Science.gov

Sample records for 293t hek293t cells

  1. Characterization of crude Echis carinatus venom-induced cytotoxicity in HEK 293T cells.

    PubMed

    Pierce, Rebecca D; Kim, Ethan S; Girton, Lance W; McMurry, Jonathan L; Francis, Joshua W; Albrecht, Eric A

    2011-01-01

    Echis carinatus (saw-scaled viper) produces potent hemorrhagic venom that causes the development of apoptotic and necrotic tissues. In this study, we used polyethyleneimine (PEI) to enhance cellular adherence, and to determine whether the substrate attachment influenced the survival of cells treated with crude E. carinatus venom. Human embryonic kidney (HEK) 293T cells were grown for 18hr in tissue culture plates with or without polyethyleneimine (PEI), and were then stimulated with crude E. carinatus venom for 3 or 12hr. HEK 293T cells grown without PEI displayed a robust oxidative response to corresponding substrate detachment, loss of plasma membrane integrity and decreased cell viability. Cells grown on PEI adsorbed substrates demonstrated prolonged substrate attachment resulting in significantly higher cell viabilities. These observations suggest that the cytotoxicity of crude E. carinatus venom is dependent upon cellular detachment. PMID:22331993

  2. Display and selection of scFv antibodies on HEK-293T cells

    PubMed Central

    Ho, Mitchell; Pastan, Ira

    2009-01-01

    We describe a human cell display strategy to isolate high affinity single chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen's sensitivity thereby allowing us to isolate high affinity scFvs. Using human cell display one could isolate and engineer scFvs, single domains, Fabs or whole IgGs for increased affinity and other biological functions. PMID:19554290

  3. Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

    PubMed Central

    2014-01-01

    Background Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data. Results A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport. Conclusions This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling. PMID:24712863

  4. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

    PubMed Central

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis. PMID:27042825

  5. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation.

    PubMed

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.

  6. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

    PubMed

    Xu, Yunpeng; Zhao, Gang; Zhou, Xiaoming; Ding, Weiping; Shu, Zhiquan; Gao, Dayong

    2014-04-01

    The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min.

  7. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T).

    PubMed

    Xu, Yunpeng; Zhao, Gang; Zhou, Xiaoming; Ding, Weiping; Shu, Zhiquan; Gao, Dayong

    2014-04-01

    The objective of this study is to determine the cryobiological characteristics of human embryonic kidney (HEK293T) cells. The cell membrane hydraulic conductivity (L(pg)) and the activation energy of water transport (E(Lp)) were determined in the absence/presence of cryoprotectant agent (CPA), while the nucleation rate kinetic and thermodynamic parameters (Ωo(SCN) and κo(SCN)) were determined in the absence of CPA. Since dehydration and intracellular ice formation (IIF) are two factors that may cause damage to cells during the freezing process, systematical freezing experiments were carried out at different cooling rates (5, 10, 15, 20, 30, and 60°C/min) under the commercial available cryomicroscopy (FDCS 196, Linkham, Waterfield, UK) to further explore the cryoinjury mechanism for HEK293T cells. By simultaneously fitting the water transport equation to the experimentally measured volumetric shrinkage data at 5, 10, and 15°C/min, the "combined best fit" membrane permeability parameters for HEK293T cells in both phosphate buffer saline (PBS) and CPA media (0.75M Me2SO in PBS) are determined. They are L(pg)=2.85×10(-14)m/s/Pa (0.17μm/min/atm), E(Lp)=142.91kJ/mol (34.13kcal/mol) (R(2)=0.990), and L(pg)[cpa]=2.73±0.44×10(-14)m/s/Pa (0.16±0.03μm/min/atm), E(Lp)[cpa]=152.52±27.69kJ/mol (36.42±6.61kcal/mol) (R(2)=0.993), respectively. An optimal cooling rate B(opt) (the highest cooling rate without IIF) was determined to be 14.24°C/min in the absence of CPA. Additionally, the ice nucleation parameters (Ωo(SCN) and κo(SCN)) were averaged to be 1.31±0.11×10(8)m(-2)s(-1) and 7.67±2.55×10(9)K(5) for the cooling rates 20, 30, and 60°C/min. PMID:24582893

  8. The role of sensitivity of ALA (PpIX)-based PDT on Human embryonic kidney cell line (HEK293T)

    NASA Astrophysics Data System (ADS)

    Fakhar-e-Alam, M.; Atif, M.; Rehman, T.; Sadia, H.; Firdous, S.

    2011-08-01

    Present study evaluates the effects of photodynamic therapy (PDT) with aminolevulinic acid (5-ALA) as photo sensitizer using Human embryonic kidney (HEK293T) cell line as an experimental model. Porphyrins derivatives are used as active cytotoxic antitumor agents in PDT. Above mentioned cell line were irradiated with red light (a diode laser, λ = 635 nm) at different doses (0-160 J/cm2) of light. The influence/effectiveness of incubation time, various concentrations of aminolevulinic acid (5-ALA) and light doses on the cellular viability was studied. HEK293T cells were deliberated by exposing the ALA-PpIX (0-1000 μg/ml) of concentrations. The optimal uptakes of photosensitizer (PS) in cell lines were investigated by means of spectro photo metric measurements. Cells viability was determined by means of neutral red assay (NRA). It was observed that alone, neither photosensitizer nor light dose have significant effect on cells viability, but optimal concentration of PS along with suitable dose of light exhibit effective impact on the viability of cell. Our results showed that light doses of 40 J/cm2 demonstrates effective PDT outcome for HEK293T cell line when incubated with 400 μg/ml, with wrapping up view that HEK293T cell line is very sensitive to ALA-mediated PDT as compared to cell line published in our data. At the end results has been verified by using reactive oxygen species (ROS) measure test.

  9. DAPIT Over-Expression Modulates Glucose Metabolism and Cell Behaviour in HEK293T Cells

    PubMed Central

    Kontro, Heidi; Cannino, Giuseppe; Rustin, Pierre; Dufour, Eric; Kainulainen, Heikki

    2015-01-01

    Introduction Diabetes Associated Protein in Insulin-sensitive Tissues (DAPIT) is a subunit of mitochondrial ATP synthase and has also been found to associate with the vacuolar H+-ATPase. Its expression is particularly high in cells with elevated aerobic metabolism and in epithelial cells that actively transport nutrients and ions. Deletion of DAPIT is known to induce loss of mitochondrial ATP synthase but the effects of its over-expression are obscure. Results In order to study the consequences of high expression of DAPIT, we constructed a transgenic cell line that constitutively expressed DAPIT in human embryonal kidney cells, HEK293T. Enhanced DAPIT expression decreased mtDNA content and mitochondrial mass, and saturated respiratory chain by decreasing H+-ATP synthase activity. DAPIT over-expression also increased mitochondrial membrane potential and superoxide level, and translocated the transcription factors hypoxia inducible factor 1α (Hif1α) and β-catenin to the nucleus. Accordingly, cells over-expressing DAPIT used more glucose and generated a larger amount of lactate compared to control cells. Interestingly, these changes were associated with an epithelial to mesenchymal (EMT)-like transition by changing E-cadherin to N-cadherin and up-regulating several key junction/adhesion proteins. At physiological level, DAPIT over-expression slowed down cell growth by G1 arrest and migration, and enhanced cell detachment. Several cancers also showed an increase in genomic copy number of Usmg5 (gene encoding DAPIT), thereby providing strong correlative evidence for DAPIT possibly having oncogenic function in cancers. Conclusions DAPIT over-expression thus appears to modulate mitochondrial functions and alter cellular regulations, promote anaerobic metabolism and induce EMT-like transition. We propose that DAPIT over-expression couples the changes in mitochondrial metabolism to physiological and pathophysiological regulations, and suggest it could play a critical

  10. Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

    PubMed Central

    Mao, Yingying; Yan, Renhe; Li, Andrew; Zhang, Yanling; Li, Jinlong; Du, Hongyan; Chen, Baihong; Wei, Wenjin; Zhang, Yi; Sumners, Colin; Zheng, Haifa; Li, Hongwei

    2015-01-01

    . Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells. Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells. PMID:26005375

  11. Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons.

    PubMed

    Costain, Willard J; Tauskela, Joseph S; Rasquinha, Ingrid; Comas, Tanya; Hewitt, Melissa; Marleau, Vincent; Soo, Evelyn C

    2016-09-01

    There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs. PMID:27260125

  12. Measuring Ca2+-Dependent Modulation of Voltage-Gated Ca2+ Channels in HEK-293T Cells.

    PubMed

    Thomas, Jessica R; Lee, Amy

    2016-01-01

    Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells. PMID:27587775

  13. Transcriptome Analysis Reveals a Signature Profile for Tick-Borne Flavivirus Persistence in HEK 293T Cells

    PubMed Central

    Lam, Jennifer; Offerdahl, Danielle K.; Martens, Craig; Sturdevant, Daniel; Turner, Charles V.; Porcella, Stephen F.; Bloom, Marshall E.

