG to CG regime on cellular activity.

  18. Oncogenic activation of the PI3K/Akt pathway promotes cellular glucose uptake by downregulating the expression of thioredoxin-interacting protein.

    PubMed

    Hong, Shin Yee; Yu, Fa-Xing; Luo, Yan; Hagen, Thilo

    2016-05-01

    Oncogenic activation of the PI3K/Akt pathway is known to play an important role to promote glucose metabolism in cancer cells. However, the molecular mechanism through which the PI3K/Akt signalling pathway promotes glucose utilisation in cancer cells is still not well understood. It has recently been shown that the oncogenic activation of the PI3K/Akt/mTOR signalling in lung adenocarcinoma is important in promoting the localisation of glucose transporter 1 (GLUT1) at the plasma membrane. We thus hypothesised that the effect of constitutive activation of the PI3K/AKT signalling on glucose metabolism is mediated by thioredoxin interacting protein (TXNIP), a known regulator of the GLUT1 plasma membrane localisation. Consistent with previous studies, inhibition of the PI3K/Akt pathway decreased cellular glucose uptake. Furthermore, inhibition of PI3K/Akt signalling in non-small cell lung cancer (NSCLC) cell lines using clinically used tyrosine kinase inhibitors (TKIs) resulted in a decrease in GLUT1 membrane localisation. We also observed that inhibition of the PI3K/Akt pathway in various cell lines, including NSCLC cells, resulted in an increase in TXNIP expression. Importantly, knockdown of TXNIP using siRNA in the NSCLC cells promoted GLUT1 to be localised at the plasma membrane and reversed the effect of PI3K/Akt inhibitors. Together, our results suggest that the oncogenic activation of PI3K/Akt signalling promotes cellular glucose uptake, at least in part, through the regulation of TXNIP expression. This mechanism may contribute to the Warburg effect in cancer cells.

  19. Enhanced Multistatic Active Sonar via Innovative Signal Processing

    DTIC Science & Technology

    2015-09-30

    Grove, CA, November, 2014. [in press, refereed]. C . Gianelli, L. Xu, and J. Li, " Active Sonar Systems in the Presence of Strong Direct Blast", Oceans...3. DATES COVERED (From - To) Oct. 01, 2014-Sept. 30, 2015 4. TITLE AND SUBTITLE Enhanced Multistatic Active Sonar via Innovative Signal... active sonar (CAS) in the presence of strong direct blast is studied for the Doppler-tolerant linear frequency modulation waveform. A receiver design

  20. Enhanced Multistatic Active Sonar via Innovative Signal Processing

    DTIC Science & Technology

    2014-09-30

    DATES COVERED (From - To) Oct. 01. 2013-Sept. 30, 2014 4. TITLE AND SUBTITLE Enhanced Multistatic Active Sonar via Innovative Signal Processing 5a...DISTRIBUTION AVAILABILITY STATEMENT Approved for Public Release; Distribution is Unlimited. 13. SUPPLEMENTARY NOTES 14. ABSTRACT Pulsed active sonar ...PAS) and continuous active sonar (CAS) in the presence of strong direct blast are studied for the Doppler-tolerant linear frequency modulation

  1. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction

    PubMed Central

    Desikan, Radhika

    2016-01-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction. PMID:27581482

  2. Linear models of activation cascades: analytical solutions and coarse-graining of delayed signal transduction.

    PubMed

    Beguerisse-Díaz, Mariano; Desikan, Radhika; Barahona, Mauricio

    2016-08-01

    Cellular signal transduction usually involves activation cascades, the sequential activation of a series of proteins following the reception of an input signal. Here, we study the classic model of weakly activated cascades and obtain analytical solutions for a variety of inputs. We show that in the special but important case of optimal gain cascades (i.e. when the deactivation rates are identical) the downstream output of the cascade can be represented exactly as a lumped nonlinear module containing an incomplete gamma function with real parameters that depend on the rates and length of the cascade, as well as parameters of the input signal. The expressions obtained can be applied to the non-identical case when the deactivation rates are random to capture the variability in the cascade outputs. We also show that cascades can be rearranged so that blocks with similar rates can be lumped and represented through our nonlinear modules. Our results can be used both to represent cascades in computational models of differential equations and to fit data efficiently, by reducing the number of equations and parameters involved. In particular, the length of the cascade appears as a real-valued parameter and can thus be fitted in the same manner as Hill coefficients. Finally, we show how the obtained nonlinear modules can be used instead of delay differential equations to model delays in signal transduction.

  3. Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

    PubMed Central

    Piloto, Obdulio; Wright, Melissa; Brown, Patrick; Kim, Kyu-Tae; Levis, Mark; Small, Donald