    2016-01-01

    ABSTRACT Tick-borne flaviviruses (TBFVs) cause febrile illnesses, which may progress to severe encephalitis and/or death in humans globally. Most people who recover from severe acute disease suffer from debilitating neurological sequelae, which may be due to viral persistence, infection-induced neurological cell damage, host response, or some combination of these. Acute TBFV infection of human embryonic kidney (HEK) 293T cells in vitro results in the death of >95% of infected cells by day 5. However, replacing cell growth medium allows surviving cells to repopulate and become persistently infected for extended periods of time. The mechanisms responsible for initiation and maintenance of viral persistence remain vague. We subjected the HEK 293T cell transcriptome to deep sequencing to identify genes differentially expressed during acute infection and persistent infection. A total of 451 genes showed unique significant differential expression levels in persistently infected cells relative to the acute phase of infection. Ingenuity Pathway Analysis results suggested that the expression of prosurvival oncogenes AKT2 and ERBB2 was upregulated in persistently infected cells, whereas proapoptotic genes, such as Bad and the beta interferon 1 (IFN-β1) gene, were downregulated. Genes encoding antiviral cytokines such as the CCL5, tumor necrosis factor alpha (TNF-α), and CXCL10 genes were upregulated during the acute phase, but the same genes were relatively quiescent in persistently infected cells. Exogenous induction of apoptosis demonstrated that persistently infected cells were resistant to apoptosis in a dose-dependent manner. In summary, the differential transcriptome profiles of acute-phase compared to persistently infected HEK 293T cells demonstrated an evasion of apoptosis, which may be critical for a chronic TBFV infection state. These results provide a basis for further study of the mechanisms of TBFV persistence. PMID:27222466

  14. Protective effect of reduced glutathione C60 derivative against hydrogen peroxide-induced apoptosis in HEK 293T cells.

    PubMed

    Huang, Jin; Zhou, Chi; He, Jun; Hu, Zheng; Guan, Wen-Chao; Liu, Sheng-Hong

    2016-06-01

    Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and (1)H nuclear magnetic resonance were used to confirm the chemical structure of C60-GSH. Our results demonstrated that C60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C60-GSH were superior to those of GSH. In conclusion, these results suggested that C60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.

  15. Protective effect of reduced glutathione C60 derivative against hydrogen peroxide-induced apoptosis in HEK 293T cells.

    PubMed

    Huang, Jin; Zhou, Chi; He, Jun; Hu, Zheng; Guan, Wen-Chao; Liu, Sheng-Hong

    2016-06-01

    Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and (1)H nuclear magnetic resonance were used to confirm the chemical structure of C60-GSH. Our results demonstrated that C60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C60-GSH were superior to those of GSH. In conclusion, these results suggested that C60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity. PMID:27376803

  16. Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells

    PubMed Central

    Mlera, Luwanika; Offerdahl, Danielle K.; Martens, Craig; Porcella, Stephen F.; Melik, Wessam

    2015-01-01

    ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. PMID:26045539

  17. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc. PMID:27563853

  18. Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

    PubMed

    Sanchez-Rodriguez, Sandra P; Sauer, Jeremy P; Stanley, Sarah A; Qian, Xi; Gottesdiener, Andrew; Friedman, Jeffrey M; Dordick, Jonathan S

    2016-10-01

    Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc.

  19. COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits

    PubMed Central

    Bailey Blackburn, Jessica; Pokrovskaya, Irina; Fisher, Peter; Ungar, Daniel; Lupashin, Vladimir V.

    2016-01-01

    The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole. PMID:27066481

  20. Impact of phosphomimetic and non-phosphorylatable mutations of phospholemman on L-type calcium channels gating in HEK 293T cells

    PubMed Central

    Guo, Kai; Wang, Yue-Peng; Zhou, Zhi-Wen; Jiang, Yi-Bo; Li, Wei; Chen, Xiao-Meng; Li, Yi-Gang

    2015-01-01

    Background: Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods: The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results: WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions: Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects. PMID:25656605

  1. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

    SciTech Connect

    Thakur, Basant Kumar; Dittrich, Tino; Chandra, Prakash; Becker, Annette; Lippka, Yannick; Selvakumar, Divakarvel; Klusmann, Jan-Henning; Reinhardt, Dirk; Welte, Karl

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer In 293T cells, p53 is considered to be inactive due to its interaction with the large T-antigen. Black-Right-Pointing-Pointer Acetylation of p53 at lysine 382 is important for its functional activation. Black-Right-Pointing-Pointer First evidence to document the presence of a functional p53 in 293T cells. Black-Right-Pointing-Pointer Inhibition of NAMPT/SIRT pathway by FK866 in 293T cells increases the functional activity of p53. Black-Right-Pointing-Pointer This activation of p53 involves reversible acetylation of p53 at lysine 382. -- Abstract: Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.

  2. Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    PubMed Central

    Yang, Liang; Wang, Xu; Deng, Weixian; Mo, Weiliang; Gao, Jie; Liu, Qing; Zhang, Chuanyu; Wang, Qin; Lin, Chentao; Zuo, Zecheng

    2016-01-01

    Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein–protein interactions. PMID:27446167

  3. Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

    PubMed

    You, Yun-Wen; Chang, Hsun-Yun; Liao, Hua-Yang; Kao, Wei-Lun; Yen, Guo-Ji; Chang, Chi-Jen; Tsai, Meng-Hung; Shyue, Jing-Jong

    2012-10-01

    Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

  4. Acute effects of pregabalin on the function and cellular distribution of Ca(V)2.1 in HEK293t cells.

    PubMed

    Weissmann, Carina; Di Guilmi, Mariano N; Urbano, Francisco J; Uchitel, Osvaldo D

    2013-01-01

    We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated Ca(V)2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the Ca(V)2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through Ca(V)2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the Ca(V)2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on Ca(V)2.1 calcium channels' currents and distribution and suggest that internalization of Ca(V)2.1 channels might be a mechanism of PGB action. PMID:23063705

  5. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

    PubMed

    Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

    2014-03-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  6. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    PubMed Central

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  7. Nephrotoxicity evaluation of a new cembrane diterpene from Euphorbiae pekinensis Radix with HEK 293T cells and the toxicokinetics study in rats using a sensitive and reliable UFLC-MS/MS.

    PubMed

    Zhang, Yuanyuan; Liu, Ziying; Zhang, Ruowen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui

    2016-02-01

    (-)-(1S)-15-Hydroxy-18-carboxycembrene, the first cembrane-type diterpenoid found in the family Euphorbiaceae, isolated from Euphorbiae pekinensis Radix, was identified to be nephrotoxic using HEK 293T cells. Tests on cell morphology, cell viability and biochemical markers about oxidation stress were carried out using inverted microscope, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and commercial kits respectively, which proved the diterpene time- and dose-dependently decreased cells proliferation. Besides, a sensitive and robust UFLC-MS/MS method was developed and fully validated to investigate the in vivo behavior in rats of the diterpene after oral administration of Euphorbiae pekinensis Radix extracts at a dosage of 9g/kg. The method showed a good linearity in tested range (3-1500ng/mL) with acceptable accuracy and precision. The recovery of the diterpene was more than 85% and the matrix effect was within ±20%. The toxicokinetics parameters indicate the diterpene reached Cmax quickly and slowly eliminate. The study proved the newly found diterpene was one of the nephrotoxic substances of Euphorbiae pekinensis Radix and revealed its toxicokinetics behavior.

  8. Effects of single N-glycosylation site knockout on folding and defibrinogenating activities of acutobin recombinants from HEK293T.

    PubMed

    Tsai, Inn-Ho; Wang, Ying-Ming; Huang, Kai-Fa

    2015-02-01

    Acutobin, the α-fibrinogenase from Deinagkistrodon acutus venom, contains four N-glycosylation sites with disialylated complex-typed glycans. Here, we explore the functional roles of each of the N-glycan by site-directed mutagenesis. The wild-type (ATB-wt) and single glycan-knockout mutants of recombinant acutobin were prepared from HEK293T, demonstrating that mutations at Asn(77), Asn(81) and Asn(100) impaired the folding while the S79A mutant and various Asn(229)-deglycosylated mutants were correctly folded. Based on homology modeling of acutobin and multiple sequence alignment with various venom thrombin-like enzymes, the importance of a hydrophilic environment at each glycosylation site to the enzyme folding could be rationalized. Remarkably, all the mutants showed similar catalytic activities for the chromogenic substrate and similar thermal stabilities as ATB-wt, suggesting that the glycan knockout did not affect the gross conformation and stability of the active sites. Although SDS-PAGE analyses revealed that ATB-wt and the D229-mutant degraded all human fibrinogen subunits faster but less specifically in vitro as compared with other mutants that cleaved only the α-subunit, ATB-wt and D229-mutant were not able to release fibrinogen-peptide A and thus coagulated human plasma slower than the other mutants did. In the mice model, the defibrinogenating effect of ATB-wt was stronger and lasting-longer than those of all the mutants. Taken together, all the glycans contribute to the pharmacokinetics of acutobin and ATB-wt in vivo, and the microenvironment around the Asn(229)-glycan appears to regulate the fibrinogen-chain specificity of acutobin while the N-glycans at positions 77, 81 and 100 are crucial for its folding. PMID:25533529

  9. An analytical strategy to investigate Semen Strychni nephrotoxicity based on simultaneous HILIC-ESI-MS/MS detection of Semen Strychni alkaloids, tyrosine and tyramine in HEK 293t cell lysates.