    2007-01-01

    Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance. PMID:17047150

  4. Functional characterization of AMP-activated protein kinase signaling in tumorigenesis.

    PubMed

    Cheng, Ji; Zhang, Tao; Ji, Hongbin; Tao, Kaixiong; Guo, Jianping; Wei, Wenyi

    2016-12-01

    AMP-activated protein kinase (AMPK) is a ubiquitously expressed metabolic sensor among various species. Specifically, cellular AMPK is phosphorylated and activated under certain stressful conditions, such as energy deprivation, in turn to activate diversified downstream substrates to modulate the adaptive changes and maintain metabolic homeostasis. Recently, emerging evidences have implicated the potential roles of AMPK signaling in tumor initiation and progression. Nevertheless, a comprehensive description on such topic is still in scarcity, especially in combination of its biochemical features with mouse modeling results to elucidate the physiological role of AMPK signaling in tumorigenesis. Hence, we performed this thorough review by summarizing the tumorigenic role of each component along the AMPK signaling, comprising of both its upstream and downstream effectors. Moreover, their functional interplay with the AMPK heterotrimer and exclusive efficacies in carcinogenesis were chiefly explained among genetically altered mice models. Importantly, the pharmaceutical investigations of AMPK relevant medications have also been highlighted. In summary, in this review, we not only elucidate the potential functions of AMPK signaling pathway in governing tumorigenesis, but also potentiate the future targeted strategy aiming for better treatment of aberrant metabolism-associated diseases, including cancer.

  5. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    SciTech Connect

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  6. Mathematical modelling unveils the essential role of cellular phosphatases in the inhibition of RAF-MEK-ERK signalling by sorafenib in hepatocellular carcinoma cells.

    PubMed

    Saidak, Zuzana; Giacobbi, Anne-Sophie; Louandre, Christophe; Sauzay, Chloé; Mammeri, Youcef; Galmiche, Antoine

    2017-04-28

    The RAS-RAF-MEK-ERK cascade is a key oncogenic signal transduction pathway activated in many types of tumours in humans. Sorafenib, the medical treatment of reference against advanced stages of hepatocellular carcinoma (HCC), inhibits the RAF-MEK-ERK cascade in HCC cells. Based on previous studies suggesting that this cascade is an important target of sorafenib in HCC cells, we explored its regulation using mathematical modelling and ordinary differential equations. We analysed the dynamic regulation of the core components of the RAF-MEK-ERK cascade in three human HCC cell lines (Huh7, Hep3B and PLC/PRF5) with heterogeneous responses to sorafenib. In silico predictions derived from our mathematical model suggested that the disappearance of phosphorylated MEK and ERK proteins catalysed by cellular phosphatases is an essential mechanism underlying the anti-ERK efficacy of sorafenib in HCC cells. This prediction was experimentally validated using specific inhibitors of the phosphatases PP2A (Protein Phosphatase 2A) and DUSP1/6 (Dual-specificity phosphatases 1/6). These findings highlight an unexpected mode of action of sorafenib on the kinome of HCC cells, and open new perspectives regarding the therapeutic targeting of the RAF-MEK-ERK cascade in this context.

  7. Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

    PubMed Central

    Redemann, N; Holzmann, B; von Rüden, T; Wagner, E F; Schlessinger, J; Ullrich, A

    1992-01-01

    Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product. Images PMID:1346334

  8. Sleep Deprivation and Divergent Toll-like Receptor-4 Activation of Cellular Inflammation in Aging

    PubMed Central

    Carroll, Judith E.; Carrillo, Carmen; Olmstead, Richard; Witarama, Tuff; Breen, Elizabeth C.; Yokomizo, Megumi; Seeman, Teresa E.; Irwin, Michael R.

    2015-01-01

    Objectives: Sleep disturbance and aging are associated with increases in inflammation, as well as increased risk of infectious disease. However, there is limited understanding of the role of sleep loss on age-related differences in immune responses. This study examines the effects of sleep deprivation on toll-like receptor activation of monocytic inflammation in younger compared to older adults. Design, Setting, and Participants: Community-dwelling adults (n = 70) who were categorized as younger (25–39 y old, n = 21) and older (60–84 y old, n = 49) participants, underwent a sleep laboratory-based experimental partial sleep deprivation (PSD) protocol including adaptation, an uninterrupted night of sleep, sleep deprivation (sleep restricted to 03:00–07:00), and recovery. Measurement and Results: Blood samples were obtained each morning to measure toll-like receptor-4 activation of monocyte intracellular production of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Partial sleep deprivation induced a significant increase in the production of IL-6 and/or TNF-α that persisted after a night of recovery sleep (F(2,121.2) = 3.8, P < 0.05). Age moderated the effects of sleep loss, such that younger adults had an increase in inflammatory cytokine production that was not present in older adults (F(2,121.2) = 4.0, P < 0.05). Conclusion: Older adults exhibit reduced toll-like receptor 4 stimulated cellular inflammation that, unlike in younger adults, is not activated after a night of partial sleep loss. Whereas sleep loss increases cellular inflammation in younger adults and may contribute to inflammatory disorders, blunted toll-like receptor activation in older adults may increase the risk of infectious disease seen with aging. Citation: Carroll JE, Carrillo C, Olmstead R, Witarama T, Breen EC, Yokomizo M, Seeman TE, Irwin MR. Sleep deprivation and divergent toll-like receptor-4 activation of cellular inflammation in aging. SLEEP

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

    PubMed

    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.