    PubMed

    Gu, Liqiang; Hou, Pengyi; Zhang, Ruowen; Liu, Ziying; Bi, Kaishun; Chen, Xiaohui

    2016-10-15

    A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150mm×2.1mm, 2.7μm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%-107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.

  10. Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

    PubMed

    Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

    2005-07-19

    The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

  11. Molecular delivery into live cells using gold nanoparticle coated substrates fabricated by pulsed laser annealing

    NASA Astrophysics Data System (ADS)

    Wu, Ting-Hsiang; Xiao, Fan; Teitell, Michael A.; Chiou, Pei-Yu

    2010-02-01

    A massively-parallel molecular delivery system for mammalian cells is demonstrated by pulsed-laser irradiation of a gold-nanoparticles-coated substrate situated below a cell monolayer. This system is capable of high throughput and spatially-targeted delivery into desired areas of a cell culture by designing the laser irradiation pattern. Large area, rapid fabrication of the gold-nanoparticle-coated substrate is achieved by pulsed laser annealing of a continuous gold thin film. Randomly distributed gold nanoparticles and periodic gold nanoparticle arrays were obtained by pulsed laser flood exposure and by polymer mold guided laser annealing respectively. Optical image patterned molecular delivery into adherent cells were demonstrated in HeLa and HEK 293T cells. Delivery efficiencies of fluorescent dye, calcein, were as high as >90% with high cell viability (>90%) in HEK 293T using this device.

  12. Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.

    PubMed

    Albrecht, Christian; Hosiner, Stefanie; Tichy, Brigitte; Aldrian, Silke; Hajdu, Stefan; Nürnberger, Sylvia

    2015-06-01

    Lentiviral transduction is a highly efficient DNA delivery method for RNA interference applications. However, obtaining high lentiviral titers of shRNA and miRNA encoding vectors is challenging, since shRNA and miRNA cassettes have been shown to reduce lentiviral titers. In this study, we compare four commercially available packaging mixes and two producer cell lines in order to optimize lentiviral production for gene silencing experiments. Lentiviral vectors encoding a miRNA sequence and emerald green fluorescence protein were co-transfected with ViraPower™, Lenti-X™ HTX, MISSION(®) Lentiviral or Trans-Lentiviral™ packaging mix in HEK-293T or 293FT cells. After transducing HeLa cells with virus-containing supernatant, lentiviral titers were determined by flow cytomerty. In both cell lines, the highest lentiviral titer was obtained with MISSION(®) Lentiviral packaging mix, followed by ViraPower™, Lenti-X™ HTX, and Trans-Lentiviral™. On average, HEK-293T cells produced 6.2-fold higher lentiviral titers than 293FT cells (p < 0.001). With the combination of MISSION(®) Lentiviral packaging mix and HEK-293T cells, an up to 48.5-fold higher lentiviral titer was reached compared to other packaging mixes and producer cell lines. The optimized selection of packaging mix and cell line described in this work should facilitate the production of high-titer lentiviruses for gene silencing experiments. PMID:25616840

  13. Effect of the ARG1 gene on arsenic resistance of 293T cells.

    PubMed

    Mu, X L; Li, L; Li, H

    2013-01-01

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells. PMID:24391030

  14. Effect of the ARG1 gene on arsenic resistance of 293T cells.

    PubMed

    Mu, X L; Li, L; Li, H

    2013-12-19

    To study the relationship between arsenic resistance of 293T cells and overexpression of ARG1, the ARG1 gene in a recombinant plasmid was transfected into 293T cells via liposomes, and then ARG1 overexpression was examined by real-time PCR and immunocytochemistry. The survival rate, arsenic accumulation and arsenic efflux, GSH level, and GST activity of 293T cells overexpressing ARG1 were assayed by MTT, atomic absorption spectrophotometry, and DTNB, and expression of MRP-2 was detected by Western blot analysis. Compared to that in the control cells, the survival rate of ARG1 gene-overexpressing cells was much higher following exposure to lower sodium arsenite (≤ 8 µM). When cells were exposed to lower sodium arsenite for 24 h, the arsenite content of ARG1 gene-overexpressing cells decreased and arsenic efflux increased. After 48 h, the GSH level, GST activity, and expression of MRP2 increased in a concentration-dependent manner. We conclude that the ARG1 gene increases arsenic resistance of 293T cells.

  15. Nek5 interacts with mitochondrial proteins and interferes negatively in mitochondrial mediated cell death and respiration.

    PubMed

    Melo Hanchuk, Talita D; Papa, Priscila Ferreira; La Guardia, Paolo G; Vercesi, Anibal E; Kobarg, Jörg

    2015-06-01

    Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2 μM). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in ROS formation after 4 h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

  16. Nek5 interacts with mitochondrial proteins and interferes negatively in mitochondrial mediated cell death and respiration.

    PubMed

    Melo Hanchuk, Talita D; Papa, Priscila Ferreira; La Guardia, Paolo G; Vercesi, Anibal E; Kobarg, Jörg

    2015-06-01

    Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2 μM). Nek5 silenced cells as well as cells expressing a "kinase dead" version of Nek5, displayed an increase in ROS formation after 4 h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells. PMID:25725288

  17. Comparison of in vitro methods for carboxylesterase activity determination in immortalized cells representative of the intestine, liver and kidney.

    PubMed

    Lamego, Joana; Ferreira, Pedro; Alves, Márcia; Matias, Ana; Simplício, Ana Luisa

    2015-08-01

    Herein we compare the fluorimetric determination of total and specific carboxylesterase activity in immortalized human derived living cells and in cell lysates. The cell lines chosen are representative of metabolism occurring in the intestine (Caco-2 and HT-29), kidney (HEK-293T) and liver (Hep G2). Caco-2 and HT-29, as cells prone to differentiation, were tested along the differentiation time. For evaluation of both methods when distinguishing activity of different carboxylesterases, HEK-293T transfected with the human carboxylestarase-2 (hCES2) were also tested. Application to Caco-2 or HT-29 cells demonstrated higher activity detected in cell lysates than in cell monolayers. The difference is most striking when comparing the methods at different stages of Caco-2 and HT-29 cell maturation, highlighting substrate accessibility as a limiting step in the in vivo hydrolysis rates (possibly limited by plasma and Endoplasmic Reticulum membrane permeability) with increasing relevance as the cells differentiate. Application to Hep G2 or to hCES2 transfected and non-transfected HEK-293T cells, demonstrated a tendency for higher sensitivity in living cell suspensions than that obtained with the cell lysates which indicates the importance of cell environment in the maintenance of enzyme activity. However, quantification of hCES2 activity relative to total esterase, or to total carboxylesterase activity, was not significantly different in any case. The results herein presented help to clarify which method is best suited for evaluation of carboxylesterase activity in vitro depending on the final goal of the study.

  18. A Lentiviral Vector Expressing Desired Gene Only in Transduced Cells: An Approach for Suicide Gene Therapy.

    PubMed

    Mohammadi, Zahra; Shariati, Laleh; Khanahmad, Hossein; Kolahdouz, Mahsa; Kianpoor, Fariborz; Ghanbari, Jahan Afrooz; Hejazi, Zahra; Salehi, Mansoor; Nikpour, Parvaneh; Tabatabaiefar, Mohammad Amin

    2015-09-01

    Suicide gene therapy is a therapeutic strategy, in which cell suicide inducing transgenes are introduced into target cells. Inserting a toxin-encoding gene into a lentiviral vector leads to decreased efficiency of virus production due to lethal effect of toxin on packaging cells. In this study, we designed and constructed a transfer vector to express the toxin in transduced cells but not in packaging cells. Plasmid pLenti-F/GFP was constructed by cutting out R 5'LTR-R 3'LTR fragment with the AflII restriction endonuclease from a plasmid pLenti4-GW/H1/TO-laminshRNA, followed by ligating R 5'LTR-R 3'LTR fragment, constructed by three PCR stages. The promoter and GFP CDS were inserted in opposite strand. For lentiviral production, the HEK293T cell line was co-transfected with the PMD2G, psPAX2, and pLenti-F/GFP plasmids (envelope, packaging, and transfer plasmids).Viral vector titers were assayed. The HEK293T cell line was transduced with this virus. PCR was performed to confirm the presence of the promoter fragment between the R and U5 in 3'LTR. The lentivirus titers were approximately 2 × 10(5). The GFP expression was seen in 51 % of the HEK293T cells transduced with lentivirus. The PCR product size was 1440 bp confirming the promoter fragment position between the R and U5 in 3'LTR. The strategy enables us to use a broad spectrum of toxin genes in gene therapy and helps avoid the death of the packaging cells with lentiviral vectors carrying a toxin-encoding gene, thereby increasing the efficiency of viral production in packaging cells.

  19. Toxicological impact of JWH-018 and its phase I metabolite N-(3-hydroxypentyl) on human cell lines.

    PubMed

    Couceiro, Joana; Bandarra, Susana; Sultan, Haider; Bell, Suzanne; Constantino, Susana; Quintas, Alexandre

    2016-07-01

    The emergence and abuse of synthetic cannabinoids has been increasing as an alternative to cannabis, mainly among youth. As their appearance on the drug market has been recent, the pharmacological and toxicological profiles of these psychoactive substances are poorly understood. Current studies suggest that they have stronger effects compared to their natural alternatives and their metabolites retain affinity towards CB1 receptors in CNS. Since studies on its toxicological properties are scarce, the effects of the drug in human derived cell lines were investigated. The present study was designed to explore the toxicological impact of parent drug versus phase I metabolites of synthetic cannabinoids on human cells with and without CB1 receptor. The human cell line of neuroblastoma SH-SY5Y and human kidney cell line HEK-293T were exposed to JWH-018 and to its N-(3-hydroxypentyl) metabolite. Cell toxicity was evaluated using the MTT and LDH assay. Additionally, a dual staining methodology with fluorescent Annexin V-FITC and propidium iodide was performed to address the question of whether JWH-018 N-(3-hydroxypentyl) metabolite is inducing cell death through apoptosis or necrosis, in HEK293T and SH-SY5Y cell lines. The obtained results show that JWH-018 does not cause a statistically significant decrease in cell viability, in contrast to its N-(3-hydroxypentyl) metabolite, which at ≥25μM causes a significant decrease in cell viability. Both cell lines are affected by JWH-018 metabolite. Our results point to higher toxicity of JWH-018 metabolite when compared to its parent drug, suggesting a non-CB1 receptor mediated toxicological mechanism. Comparing the results from Annexin V/PI with MTT and LDH assays of SH-SY5Y and HEK293T in the presence of the synthetic cannabinoid metabolite, emerges the picture that cellular viability decreases and associated death is occurring through necrosis.

  20. Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus

    PubMed Central

    Kato, Tatsuya; Sugioka, Saki; Itagaki, Kohei; Park, Enoch Y.

    2016-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an alphabaculovirus, has been widely utilized for protein expression in not only insect cells but also mammalian cells. AcMNPV is closely related to Bombyx mori nucleopolyhedrovirus (BmNPV), and nucleotide sequences of AcMNPV genes have high similarity with those of BmNPV. However, the transduction of BmNPV into mammalian cells has not been reported. In this study, we constructed a recombinant BmNPV (BmNPVΔbgp/AcGP64/EGFP) whose surface 64 kDa glycoprotein (BmGP64) was substituted with that from AcMNPV (AcGP64). BmNPVΔbgp/AcGP64/EGFP also carried an EGFP gene under the control of the CMV promoter. BmNPVΔbgp/AcGP64/EGFP successfully transduced HEK293T cells. In comparison, a control construct (BmNPVΔbgp/BmGP64/EGFP) which possessed BmGP64 instead of AcGP64 did not express EGFP in HEK293T cells. The transduction efficiency of BmNPVΔbgp/AcGP64/EGFP was lower than that of an AcMNPV based-BacMam GFP transduction control. This result indicates that AcGP64 facilitates BmNPV transduction into HEK293T cells. BmNPV can be prepared easily on a large scale because BmNPV can infect silkworm larvae without any special equipment, even though specific diet is needed for silkworm rearing. BmNPV gene transduction into mammalian cells can potentially be applied easily for gene delivery into mammalian cells. PMID:27562533

  1. Gene transduction in mammalian cells using Bombyx mori nucleopolyhedrovirus assisted by glycoprotein 64 of Autographa californica multiple nucleopolyhedrovirus.

    PubMed

    Kato, Tatsuya; Sugioka, Saki; Itagaki, Kohei; Park, Enoch Y

    2016-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an alphabaculovirus, has been widely utilized for protein expression in not only insect cells but also mammalian cells. AcMNPV is closely related to Bombyx mori nucleopolyhedrovirus (BmNPV), and nucleotide sequences of AcMNPV genes have high similarity with those of BmNPV. However, the transduction of BmNPV into mammalian cells has not been reported. In this study, we constructed a recombinant BmNPV (BmNPVΔbgp/AcGP64/EGFP) whose surface 64 kDa glycoprotein (BmGP64) was substituted with that from AcMNPV (AcGP64). BmNPVΔbgp/AcGP64/EGFP also carried an EGFP gene under the control of the CMV promoter. BmNPVΔbgp/AcGP64/EGFP successfully transduced HEK293T cells. In comparison, a control construct (BmNPVΔbgp/BmGP64/EGFP) which possessed BmGP64 instead of AcGP64 did not express EGFP in HEK293T cells. The transduction efficiency of BmNPVΔbgp/AcGP64/EGFP was lower than that of an AcMNPV based-BacMam GFP transduction control. This result indicates that AcGP64 facilitates BmNPV transduction into HEK293T cells. BmNPV can be prepared easily on a large scale because BmNPV can infect silkworm larvae without any special equipment, even though specific diet is needed for silkworm rearing. BmNPV gene transduction into mammalian cells can potentially be applied easily for gene delivery into mammalian cells. PMID:27562533

  2. A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1.

    PubMed

    Gonçalves, Cristine; Gross, Fabian; Guégan, Philippe; Cheradame, Hervé; Midou, Patrick

    2014-11-01

    Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 × 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 μg/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000™, and gene expression is higher than observed with FreeStyle™ and JetPEI®. Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications. PMID:25215936

  3. Increasing cellular uptake of mesoporous silica nanoparticles in human embryonic kidney cell line 293T cells by using Lipofectamine 2000.

    PubMed

    Wang, Jiandong; Teng, Zhaogang; Tian, Ying; Fang, Tian; Ma, Jie; Sun, Jin; Zhu, Feipeng; Wu, Jinrong; Wang, Xin; Yang, Nannan; Zhou, Xiaojun; Yun, Shifeng; Lu, Guangming

    2013-11-01

    Mesoporous silica nanoparticles (MSNs) are ideal nanocarriers that have recently gained attention in important bioapplications such as drug, gene, and protein delivery. The efficacy of endocytosis greatly affects the biological functions of MSNs. In the present study, we investigated the effect of cationic liposomes of Lipofectamine 2000 on cellular uptake of MSNs and the cytotoxicity of cationic liposomes combining with MSNs both in vitro and in vivo. Therefore, mesoporous silica nanoparticles with an average diameter of 130 nm and negative surface charge were synthesized and characterized. The possible role of Lipofectamine 2000 in cellular uptake of MSNs was evaluated in human embryonic kidney cell line 293T cells by transmission electron microscopy (TEM) and with inductively coupled plasma (ICP) analysis. The toxicities of liposomes combining with MSNs were tested in vitro via cell apoptosis assay and MTT cell viability assay, and in vivo by histological examination of six organs of mice after intravenous injection. The endocytosis efficiency of MSNs in human embryonic kidney 293T cells was greatly increased using Lipofectamine 2000 compared with controls (P < 0.001). No apparent in vitro or in vivo cytotoxicity was found for Lipofectamine 2000 combining with MSNs. Our data indicate that cationic liposomes of Lipofectamine 2000 has the potential to greatly increase cellular uptake of MSNs with negative surface charge in human renal 293T cells without apparent toxicity. PMID:24059087

  4. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

    PubMed

    Li, Jing; Chen, Xiulan; Yi, Jiao; Liu, Yuchen; Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu; Zhang, Yujing

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  5. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components

    PubMed Central

    Li, Dameng; Wang, Jifeng; Hou, Dongxia; Jiang, Xiaohong; Zhang, Junfeng; Wang, Jin; Zen, Ke; Yang, Fuquan; Zhang, Chen-Yu

    2016-01-01

    Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects. PMID:27649079

  6. Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells.

    PubMed

    Dawaliby, Rosie; Trubbia, Cataldo; Delporte, Cédric; Noyon, Caroline; Ruysschaert, Jean-Marie; Van Antwerpen, Pierre; Govaerts, Cédric

    2016-02-12

    Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.

  7. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  8. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    PubMed Central

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  9. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  10. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation.

    PubMed

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  11. Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction

    PubMed Central

    Shurtleff, Matthew J; Temoche-Diaz, Morayma M; Karfilis, Kate V; Ri, Sayaka; Schekman, Randy

    2016-01-01

    Exosomes are small vesicles that are secreted from metazoan cells and may convey selected membrane proteins and small RNAs to target cells for the control of cell migration, development and metastasis. To study the mechanisms of RNA packaging into exosomes, we devised a purification scheme based on the membrane marker CD63 to isolate a single exosome species secreted from HEK293T cells. Using immunoisolated CD63-containing exosomes we identified a set of miRNAs that are highly enriched with respect to their cellular levels. To explore the biochemical requirements for exosome biogenesis and RNA packaging, we devised a cell-free reaction that recapitulates the species-selective enclosure of miR-223 in isolated membranes supplemented with cytosol. We found that the RNA-binding protein Y-box protein I (YBX1) binds to and is required for the sorting of miR-223 in the cell-free reaction. Furthermore, YBX1 serves an important role in the secretion of miRNAs in exosomes by HEK293T cells. DOI: http://dx.doi.org/10.7554/eLife.19276.001 PMID:27559612

  12. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    PubMed

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

  13. A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells

    PubMed Central

    Durham, Sarah E.; Tarasov, Sergey G.; Walters, Kylie J.