  10. Stress-activated signaling responses leading to apoptosis following photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Oleinick, Nancy L.; He, Jin; Xue, Liang-yan; Separovic, Duska

    1998-05-01

    Photodynamic treatment with the phthalocyanine Pc 4, a mitochondrially localizing photosensitizer, is an efficient inducer of cell death by apoptosis, a cell suicide pathway that can be triggered by physiological stimuli as well as by various types of cellular damage. Upon exposure of the dye- loaded cells to red light, several stress signalling pathways are rapidly activated. In murine L5178Y-R lymphoblasts, caspase activation and other hallmarks of the final phase of apoptosis are observed within a few minutes post-PDT. In Chinese hamster CHO-K1 cells, the first signs of apoptosis are not observed for 1 - 2 hours. The possible involvement of three parallel mitogen-activated protein kinase (MAPK) signalling pathways has been investigated. The extracellular- regulated kinases (ERK-1 and ERK-2), that are thought to promote cell growth, are not appreciably altered by PDT. However, PDT causes marked activation of the stress-activated protein kinase (SAPK) cascade in both cell types and of the p38/HOG-type kinase in CHO cells. Both of these latter pathways have been demonstrated to be associated with apoptosis. A specific inhibitor of the ERK pathway did not alter PDT-induced apoptosis; however, an inhibitor of the p38 pathway partially blocked PDT-induced apoptosis. Blockage of the SAPK pathway is being pursued by a genetic approach. It appears that the SAPK and p38 pathways may participate in signaling apoptosis in response to PDT with Pc 4.

  11. The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter.

    PubMed Central

    Pereira, D J; Muzyczka, N

    1997-01-01

    Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions -50 and -130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position -140. Mutational analysis of the p19 promoter suggested that the SP1 site at -50 and the cAAP site at -140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA. PMID:9032303

  12. Blood biochemical and cellular changes during decompression and simulated extravehicular activity

    NASA Technical Reports Server (NTRS)

    Jauchem, J. R.; Waligora, J. M.; Johnson, P. C. Jr

    1990-01-01

    Blood biochemical and cellular parameters were measured in human subjects before and after exposure to a decompression schedule involving 6 h of oxygen prebreathing. The exposure was designed to simulate extravehicular activity for 6 h (subjects performed exercise while exposed to 29.6 kPa). There were no significant differences between blood samples from subjects who were susceptible (n = 11) versus those who were resistant (n = 27) to formation of venous gas emboli. Although several statistically significant (P less than 0.05) changes in blood parameters were observed following the exposure (increases in white blood cell count, prothrombin time, and total bilirubin, and decreases in triglycerides, very-low-density lipoprotein cholesterol, and blood urea nitrogen), the changes were small in magnitude and blood factor levels remained within normal clinical ranges. Thus, the decompression schedule used in this study is not likely to result in blood changes that would pose a threat to astronauts during extravehicular activity.

  13. Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

    PubMed

    Neufer, P Darrell; Bamman, Marcas M; Muoio, Deborah M; Bouchard, Claude; Cooper, Dan M; Goodpaster, Bret H; Booth, Frank W; Kohrt, Wendy M; Gerszten, Robert E; Mattson, Mark P; Hepple, Russell T; Kraus, William E; Reid, Michael B; Bodine, Sue C; Jakicic, John M; Fleg, Jerome L; Williams, John P; Joseph, Lyndon; Evans, Mary; Maruvada, Padma; Rodgers, Mary; Roary, Mary; Boyce, Amanda T; Drugan, Jonelle K; Koenig, James I; Ingraham, Richard H; Krotoski, Danuta; Garcia-Cazarin, Mary; McGowan, Joan A; Laughlin, Maren R

    2015-07-07

    The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

  14. [Stimulating effect of cellular RNA on the in vitro polymerizing activity of influenza virus ribonucleoprotein].

    PubMed

    Tentsov, Iu Iu; Bukrinskaia, A G

    1981-01-01

    The stimulating effect of RNAs isolated from noninfected and influenza virus-infected chick fibroblasts on the polymerase activity of influenza virus intracellular ribonucleoprotein (RNP) was studied in vitro. The infected cells were shown to contain two classes of RNAs which stimulated well the polymerase activity of influenza virus RNP. One class seemed to be represented by a heterogenous cellular 10-20 S mRNA since it contained poly (A)-sequences and was present in noninfected cells. The other RNA class was induced during the infection and differed in number of properties from the RNA isolated from noninfected cells. This class RNA was smaller (4-10 S) and appeared not to contain poly(A)-sequences. Treatment of both noninfected and infected cells with actinomycin D resulted in inhibition of synthesis of both classes of RNA-primers.

  15. Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

    PubMed

    Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

    2016-12-09

    The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells.

  16. Anti-Inflammatory Effect of Streptochlorin via TRIF-Dependent Signaling Pathways in Cellular and Mouse Models

    PubMed Central

    Shim, Do-Wan; Shin, Hee Jae; Han, Ji-Won; Shin, Woo-Young; Sun, Xiao; Shim, Eun-Jeong; Kim, Tack-Joong; Kang, Tae-Bong; Lee, Kwang-Ho

    2015-01-01

    Streptochlorin, a small compound derived from marine actinomycete, has been shown to have anti-angiogenic, anti-tumor, and anti-allergic activities. However, the anti-inflammatory effects and underlying mechanisms have not yet been reported. In the present study, we investigated the effect of streptochlorin on lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. Streptochlorin attenuated the production of proinflammatory mediators such as nitric oxide, cyclooxygenase-2, pro-interleukin (IL)-1β, and IL-6 in LPS-stimulated RAW264.7 cells through inhibition of the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF)-dependent signaling pathway. Furthermore, streptochlorin suppressed the infiltration of immune cells such as neutrophils into the lung and proinflammatory cytokine production such as IL-6 and TNF-α in broncho-alveolar lavage fluid (BALF) in the LPS-induced acute lung injury (ALI) mouse model. Streptochlorin has potent anti-inflammatory effects through regulating TRIF-dependent signaling pathways, suggesting that streptochlorin may provide a valuable therapeutic strategy in treating various inflammatory diseases. PMID:25822875

  17. Ethanol alters cellular activation and CD14 partitioning in lipid rafts

    SciTech Connect

    Dai Qun; Zhang Jun; Pruett, Stephen B. . E-mail: spruet@lsuhsc.edu

    2005-06-24

    Alcohol consumption interferes with innate immunity. In vivo EtOH administration suppresses cytokine responses induced through Toll-like receptor 4 (TLR4) and inhibits TLR4 signaling. Actually, EtOH exhibits a generalized suppressive effect on signaling and cytokine responses induced by through most TLRs. However, the underlying mechanism remains unknown. RAW264.7 cells were treated with LPS or co-treated with EtOH or with lipid raft-disrupting drugs. TNF-{alpha} production, IRAK-1 activation, and CD14 partition were evaluated. EtOH or nystatin, a lipid raft-disrupting drug, suppressed LPS-induced production of TNF-{alpha}. The suppressive effect of EtOH on LPS-induced TNF-{alpha} production was additive with that of methyl-{beta}-cyclodextrin (MCD), another lipid raft-disrupting drug. EtOH interfered with IRAK-1 activation, an early TLR4 intracellular signaling event. Cell fractionation analyses show that acute EtOH altered LPS-related partition of CD14, a critical component of the LPS receptor complex. These results suggest a novel mechanism of EtOH action that involves interference with lipid raft clustering induced by LPS. This membrane action of EtOH might be one of the mechanisms by which EtOH acts as a generalized suppressor for TLR signaling.

  18. Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes.

    PubMed

    Rudolf, Emil; Cervinka, Miroslav

    2010-04-01

    Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

  19. Decoding Cellular Dynamics in Epidermal Growth Factor Signaling Using a New Pathway-Based Integration Approach for Proteomics and Transcriptomics Data

    PubMed Central

    Wachter, Astrid; Beißbarth, Tim

    2016-01-01

    Identification of dynamic signaling mechanisms on different cellular layers is now facilitated as the increased usage of various high-throughput techniques goes along with decreasing costs for individual experiments. A lot of these signaling mechanisms are known to be coordinated by their dynamics, turning time-course data sets into valuable information sources for inference of regulatory mechanisms. However, the combined analysis of parallel time-course measurements from different high-throughput platforms still constitutes a major challenge requiring sophisticated bioinformatic tools in order to ease biological interpretation. We developed a new pathway-based integration approach for the analysis of coupled omics time-series data, which we implemented in the R package pwOmics. Unlike many other approaches, our approach acknowledges the role of the different cellular layers of measurement and infers consensus profiles and time profile clusters for further biological interpretation. We investigated a time-course data set on epidermal growth factor stimulation of human mammary epithelial cells generated on the two layers of RNA and proteins. The data was analyzed using our new approach with a focus on feedback signaling and pathway crosstalk. We could confirm known regulatory patterns relevant in the physiological cellular response to epidermal growth factor stimulation as well as identify interesting new interactions in this signaling context, such as the regulatory influence of the connective tissue growth factor on transferrin receptor or the influence of growth arrest and DNA-damage-inducible alpha on the connective tissue growth factor. Thus, we show that integrated cross-platform analysis provides a deeper understanding of regulatory signaling mechanisms. Combined with time-course information it enables the characterization of dynamic signaling processes and leads to the identification of important regulatory interactions which might be dysregulated in disease

  20. Zinc modulates PPARgamma signaling and activation of porcine endothelial cells.

    PubMed

    Meerarani, Purushothaman; Reiterer, Gudrun; Toborek, Michal; Hennig, Bernhard

    2003-10-01

    Dietary zinc has potent antioxidant and anti-inflammatory properties and is a critical component of peroxisome proliferator-activated receptor (PPAR) gene expression and regulation. To assess the protective mechanisms of PPARgamma in endothelial cell dysfunction and the role of zinc in the modulation of PPARgamma signaling, cultured porcine pulmonary artery endothelial cells were exposed to the membrane-permeable zinc chelator N,N,N'N'-tetrakis (2-pyridylmethyl)-ethylene diamine (TPEN), thiazolidinedione (TZD; PPARgamma agonist) or bisphenol A diglycidyl ether (BADGE; PPARgamma antagonist). Subsequently, endothelial cells were activated by treatment with linoleic acid (90 micro mol/L) for 6 h. Zinc chelation by TPEN increased the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1, decreased PPARgamma expression and activation as well as up-regulated interleukin (IL)-6 expression and production. These effects were fully reversed by zinc supplementation. In addition, exposure to TZD down-regulated linoleic acid-induced DNA binding activity of NF-kappaB and AP-1, whereas BADGE further induced activation of these oxidative stress-sensitive transcription factors. Most importantly, the TZD-mediated down-regulation of NF-kappaB and AP-1 and reduced inflammatory response were impaired during zinc chelation. These data suggest that zinc plays a critical role in PPARgamma signaling in linoleic acid-induced endothelial cell activation and indicate that PPARgamma signaling is impaired during zinc deficiency.