    2015-01-01

    Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome. PMID:26466095

  14. Use of optogenetic technology in cell culture models

    NASA Astrophysics Data System (ADS)

    Erofeev, A. I.; Zakharova, O. A.; Matveev, M. V.; Terekhin, S. G.; Bezprozvanny, I. B.; Vlasova, O. L.

    2016-08-01

    Optogenetic is a powerful method that allows to modulate cellular physiological properties. In our article, we demonstrate changes of electrical properties of cellular membranes on HEK-293T and hippocampal neurons transfected with channelrhodopsins and halorhodopsins induced by blue and orange light stimulation.

  15. Mutational Analysis of the C8-Guanine Adduct of the Environmental Carcinogen 3-Nitrobenzanthrone in Human Cells: Critical Roles of DNA Polymerases η and κ and Rev1 in Error-Prone Translesion Synthesis

    PubMed Central

    2015-01-01

    3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G → T > G → A > G → C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct. PMID:25080294

  16. Effects of a brain-engraftable microglial cell line expressing anti-prion scFv antibodies on survival times of mice infected with scrapie prions.

    PubMed

    Fujita, Koji; Yamaguchi, Yoshitaka; Mori, Tsuyoshi; Muramatsu, Naomi; Miyamoto, Takahito; Yano, Masashi; Miyata, Hironori; Ootsuyama, Akira; Sawada, Makoto; Matsuda, Haruo; Kaji, Ryuji; Sakaguchi, Suehiro

    2011-10-01

    We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.

  17. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

    PubMed

    Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

    2014-10-16

    Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal.

  18. An efficient platform for screening expression and crystallization of glycoproteins produced in human cells

    PubMed Central

    Lee, Jeffrey E.; Fusco, Marnie L.; Saphire, Erica Ollmann

    2010-01-01

    Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks. PMID:19373230

  19. Actin and Arp2/3 localize at the centrosome of interphase cells

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  20. Dopamine D1 and corticotrophin-releasing hormone type-2α receptors assemble into functionally interacting complexes in living cells

    PubMed Central

    Fuenzalida, J; Galaz, P; Araya, K A; Slater, P G; Blanco, E H; Campusano, J M; Ciruela, F; Gysling, K

    2014-01-01

    Background and Purpose Dopamine and corticotrophin-releasing hormone (CRH; also known as corticotrophin-releasing factor) are key neurotransmitters in the interaction between stress and addiction. Repeated treatment with cocaine potentiates glutamatergic transmission in the rat basolateral amygdala/cortex pathway through a synergistic action of D1-like dopamine receptors and CRH type-2α receptors (CRF2α receptors). We hypothesized that this observed synergism could be instrumented by heteromers containing the dopamine D1 receptor and CRF2α receptor. Experimental Approach D1/CRF2α receptor heteromerization was demonstrated in HEK293T cells using co-immunoprecipitation, BRET and FRET assays, and by using the heteromer mobilization strategy. The ability of D1 receptors to signal through calcium, when singly expressed or co-expressed with CRF2α receptors, was evaluated by the calcium mobilization assay. Key Results D1/CRF2α receptor heteromers were observed in HEK293T cells. When singly expressed, D1 receptors were mostly located at the cell surface whereas CRF2α receptors accumulated intracellularly. Interestingly, co-expression of both receptors promoted D1 receptor intracellular and CRF2α receptor cell surface targeting. The heteromerization of D1/CRF2α receptors maintained the signalling through cAMP of both receptors but switched D1 receptor signalling properties, as the heteromeric D1 receptor was able to mobilize intracellular calcium upon stimulation with a D1 receptor agonist. Conclusions and Implications D1 and CRF2α receptors are capable of heterodimerization in living cells. D1/CRF2α receptor heteromerization might account, at least in part, for the complex physiological interactions established between dopamine and CRH in normal and pathological conditions such as addiction, representing a new potential pharmacological target. PMID:25073922

  1. Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

    PubMed Central

    Le, Anne; Stine, Zachary E.; Nguyen, Christopher; Afzal, Junaid; Sun, Peng; Hamaker, Max; Siegel, Nicholas M.; Gouw, Arvin M.; Kang, Byung-hak; Yu, Shu-Han; Cochran, Rory L.; Sailor, Kurt A.; Song, Hongjun; Dang, Chi V.

    2014-01-01

    Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity. PMID:25114222

  2. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    SciTech Connect

    Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  3. EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease.

    PubMed

    Brennaman, Leann H; Moss, Marcia L; Maness, Patricia F

    2014-01-01

    EphrinA/EphA-dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid-NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase-inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E-F loop of the second fibronectin type III domain (Leu(671) -Lys(672) /Ser(673) -Leu(674) ) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3-induced shedding of NCAM in HEK293T cells. EphrinA5-induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia. PSA-NCAM and ephrinA/EphA3 coordinately regulate inhibitory synapse development. Here, we have found that ephrinA5 stimulates EphA3 kinase and ADAM10 activity to promote PSA-NCAM cleavage at a site in its second FNIII repeat, which regulates ephrinA5-induced growth cone collapse in GABAergic and non-GABAergic neurons. These findings identify a new regulatory mechanism which may contribute to inhibitory connectivity.

  4. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  5. Overexpression of YY1 increases the protein production in mammalian cells.

    PubMed

    Tastanova, Aizhan; Schulz, Alexandra; Folcher, Marc; Tolstrup, Anne; Puklowski, Anja; Kaufmann, Hitto; Fussenegger, Martin

    2016-02-10

    The production of therapeutic antibodies using mammalian cells remains a high-priority in the biopharmaceutical manufacturing industry. Bioengineers have targeted different cellular processes, including transcription, translation, secretion and post-translational modifications, to overcome the metabolic bottlenecks limiting production capacity and create high-producing mammalian cell lines. The polycomb group (PcG) proteins belong to a family of chromatin regulators with important roles in multicellular development. By overexpressing and screening genes from the PcG family, we have identified an epigenetic key player for biopharmaceutical manufacturing enhancement: the transcription factor Yin Yang 1 (YY1). The overexpression of YY1 led to an increase in the production of several product genes (SEAP, VEGF165, IgG including Rituximab), provided that human YY1 (hYY1) was expressed in human cells (HeLa, HT-1080, HEK-293T, FreeStyle™ 293-F) and Chinese hamster ovary cell-derived YY1 (cYY1) was expressed in CHO cells (CHO-K1, CHO-easyC, FreeStyle™ CHO-S, CHO-B13-24, CHO-IgG1). Ectopic expression of cYY1 in the stable CHO-derived IgG producer cell lines CHO-B13-24 and CHO-IgG1 increased the antibody titer up to 6-fold, suggesting that epigenetic engineering of mammalian production cell lines could become a new strategy to improve the manufacturing of complex protein pharmaceuticals. PMID:26686315

  6. Telomere uncapping by the G-quadruplex ligand RHPS4 inhibits clonogenic tumour cell growth in vitro and in vivo consistent with a cancer stem cell targeting mechanism

    PubMed Central

    Phatak, P; Cookson, J C; Dai, F; Smith, V; Gartenhaus, R B; Stevens, M F G; Burger, A M

    2007-01-01

    The pentacyclic acridinium methosulfate salt RHPS4 induces the 3′single-stranded guanine-rich telomeric overhang to fold into a G-quadruplex structure. Stabilisation of the latter is incompatible with an attachment of telomerase to the telomere and thus G-quadruplex ligands can effectively inhibit both the catalytic and capping functions of telomerase. In this study, we examined mechanisms underlying telomere uncapping by RHPS4 in uterus carcinoma cells (UXF1138L) with short telomeres and compared the susceptibility of bulk and clonogenic cancer cells to the G-quadruplex ligand. We show that treatment of UXF1138L cells with RHPS4 leads to the displacement of the telomerase catalytic subunit (hTERT) from the nucleus, induction of telomere-initiated DNA-damage signalling and chromosome fusions. We further report that RHPS4 is more potent against cancer cells that grow as colonies in soft agar than cells growing as monolayers. Human cord blood and HEK293T embryonic kidney cell colony forming units, however, were more resistant to RHPS4. RHPS4-treated UXF1138L xenografts had a decreased clonogenicity, showed loss of nuclear hTERT expression and an induction of mitotic abnormalities compared with controls. Although single-agent RHPS4 had limited in vivo efficacy, a combination of RHPS4 with the mitotic spindle poison Taxol caused tumour remissions and further enhancement of telomere dysfunction. PMID:17406367

  7. SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD.

    PubMed

    Cramer-Morales, Kimberly; Heer, Collin D; Mapuskar, Kranti A; Domann, Frederick E

    2015-12-01

    To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells. PMID:26208779

  8. Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

    SciTech Connect

    Fu Guo; Yang Huayan; Wang Chen; Zhang Feng; You Zhendong; Wang Guiying; He Cheng; Chen Yizhang . E-mail: yzchen0928@yahoo.com; Xu Zhihan . E-mail: zzxu@mail.shcnc.ac.cn

    2006-08-04

    Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to {beta}{sub 2} subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.