  1. Biochemical and Cellular Analysis Reveals Ligand Binding Specificities, a Molecular Basis for Ligand Recognition, and Membrane Association-dependent Activities of Cripto-1 and Cryptic.

    PubMed

    Aykul, Senem; Parenti, Anthony; Chu, Kit Yee; Reske, Jake; Floer, Monique; Ralston, Amy; Martinez-Hackert, Erik

    2017-03-10

    Transforming growth factor β (TGF-β) pathways are key determinants of cell fate in animals. Their basic mechanism of action is simple. However, to produce cell-specific responses, TGF-β pathways are heavily regulated by secondary factors, such as membrane-associated EGF-CFC family proteins. Cellular activities of EGF-CFC proteins have been described, but their molecular functions, including how the mammalian homologs Cripto-1 and Cryptic recognize and regulate TGF-β family ligands, are less clear. Here we use purified human Cripto-1 and mouse Cryptic produced in mammalian cells to show that these two EGF-CFC homologs have distinct, highly specific ligand binding activities. Cripto-1 interacts with BMP-4 in addition to its known partner Nodal, whereas Cryptic interacts only with Activin B. These interactions depend on the integrity of the protein, as truncated or deglycosylated Cripto-1 lacked BMP-4 binding activity. Significantly, Cripto-1 and Cryptic blocked binding of their cognate ligands to type I and type II TGF-β receptors, indicating that Cripto-1 and Cryptic contact ligands at their receptor interaction surfaces and, thus, that they could inhibit their ligands. Indeed, soluble Cripto-1 and Cryptic inhibited ligand signaling in various cell-based assays, including SMAD-mediated luciferase reporter gene expression, and differentiation of a multipotent stem cell line. But in agreement with previous work, the membrane bound form of Cripto-1 potentiated signaling, revealing a critical role of membrane association for its established cellular activity. Thus, our studies provide new insights into the mechanism of ligand recognition by this enigmatic family of membrane-anchored TGF-β family signaling regulators and link membrane association with their signal potentiating activities.

  2. Notch signaling promotes osteoclast maturation and resorptive activity

    PubMed Central

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-01-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. PMID:25914241

  3. Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors.

    PubMed

    Fung, Chak-Hei; Cheung, Wing-Hoi; Pounder, Neill M; Harrison, Andrew; Leung, Kwok-Sui

    2014-07-01

    Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte-osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5MHz sine wave, ISATA=30 mW/cm(2)) was applied to osteocytes (mechanosensor) at three axial distances: 0mm (near field), 60mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more β-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the

  4. TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling

    PubMed Central

    Lascano, Josefina; Uchil, Pradeep D.; Mothes, Walther

    2015-01-01

    biology. TRIM5 is a cellular protein that protects host genome integrity by disrupting the retroviral capsid as it transports viral nucleic acid to the host cell nucleus. Previous data suggest that innate immune signaling contributes to TRIM5-mediated restriction. Here, we show that activation of innate immune signaling is conserved among primate and carnivore TRIM5 orthologues and among 3 of the 7 mouse Trim5 homologues and that such activity is required for TRIM5-mediated restriction activity. PMID:26468522

  5. Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

    PubMed Central

    Heusinger, Elena; Kirchhoff, Frank

    2017-01-01

    The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

  6. VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling

    PubMed Central

    Afroz, Sharmin; Brownlie, Robert; Fodje, Michel

    2016-01-01

    ABSTRACT The UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo. In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells. IMPORTANCE Since VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication

  7. Signal transduction induced by activated protein C: no role in protection against sepsis?

    PubMed

    Slofstra, Sjoukje H; ten Cate, Hugo; Spek, C Arnold

    2006-08-01

    The anticoagulant activated protein C (APC) is historically known as a risk factor for venous thrombosis. However, after the positive results of the protein C worldwide evaluation in severe sepsis (PROWESS) trial, which showed that APC was the first drug that considerably reduced sepsis-related mortality, APC is considered a pleiotropic protein with both anticoagulant and anti-inflammatory properties. In addition, in vitro studies have suggested that APC-induced intracellular signal transduction is a potential mechanism by which APC might be protective against sepsis. Recently, however, the efficacy of APC in sepsis has been argued, and also the extent to which the signal transduction capacity of APC contributes to its pro-survival effects is debated. Here, we review the role of APC in the body natural defense against sepsis and discuss the mechanism by which APC might act at a cellular level.

  8. Vanderbilt University Study Creates New Roadmap for Cellular Activity - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    Scientists studying cellular processes have long sought to measure redox modifications because they provide one of the normal layers of cell control. But redox disruption or oxidative stress at the cellular level can also create a pathway to diseases like

  9. Reverting p53 activation after recovery of cellular stress to resume with cell cycle progression.

    PubMed

    Lazo, Pedro A

    2017-05-01

    The activation of p53 in response to different types of cellular stress induces several protective reactions including cell cycle arrest, senescence or cell death. These protective effects are a consequence of the activation of p53 by specific phosphorylation performed by several kinases. The reversion of the cell cycle arrest, induced by p53, is a consequence of the phosphorylated and activated p53, which triggers its own downregulation and that of its positive regulators. The different down-regulatory processes have a sequential and temporal order of events. The mechanisms implicated in p53 down-regulation include phosphatases, deacetylases, and protein degradation by the proteasome or autophagy, which also affect different p53 protein targets and functions. The necessary first step is the dephosphorylation of p53 to make it available for interaction with mdm2 ubiquitin-ligase, which requires the activation of phosphatases targeting both p53 and p53-activating kinases. In addition, deacetylation of p53 is required to make lysine residues accessible to ubiquitin ligases. The combined action of these downregulatory mechanisms brings p53 protein back to its basal levels, and cell cycle progression can resume if cells have overcome the stress or damage situation. The specific targeting of these down-regulatory mechanisms can be exploited for therapeutic purposes in cancers harbouring wild-type p53.

  10. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  11. Human immunodeficiency virus long terminal repeat responds to T-cell activation signals

    SciTech Connect

    Tong-Starksen, S.E.; Luciw, P.A.; Peterlin, B.M.

    1987-10-01

    Human immunodeficiency virus (HIV), the causative agent of AIDS, infects and kills lymphoid cells bearing the CD4 antigen. In an infected cell, a number of cellular as well as HIV-encoded gene products determine the levels of viral gene expression and HIV replication. Efficient HIV replication occurs in activated T cells. Utilizing transient expression assays, the authors show that gene expression directed by the HIV long terminal repeat (LTR) increases in response to T-cell activation signals. The effects of T-cell activation and of the HIV-encoded trans-activator (TAT) are multiplicative. Analysis of mutations and deletions in the HIV LTR reveals that the region responding to T-cell activation signals is located at positions -105 to -80. These sequences are composed of two direct repeats, which are homologous to the core transcriptional enhancer elements in the simian virus 40 genome. The studies reveal that these elements function as the HIV enhancer. By acting directly on the HIV LTR, T-cell activation may play an important role in HIV gene expression and in the activation of latent HIV.

  12. Activating adaptive cellular mechanisms of resistance following sublethal cytotoxic chemotherapy: implications for diagnostic microdosing.

    PubMed

    Wurz, Gregory T; DeGregorio, Michael W

    2015-04-01

    As Phase 0 studies have proven to be reasonably predictive of therapeutic dose pharmacokinetics, the application of microdosing has expanded into metabolism, drug-drug interactions and now diagnostics. One potentially serious issue with this application of microdosing that has not been previously discussed is the possibility of activating cellular mechanisms of drug resistance. Here, we provide an overview of Phase 0 microdosing and drug resistance, with an emphasis on cisplatin resistance, followed by a discussion of the potential for inducing acquired resistance to platinum-based or other types of chemotherapy in cancer patients participating in Phase 0 diagnostic microdosing studies. A number of alternative approaches to diagnostic microdosing, such as the human tumor cloning assay and the use of peripheral blood mononuclear cells as a surrogate for measuring DNA adducts, are discussed that would avoid exposing cancer patients to low doses of first-line chemotherapy and the possible risk of triggering cellular mechanisms of acquired resistance. Until it has been established that diagnostic microdosing in cancer patients poses no risk of acquired drug resistance, such studies should be approached with caution.

  13. Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture.

    PubMed

    Harak, Christian; Meyrath, Max; Romero-Brey, Inés; Schenk, Christian; Gondeau, Claire; Schult, Philipp; Esser-Nobis, Katharina; Saeed, Mohsan; Neddermann, Petra; Schnitzler, Paul; Gotthardt, Daniel; Perez-Del-Pulgar, Sofia; Neumann-Haefelin, Christoph; Thimme, Robert; Meuleman, Philip; Vondran, Florian W R; Francesco, Raffaele De; Rice, Charles M; Bartenschlager, Ralf; Lohmann, Volker

    2016-12-19

    With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

  14. Real-time observation of signal recognition particle binding to actively translating ribosomes.

    PubMed

    Noriega, Thomas R; Chen, Jin; Walter, Peter; Puglisi, Joseph D

    2014-10-30

    The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

  15. Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1.

    PubMed

    Hauri, Simon; Wepf, Alexander; van Drogen, Audrey; Varjosalo, Markku; Tapon, Nic; Aebersold, Ruedi; Gstaiger, Matthias

    2013-01-01

    Tissue homeostasis is controlled by signaling systems that coordinate cell proliferation, cell growth and cell shape upon changes in the cellular environment. Deregulation of these processes is associated with human cancer and can occur at multiple levels of the underlying signaling systems. To gain an integrated view on signaling modules controlling tissue growth, we analyzed the interaction proteome of the human Hippo pathway, an established growth regulatory signaling system. The resulting high-resolution network model of 480 protein-protein interactions among 270 network components suggests participation of Hippo pathway components in three distinct modules that all converge on the transcriptional co-activator YAP1. One of the modules corresponds to the canonical Hippo kinase cassette whereas the other two both contain Hippo components in complexes with cell polarity proteins. Quantitative proteomic data suggests that complex formation with cell polarity proteins is dynamic and depends on the integrity of cell-cell contacts. Collectively, our systematic analysis greatly enhances our insights into the biochemical landscape underlying human Hippo signaling and emphasizes multifaceted roles of cell polarity complexes in Hippo-mediated tissue growth control.