  9. Proteomic analysis of the human cyclin-dependent kinase family reveals a novel CDK5 complex involved in cell growth and migration.

    PubMed

    Xu, Shuangbing; Li, Xu; Gong, Zihua; Wang, Wenqi; Li, Yujing; Nair, Binoj Chandrasekharan; Piao, Hailong; Yang, Kunyu; Wu, Gang; Chen, Junjie

    2014-11-01

    Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases. PMID:25096995

  10. Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.

    PubMed

    Khandelia, Piyush; Yap, Karen; Makeyev, Eugene V

    2011-08-01

    Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest. PMID:21768390

  11. Interaction of tRNA with MEK2 in pancreatic cancer cells

    PubMed Central

    Wang, Xiaoyun; Chow, Christina R.; Ebine, Kazumi; Lee, Jiyoung; Rosner, Marsha R.; Pan, Tao; Munshi, Hidayatullah G.

    2016-01-01

    Although the translational function of tRNA has long been established, extra translational functions of tRNA are still being discovered. We previously developed a computational method to systematically predict new tRNA-protein complexes and experimentally validated six candidate proteins, including the mitogen-activated protein kinase kinase 2 (MEK2), that interact with tRNA in HEK293T cells. However, consequences of the interaction between tRNA and these proteins remain to be elucidated. Here we tested the consequence of the interaction between tRNA and MEK2 in pancreatic cancer cell lines. We also generated disease and drug resistance-derived MEK2 mutants (Q60P, P128Q, S154F, E207K) to evaluate the function of the tRNA-MEK2 interaction. Our results demonstrate that tRNA interacts with the wild-type and mutant MEK2 in pancreatic cancer cells; furthermore, the MEK2 inhibitor U0126 significantly reduces the tRNA-MEK2 interaction. In addition, tRNA affects the catalytic activity of the wild type and mutant MEK2 proteins in different ways. Overall, our findings demonstrate the interaction of tRNA with MEK2 in pancreatic cancer cells and suggest that tRNA may impact MEK2 activity in cancer cells. PMID:27301426

  12. Soluble VEGF receptor 1 (sFLT1) induces non-apoptotic death in ovarian and colorectal cancer cells

    PubMed Central

    Miyake, Tatsuya; Kumasawa, Keiichi; Sato, Noriko; Takiuchi, Tsuyoshi; Nakamura, Hitomi; Kimura, Tadashi

    2016-01-01

    Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFLT1) is an angiogenesis inhibitor that competes with angiogenic factors such as VEGF and Placental Growth Factor (PlGF). Imbalances of VEGF and sFLT1 levels can cause pathological conditions such as tumour growth or preeclampsia. We observed direct damage caused by sFLT1 in tumour cells. We exposed several kinds of cells derived from ovarian and colorectal cancers as well as HEK293T cells to sFLT1 in two ways, transfection and exogenous application. The cell morphology and an LDH assay revealed cytotoxicity. Additional experiments were performed to clarify how sFLT1 injured cells. In this study, non-apoptotic cell damage was found to be induced by sFLT1. Moreover, sFLT1 showed an anti-tumour effect in a mouse model of ovarian cancer. Our results suggest that sFLT1 has potential as a cancer therapeutic candidate. PMID:27103202

  13. A New Fluorescence-Based Reporter Gene Vector as a Tool for Analyzing and Fishing Cells with Activated Wnt Signaling Pathway

    PubMed Central

    Reischmann, Patricia; Müller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β-galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way. PMID:24066239

  14. A new fluorescence-based reporter gene vector as a tool for analyzing and fishing cells with activated wnt signaling pathway.

    PubMed

    Apfel, Johanna; Reischmann, Patricia; Müller, Oliver

    2013-01-01

    The dysregulated Wnt pathway is a major cause for the activation of cell proliferation and reduced differentiation in tumor cells. Therefore the Wnt signaling pathway is the on-top target in searching for new anticancer drugs or therapeutic strategies. Although the key players of the pathway are known, no specific anti-Wnt drug entered a clinical trial by now. Several screening approaches for potential compounds have been performed with a reporter gene assay using multiple T-cell factor/lymphoid enhancer factor (TCF/LEF) binding motifs as promoters which control luciferase or β -galactosidase as reporter genes. In our work, we designed a reporter gene construct which anchors the enhanced green fluorescent protein (eGFP) to the plasma membrane. HEK 293T cells, which were stably transfected with this construct, express eGFP on the outer membrane after activation with either LiCl or WNT3A protein. Thus, cells with activated Wnt pathway could be identified and fished out of a heterogeneous cell pool by the use of magnetic-labeled anti-GFP antibodies. In summary, we present a new tool to easily detect, quantify, and sort cells with activated Wnt signaling pathway in a simple, fast, and cost-effective way.

  15. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry

    PubMed Central

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun’ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  16. 7SK small nuclear RNA inhibits cancer cell proliferation through apoptosis induction.

    PubMed

    Keramati, Farid; Seyedjafari, Ehsan; Fallah, Parviz; Soleimani, Masoud; Ghanbarian, Hossein

    2015-04-01

    7SK small nuclear RNA (snRNA) is a 331-333-bp non-coding RNA, which recruits HEXIM 1/2 protein to inhibit positive elongation factor b (P-TEFb) activity. P-TEFb is an essential factor in alleviating promoter-proximal paused RNA polymerase II (Pol II) and initiating the productive elongation phase of gene transcription. Without this protein, Pol II will remain in its hypophosphorylated state, and no transcription occurs. In this study, we inhibited P-TEFb activity by over-expressing 7SK snRNA in human embryonic kidney (HEK) 293T cancer cell line. This inhibition led to a significant decrease in cell viability, which can be due to the transcription inhibition. Moreover, 7SK snRNA over-expression promoted apoptosis in cancerous cells. Our results suggest 7SK snRNA as a potential endogenous anti-cancer agent, and to the best of our knowledge, this is the first study that uses a long non-coding RNA's over-expression against cancer cell growth and proliferation.

  17. Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei

    PubMed Central

    Sinclair, Sara H. G.; Garcia-Garcia, Jose C.; Dumler, J. Stephen

    2015-01-01

    Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated. PMID:25705208

  18. Deciphering the Receptor Repertoire Encoding Specific Odorants by Time-Lapse Single-Cell Array Cytometry.

    PubMed

    Suzuki, Masato; Yoshimoto, Nobuo; Shimono, Ken; Kuroda, Shun'ichi

    2016-01-01

    Mammals can recognize a vast number of odorants by using olfactory receptors (ORs) known as G protein-coupled receptors. The OR gene family is one of the most diverse gene families in mammalian genomes. Because of the vast combinations of ORs and odorants, few ORs have thus far been linked to specific odorants. Here, we established a functional screening method for OR genes by using a microchamber array containing >5,400 single olfactory epithelium-derived cells from mice applied to time-lapse single-cell array cytometry. This method facilitated the prompt isolation of single olfactory sensory neurons (OSNs) responding to the odorant of interest. Subsequent single-cell RT-PCR allowed us to isolate the genes encoding respective ORs. By using volatile molecules recognized as biomarkers for lung cancers, this method could deorphanize ORs and thereby reconstitute the OR-mediated signaling cascade in HEK293T cells. Thus, our system could be applied to identify any receptor by using specific ligands in the fields of physiopathology and pharmacology. PMID:26832639

  19. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations.

    PubMed

    Ramakrishna, Suresh; Cho, Seung Woo; Kim, Sojung; Song, Myungjae; Gopalappa, Ramu; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    RNA-guided endonucleases (RGENs), which are based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system, have recently emerged as a simple and efficient tool for genome editing. However, the activities of prepared RGENs are sometimes low, hampering the generation of cells containing RGEN-induced mutations. Here we report efficient methods to enrich cells containing RGEN-induced mutations by using surrogate reporters. HEK293T cells are cotransfected with the reporter plasmid, a plasmid encoding Cas9 and a plasmid encoding crRNA and tracrRNA, and subjected to flow cytometric sorting, magnetic separation or hygromycin selection. The selected cell populations are highly enriched with cells containing RGEN-induced mutations, by a factor of up to 11-fold as compared with the unselected population. The fold enrichment tends to be high when RGEN activity is low. We envision that these reporters will facilitate the use of RGEN in a wide range of biomedical research. PMID:24569644

  20. Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

    NASA Astrophysics Data System (ADS)

    Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

    2015-12-01

    Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure.

  1. Compact Wireless Microscope for In-Situ Time Course Study of Large Scale Cell Dynamics within an Incubator

    PubMed Central

    Jin, Di; Wong, Dennis; Li, Junxiang; Luo, Zhang; Guo, Yiran; Liu, Bifeng; Wu, Qiong; Ho, Chih-Ming; Fei, Peng

    2015-01-01

    Imaging of live cells in a region of interest is essential to life science research. Unlike the traditional way that mounts CO2 incubator onto a bulky microscope for observation, here we propose a wireless microscope (termed w-SCOPE) that is based on the “microscope-in-incubator” concept and can be easily housed into a standard CO2 incubator for prolonged on-site observation of the cells. The w-SCOPE is capable of tunable magnification, remote control and wireless image transmission. At the same time, it is compact, measuring only ~10 cm in each dimension, and cost-effective. With the enhancement of compressive sensing computation, the acquired images can achieve a wide field of view (FOV) of ~113 mm2 as well as a cellular resolution of ~3 μm, which enables various forms of follow-up image-based cell analysis. We performed 12 hours time-lapse study on paclitaxel-treated MCF-7 and HEK293T cell lines using w-SCOPE. The analytic results, such as the calculated viability and therapeutic window, from our device were validated by standard cell detection assays and imaging-based cytometer. In addition to those end-point detection methods, w-SCOPE further uncovered the time course of the cell’s response to the drug treatment over the whole period of drug exposure. PMID:26681552

  2. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

    PubMed

    Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B; Mayerl, Steven J; Chan, Amanda H; Zhang, Bin; Karlin-Neumann, George A; Conklin, Bruce R

    2016-01-01

    Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology. PMID:27030102

  3. Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

    PubMed

    Sala-Rabanal, Monica; Yurtsever, Zeynep; Nichols, Colin G; Brett, Tom J

    2015-03-17

    Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

  4. A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death.

    PubMed

    Wetzel-Smith, Monica K; Hunkapiller, Julie; Bhangale, Tushar R; Srinivasan, Karpagam; Maloney, Janice A; Atwal, Jasvinder K; Sa, Susan M; Yaylaoglu, Murat B; Foreman, Oded; Ortmann, Ward; Rathore, Nisha; Hansen, David V; Tessier-Lavigne, Marc; Mayeux, Richard; Pericak-Vance, Margaret; Haines, Jonathan; Farrer, Lindsay A; Schellenberg, Gerard D; Goate, Alison; Behrens, Timothy W; Cruchaga, Carlos; Watts, Ryan J; Graham, Robert R

    2014-12-01

    We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including β-amyloid (Aβ), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain. PMID:25419706

  5. Differential expression and HIV-1 regulation of μ-opioid receptor splice variants across human central nervous system cell types

    PubMed Central

    Dever, Seth M.; Xu, Ruqiang; Fitting, Sylvia; Knapp, Pamela E.; Hauser, Kurt F.

    2013-01-01

    The μ-opioid receptor (MOR) is known to undergo extensive alternative splicing as numerous splice variants of MOR have been identified. However, the functional significance of MOR variants, as well as how splice variants other than MOR-1 might differentially regulate HIV-1 pathogenesis in the CNS, or elsewhere, has been largely ignored. Our findings suggest that there are specific differences in the MOR variant expression profile among CNS cell types, and that the expression levels of these variants are differentially regulated by HIV-1. While MOR-1A mRNA was detected in astroglia, microglia and neurons, MOR-1 and MOR-1X were only found in astroglia. Expression of the various forms of MOR along with the chimeric G protein qi5 in HEK-293T cells resulted in differences in calcium/NFAT signaling with morphine treatment, suggesting that MOR variant expression might underlie functional differences in MOR-effector coupling and intracellular signaling across different cell types. Furthermore, the data suggest that the expression of MOR-1 and other MOR variants may also be differentially regulated in the brains of HIV infected subjects with varying levels of neurocognitive impairment. Overall, the results reveal an unexpected finding, that MOR-1 may not be the predominant form of MOR expressed by some CNS cell types and that other splice variants of MOR-1, with possible differing functions, may contribute to the diversity of MOR-related processes in the CNS. PMID:22528479

  6. A Tumor-specific MicroRNA Recognition System Facilitates the Accurate Targeting to Tumor Cells by Magnetic Nanoparticles

    PubMed Central

    Yu, Yingting; Yao, Yi; Yan, Hao; Wang, Rui; Zhang, Zhenming; Sun, Xiaodan; Zhao, Lingyun; Ao, Xiang; Xie, Zhen; Wu, Qiong

    2016-01-01

    Targeted therapy for cancer is a research area of great interest, and magnetic nanoparticles (MNPs) show great potential as targeted carriers for therapeutics. One important class of cancer biomarkers is microRNAs (miRNAs), which play a significant role in tumor initiation and progression. In this study, a cascade recognition system containing multiple plasmids, including a Tet activator, a lacI repressor gene driven by the TetOn promoter, and a reporter gene repressed by the lacI repressor and influenced by multiple endogenous miRNAs, was used to recognize cells that display miRNA signals that are characteristic of cancer. For this purpose, three types of signal miRNAs with high proliferation and metastasis abilities were chosen (miR-21, miR-145, and miR-9). The response of this system to the human breast cancer MCF-7 cell line was 3.2-fold higher than that to the human breast epithelial HBL100 cell line and almost 7.5-fold higher than that to human embryonic kidney HEK293T cells. In combination with polyethyleneimine-modified MNPs, this recognition system targeted the tumor location in situ in an animal model, and an ~42% repression of tumor growth was achieved. Our study provides a new combination of magnetic nanocarrier and gene therapy based on miRNAs that are active in vivo, which has potential for use in future cancer therapies. PMID:27138178

  7. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing

    PubMed Central

    Miyaoka, Yuichiro; Berman, Jennifer R.; Cooper, Samantha B.; Mayerl, Steven J.; Chan, Amanda H.; Zhang, Bin; Karlin-Neumann, George A.; Conklin, Bruce R.

    2016-01-01

    Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR–based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator–like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology. PMID:27030102

  8. p53 directly regulates the transcription of the human frataxin gene and its lack of regulation in tumor cells decreases the utilization of mitochondrial iron.

    PubMed

    Shimizu, Rina; Lan, Nguyen Ngoc; Tai, Tran Tien; Adachi, Yuka; Kawazoe, Asako; Mu, Anfeng; Taketani, Shigeru

    2014-11-01

    Mitochondrial frataxin functions in iron homeostasis, biogenesis of iron-sulfur clusters, protection from oxidative stress and apoptosis, and as a tumor suppressor protein. We examined regulation of the expression of the human frataxin by p53. Pifithrin-α, an inhibitor of p53 function, and knockdown of p53 decreased the level of frataxin mRNA in human kidney HEK 293T cells. The transcriptional activity of the human frataxin gene is enhanced by the proximal promoter containing the p53-responsive element (p53RE) on the gene. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay confirmed the binding of p53 to the human frataxin p53RE. The expression of wild-type p53 in human cancer HeLa cells increased the reporter activity carrying p53RE at the region of -209 to -200bp of the frataxin promoter. Finally, when the HeLa cells overexpressing frataxin were treated with 5-aminolevulinic acid (ALA), there was less accumulation of protoporphyrin than HeLa control cells, and it was sharply decreased by the addition of iron citrate, suggesting that the utilization of mitochondrial iron for heme biosynthesis can be dependent on the level of frataxin. Alternatively, the low expression of frataxin not regulated by p53 in tumor cells lowers the utilization of iron in mitochondria, causing the tumor-specific ALA-induced accumulation of protoporphyrin.

  9. Measuring the dynamics of cyclic adenosine monophosphate level in living cells induced by low-level laser irradiation using bioluminescence resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Zheng, Liqin; Yang, Hongqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen; Zeng, Haishan

    2015-05-01

    Several studies demonstrated that the cyclic adenosine monophosphate (cAMP), an important second messenger, is involved in the mechanism of low-level laser irradiation (LLLI) treatment. However, most of these studies obtained the cAMP level in cell culture extracts or supernatant. In this study, the cAMP level in living cells was measured with bioluminescence resonance energy transfer (BRET). The effect of LLLI on cAMP level in living cells with adenosine receptors blocked was explored to identify the role of adenosine receptors in LLLI. The results showed that LLLI increased the cAMP level. Moreover, the rise of cAMP level was light dose dependent but wavelength independent for 658-, 785-, and 830-nm laser light. The results also exhibited that the adenosine receptors, a class of G protein-coupled receptor (GPCR), modulated the increase of cAMP level induced by LLLI. The cAMP level increased more significantly when the A3 adenosine receptors (A3R) were blocked by A3R antagonist compared with A1 adenosine receptor or A2a adenosine receptor blocked in HEK293T cells after LLLI, which was in good agreement with the adenosine receptors' expressions. All these results suggested that measuring the cAMP level with BRET could be a useful technique to study the role of GPCRs in living cells under LLLI.

  10. RD2-MolPack-Chim3, a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

    PubMed Central

    Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo

    2013-01-01

    Abstract Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology. PMID:23767932

  11. RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

    PubMed

    Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

    2013-08-01

    Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

  12. Tombusvirus P19 RNA silencing suppressor (RSS) activity in mammalian cells correlates with charged amino acids that contribute to direct RNA-binding

    PubMed Central

    2012-01-01

    Background Tombusvirus P19 is a protein encoded by tomato bushy stunt virus and related tombusviruses. Earlier studies have demonstrated that P19 is an RNA silencing suppressor (RSS) in plant cells. However, it has not been systematically investigated how P19 suppresses RNA interference in various mammalian cell settings. Results We have studied the RSS effect of P19 in mammalian cells, HEK293T, HeLa, and mouse embryonic fibroblasts. We have individually mutated 18 positively charged residues in P19 and found that 6 of these charged residues in P19 reduce its ability to suppress RNA interference. In each case, the reduction of silencing of RNA interference correlated with the reduced ability by these P19 mutants to bind siRNAs (small interfering RNAs). Conclusions Our findings characterize a class of RNA-binding proteins that function as RSS moieties. We find a tight correlation between positively charged residues in P19 accounting for siRNA-binding and their RSS activity. Because P19’s activity is conserved in plant and animal cells, we conclude that its RSS function unlikely requires cell type-specific co-factors and likely arises from direct RNA-binding. PMID:23216864

  13. A Mammalian Cell Based FACS-Panning Platform for the Selection of HIV-1 Envelopes for Vaccine Development

    PubMed Central

    Bruun, Tim-Henrik; Mühlbauer, Katharina; Benen, Thomas; Kliche, Alexander; Wagner, Ralf

    2014-01-01

    An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates. PMID:25279768

  14. Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway.