  16. Characteristics of Middle School Students Learning Actions in Outdoor Mathematical Activities with the Cellular Phone

    ERIC Educational Resources Information Center

    Daher, Wajeeh; Baya'a, Nimer

    2012-01-01

    Learning in the cellular phone environment enables utilizing the multiple functions of the cellular phone, such as mobility, availability, interactivity, verbal and voice communication, taking pictures or recording audio and video, measuring time and transferring information. These functions together with mathematics-designated cellular phone…

  17. Sam68 Mediates the Activation of Insulin and Leptin Signalling in Breast Cancer Cells

    PubMed Central

    Pérez-Pérez, Antonio; Sánchez-Jiménez, Flora; Vilariño-García, Teresa; de la Cruz, Luis; Virizuela, Juan A.; Sánchez-Margalet, Víctor

    2016-01-01

    Obesity is a well-known risk factor for breast cancer development in postmenopausal women. High insulin and leptin levels seem to have a role modulating the growth of these tumours. Sam68 is an RNA-binding protein with signalling functions that has been found to be overexpressed in breast cancer. Moreover, Sam68 may be recruited to insulin and leptin signalling pathways, mediating its effects on survival, growth and proliferation in different cellular types. We aimed to study the expression of Sam68 and its phosphorylation level upon insulin and leptin stimulation, and the role of Sam68 in the proliferative effect and signalling pathways that are activated by insulin or leptin in human breast adenocarcinoma cells. In the human breast adenocarcinoma cell lines MCF7, MDA-MB-231 and BT-474, Sam68 protein quantity and gene expression were increased upon leptin or insulin stimulation, as it was checked by qPCR and immunoblot. Moreover, both insulin and leptin stimulation promoted an increase in Sam68 tyrosine phosphorylation and negatively regulated its RNA binding capacity. siRNA was used to downregulate Sam68 expression, which resulted in lower proliferative effects of both insulin and leptin, as well as a lower activation of MAPK and PI3K pathways promoted by both hormones. These effects may be partly explained by the decrease in IRS-1 expression by down-regulation of Sam68. These results suggest the participation of Sam68 in both leptin and insulin receptor signaling in human breast cancer cells, mediating the trophic effects of these hormones in proliferation and cellular growth. PMID:27415018

  18. 5'-AMP-activated protein kinase signaling in Caenorhabditis elegans.

    PubMed

    Beale, Elmus G

    2008-01-01

    5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.

  19. Convergence of dopamine and glutamate signaling onto striatal ERK activation in response to drugs of abuse

    PubMed Central

    Cahill, Emma; Salery, Marine; Vanhoutte, Peter; Caboche, Jocelyne

    2014-01-01

    Despite their distinct targets, all addictive drugs commonly abused by humans evoke increases in dopamine (DA) concentration within the striatum. The main DA Guanine nucleotide binding protein couple receptors (GPCRs) expressed by medium-sized spiny neurons of the striatum are the D1R and D2R, which are positively and negatively coupled to cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling, respectively. These two DA GPCRs are largely segregated into distinct neuronal populations, where they are co-expressed with glutamate receptors in dendritic spines. Direct and indirect interactions between DA GPCRs and glutamate receptors are the molecular basis by which DA modulates glutamate transmission and controls striatal plasticity and behavior induced by drugs of abuse. A major downstream target of striatal D1R is the extracellular signal-regulated kinase (ERK) kinase pathway. ERK activation by drugs of abuse behaves as a key integrator of D1R and glutamate NMDAR signaling. Once activated, ERK can trigger chromatin remodeling and induce gene expression that permits long-term cellular alterations and drug-induced morphological and behavioral changes. Besides the classical cAMP/PKA pathway, downstream of D1R, recent evidence implicates a cAMP-independent crosstalk mechanism by which the D1R potentiates NMDAR-mediated calcium influx and ERK activation. The mounting evidence of reciprocal modulation of DA and glutamate receptors adds further intricacy to striatal synaptic signaling and is liable to prove relevant for addictive drug-induced signaling, plasticity, and behavior. Herein, we review the evidence that built our understanding of the consequences of this synergistic signaling for the actions of drugs of abuse. PMID:24409148

  20. Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3

    PubMed Central

    Selvendiran, Karuppaiyah; Bratasz, Anna; Kuppusamy, M. Lakshmi; Tazi, Mia F.; Rivera, Brian K.; Kuppusamy, Periannan

    2010-01-01

    Signal transducer and activator of transcription 3 (STAT3) is activated in a variety of human cancers, including ovarian cancer. The molecular mechanism by which the STAT3 is activated in cancer cells is poorly understood. We observed that human ovarian xenograft tumors (A2780) in mice were severely hypoxic (pO2 ∼ 2 mmHg). We further observed that hypoxic exposure significantly increased the phosphorylation of STAT3 (pSTAT3) at the Tyr705 residue in A2780 cell line. The pSTAT3 (Tyr705) level was highly dependent on cellular oxygenation levels, with a significant increase at <2% O2, and without any change in the pSTAT3 (Ser727) or total STAT3 levels. The pSTAT3 (Tyr705) elevation following hypoxic exposure could be reversed within 12 hr after returning the cells to normoxia. The increased level of pSTAT3 was partly mediated by increased levels of reactive oxygen species generation in the hypoxic cancer cells. Conventional chemotherapeutic drugs cisplatin and taxol were far less effective in eliminating the hypoxic ovarian cancer cells suggesting a role for pSTAT3 in cellular resistance to chemotherapy. Inhibition of STAT3 by AG490 followed by treatment with cisplatin or taxol resulted in a significant increase in apoptosis suggesting that hypoxia-induced STAT3 activation is responsible for chemoresistance. The results have important clinical implications for the treatment of hypoxic ovarian tumors using STAT3-specific inhibitors. PMID:19623660

  1. Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival

    PubMed Central

    Rajala, Ammaji; Wang, Yuhong; Rajala, Raju V.S.

    2016-01-01

    In humans, daylight vision is primarily mediated by cone photoreceptors. These cells die in age-related retinal degenerations. Prolonging the life of cones for even one decade would have an enormous beneficial effect on usable vision in an aging population. Photoreceptors are postmitotic, but shed 10% of their outer segments daily, and must synthesize the membrane and protein equivalent of a proliferating cell each day. Although activation of oncogenic tyrosine kinase and inhibition of tyrosine phosphatase signaling is known to be essential for tumor progression, the cellular regulation of this signaling in postmitotic photoreceptor cells has not been studied. In the present study, we report that a novel G-protein coupled receptor–mediated insulin receptor (IR) signaling pathway is regulated by non-receptor tyrosine kinase Src through the inhibition of protein tyrosine phosphatase IB (PTP1B). We demonstrated the functional significance of this pathway through conditional deletion of IR and PTP1B in cones, in addition to delaying the death of cones in a mouse model of cone degeneration by activating the Src. This is the first study demonstrating the molecular mechanism of a novel signaling pathway in photoreceptor cells, which provides a window of opportunity to save the dying cones in retinal degenerative diseases. PMID:27391439

  2. Structures and Mechanisms of Antitumor Agents - Xestoquinones Uncouple Cellular Respiration and Disrupt HIF Signaling in Human Breast Tumor Cells

    PubMed Central

    Du, Lin; Mahdi, Fakhri; Datta, Sandipan; Jekabsons, Mika B.; Zhou, Yu-Dong; Nagle, Dale G.

    2012-01-01

    The organic extract of a marine sponge Petrosia alfiani selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1 – 7) including three new compounds 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1 – 7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), that possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC50 value of 0.2 μM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration. PMID:22938093

  3. Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells.

    PubMed

    Zhu, Yingting; Zhu, Min; Lance, Peter

    2012-08-31

    COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.

  4. The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

    PubMed

    Salari, Vahid; Scholkmann, Felix; Bokkon, Istvan; Shahbazi, Farhad; Tuszynski, Jack

    2016-01-01

    For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to 'internal photons' inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350-700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation.

  5. Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

    SciTech Connect

    Herr, Michael J.; Longhurst, Celia M.; Baker, Benjamin; Homayouni, Ramin; Speich, Henry E.; Kotha, Jayaprakash; Jennings, Lisa K.

    2014-05-16

    Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

  6. The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

    PubMed Central

    Salari, Vahid; Scholkmann, Felix; Bokkon, Istvan; Shahbazi, Farhad; Tuszynski, Jack

    2016-01-01

    For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to ‘internal photons’ inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350–700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation. PMID:26950936

  7. Cellular prion protein in the bovine mammary gland is selectively expressed in active lactocytes.

    PubMed

    Didier, Andrea; Dietrich, Richard; Steffl, Martin; Gareis, Manfred; Groschup, Martin H; Müller-Hellwig, Simone; Märtlbauer, Erwin; Amselgruber, Werner M

    2006-11-01

    The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.

  8. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    PubMed

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

  9. Chemical, enzymatic and cellular antioxidant activity studies of Agaricus blazei Murrill.

    PubMed

    Hakime-Silva, Ricardo A; Vellosa, José C R; Khalil, Najeh M; Khalil, Omar A K; Brunetti, Iguatemy L; Oliveira, Olga M M F

    2013-09-01

    Mushrooms possess nutritional and medicinal properties that have long been used for human health preservation and that have been considered by researchers as possible sources of free radical scavengers. In this work, the antioxidant properties of water extracts from Agaricus blazei Murill, produced by maceration and decoction, are demonstrated in vitro. Resistance to oxidation is demonstrated through three mechanisms: i) inhibition of enzymatic oxidative process, with 100% inhibition of HRP (horseradish peroxidase) and MPO (myeloperoxidase); ii) inhibition of cellular oxidative stress, with 80% inhibition of the oxidative burst of polymorphonuclear neutrophils (PMNs); and iii) direct action over reactive species, with 62% and 87% suppression of HOCl and superoxide anion radical (O2• -), respectively. From the data, it was concluded that the aqueous extract of A. blazei has significant antioxidant activity, indicating its possible application for nutraceutical and medicinal purposes.

  10. [Activators, receptors and signal transduction pathways of blood platelets].

    PubMed

    Shaturnyĭ, V I; Shakhidzhanov, S S; Sveshnikova, A N; Panteleev, M A

    2014-01-01

    Platelet participation in hemostatic plug formation requires transition into an activated state (or, rather, variety of states) upon action of agonists like ADP, thromboxane A , collagen, thrombin, and others. The mechanisms of action for different agonists, their receptors and signaling pathways associated with them, as well as the mechanisms of platelet response inhibition are the subject of the present review. Collagen exposed upon vessel wall damage induced initial platelet attachment and start of thrombus formation, which involves numerous processes such as aggregation, activation of integrins, granule secretion and increase of intracellular Ca2+. Thrombin