    PubMed

    Xiao, Gang; Miyazato, Akiko; Abe, Yuzuru; Zhang, Tiantuo; Nakamura, Kiwamu; Inden, Ken; Tanaka, Misuzu; Tanno, Daiki; Miyasaka, Tomomitsu; Ishii, Keiko; Takeda, Kiyoshi; Akira, Shizuo; Saijo, Shinobu; Iwakura, Yoichiro; Adachi, Yoshiyuki; Ohno, Naohito; Yamamoto, Natsuo; Kunishima, Hiroyuki; Hirakata, Yoichi; Kaku, Mitsuo; Kawakami, Kazuyoshi

    2010-01-01

    The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-alpha and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-kappaB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner. PMID:20451901

  15. Agonist-dependent modulation of cell surface expression of the cold receptor TRPM8.

    PubMed

    Toro, Carlos A; Eger, Stephanie; Veliz, Luis; Sotelo-Hitschfeld, Pamela; Cabezas, Deny; Castro, Maite A; Zimmermann, Katharina; Brauchi, Sebastian

    2015-01-14

    The spatial and temporal distribution of receptors constitutes an important mechanism for controlling the magnitude of cellular responses. Several members of the transient receptor potential (TRP) ion channel family can regulate their function by modulating their expression at the plasma membrane (PM) through rapid vesicular translocation and fusion. The mechanisms underlying this regulation are not completely understood, and the contribution of vesicular trafficking to physiological function is unknown. TRPM8 receptors are expressed in mammalian peripheral sensory neurons and are essential for the detection of cold temperatures. Previously, we showed that TRPM8-containing vesicles are segregated into three main pools, immobile at the PM, simple diffusive and corralled-hopping. Here, we show that channel expression at the PM is modulated by TRPM8 agonists in F11 and HEK293T cells. Our results support a model in which the activation of TRPM8 channels, located at the PM, induces a short-lived recruitment of a TRPM8-containing vesicular pool to the cell surface causing a transitory increase in the number of functional channels, affecting intrinsic properties of cold receptor responses. We further demonstrate the requirement of intact vesicular trafficking to support sustained cold responses in the skin of mice. PMID:25589752

  16. Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial.

    PubMed

    Pharo, Elizabeth A; Renfree, Marilyn B; Cane, Kylie N

    2016-11-15

    The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP. PMID:27452799

  17. Mammary cell-activating factor regulates the hormone-independent transcription of the early lactation protein (ELP) gene in a marsupial.

    PubMed

    Pharo, Elizabeth A; Renfree, Marilyn B; Cane, Kylie N

    2016-11-15

    The regulation of the tammar wallaby (Macropus eugenii) early lactation protein (ELP) gene is complex. ELP is responsive to the lactogenic hormones; insulin (I), hydrocortisone (HC) and prolactin (PRL) in mammary gland explants but could not be induced with lactogenic hormones in tammar primary mammary gland cells, nor in KIM-2 conditionally immortalised murine mammary epithelial cells. Similarly, ELP promoter constructs transiently-transfected into human embryonic kidney (HEK293T) cells constitutively expressing the prolactin receptor (PRLR) and Signal Transducer and Activator of Transcription (STAT)5A were unresponsive to prolactin, unlike the rat and mouse β-casein (CSN2) promoter constructs. Identification of the minimal promoter required for the hormone-independent transcription of tammar ELP in HEK293Ts and comparative analysis of the proximal promoters of marsupial ELP and the orthologous eutherian colostrum trypsin inhibitor (CTI) gene suggests that mammary cell-activating factor (MAF), an E26 transformation-specific (ETS) factor, may bind to an AGGAAG motif and activate tammar ELP.

  18. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    PubMed

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

  19. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

    PubMed Central

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  20. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    PubMed

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  1. Identification of small molecule agonists of human relaxin family receptor 1 (RXFP1) by utilizing a homogenous cell-based cAMP assay

    PubMed Central

    Chen, Catherine Z.; Southall, Noel; Xiao, Jingbo; Marugan, Juan J.; Ferrer, Marc; Hu, Xin; Jones, Raisa E.; Feng, Shu; Agoulnik, Irina U.

    2016-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the receptor could have a wide range of pharmacological utility, up to date, there are no reported small molecule agonists for relaxin receptors. Here, we report the development of quantitative high-throughput platform for RXFP1 agonist screen based on homogenous cell-based HTRF cAMP assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365,677 compounds. Neither compound showed activity in a counter screen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  2. Observation of "wired" cell communication over 10-μm and 20-μm poly(dimethylsiloxane) barriers in tetracycline inducible expression systems

    NASA Astrophysics Data System (ADS)

    Kuo, Ching-Te; Chi, Cheng-Yu; Wu, Pei-Yi; Chuang, Fang-Tzu; Lin, Yueh-Chien; Liu, Hao-Kai; Huang, Guan-Syuan; Tsai, Tzu-Ching; Wo, Andrew M.; Lee, Hsinyu; Lee, Si-Chen

    2016-01-01

    Communication between cells and extracellular environments is of interest because of its critical roles in cell development and differentiation. Particularly, this signal transduction is commonly believed to rely on the contact and binding of the participating molecules/proteins, suggesting that the binding distance needed is less than a few nanometers. However, it is difficult to precisely match the rapidly binding interaction which depends on the probability of molecular collision in living systems, raising a hypothesis that another mechanism exists, could promote this signal communication, and remains unknown. Here we report that a long-range signal delivery over 10-μm and 20-μm polydimethylsiloxane (PDMS) barriers can be observed in microfluidically tetracycline (Tet) inducible expression systems. Results show that a significant increment of the long-range induced green fluorescent protein in human embryonic kidney 293T (HEK 293T) cells by the stimulation of Tet is demonstrated, and that such a signal induction is not dominated by Tet diffusion and displays a specific bindingless property. In addition, our experimental results, combined with theoretical modeling, suggest that this communication exhibits a bump-shaped characteristic depending on barrier thickness, materially structural property, surface roughness, and agonist concentration. It strongly relies on the PDMS barrier to delivery signal; therefore, we call such a mechanism as "wired" cell communication instead of wireless. These results could ignite interests in the novel and "wired" cell communication, which we call it X-signal, and in the use of such systems for the study of cellular biology and development of new drug.

  3. Soluble THSD7A Is an N-Glycoprotein That Promotes Endothelial Cell Migration and Tube Formation in Angiogenesis

    PubMed Central

    Kuo, Meng-Wei; Wang, Chian-Huei; Wu, Hsiao-Chun; Chang, Shing-Jyh; Chuang, Yung-Jen

    2011-01-01

    Background Thrombospondin type I domain containing 7A (THSD7A) is a novel neural protein that is known to affect endothelial migration and vascular patterning during development. To further understand the role of THSD7A in angiogenesis, we investigated the post-translational modification scheme of THS7DA and to reveal the underlying mechanisms by which this protein regulates blood vessel growth. Methodology/Principal Findings Full-length THSD7A was overexpressed in human embryonic kidney 293T (HEK293T) cells and was found to be membrane associated and N-glycosylated. The soluble form of THSD7A, which is released into the cultured medium, was harvested for further angiogenic assays. We found that soluble THSD7A promotes human umbilical vein endothelial cell (HUVEC) migration and tube formation. HUVEC sprouts and zebrafish subintestinal vessel (SIV) angiogenic assays further revealed that soluble THSD7A increases the number of branching points of new vessels. Interestingly, we found that soluble THSD7A increased the formation of filopodia in HUVEC. The distribution patterns of vinculin and phosphorylated focal adhesion kinase (FAK) were also affected, which implies a role for THSD7A in focal adhesion assembly. Moreover, soluble THSD7A increased FAK phosphorylation in HUVEC, suggesting that THSD7A is involved in regulating cytoskeleton reorganization. Conclusions/Significance Taken together, our results indicate that THSD7A is a membrane-associated N-glycoprotein with a soluble form. Soluble THSD7A promotes endothelial cell migration during angiogenesis via a FAK-dependent mechanism and thus may be a novel neuroangiogenic factor. PMID:22194972

  4. Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1.

    PubMed

    Takayama, Yasunori; Shibasaki, Koji; Suzuki, Yoshiro; Yamanaka, Akihiro; Tominaga, Makoto

    2014-05-01

    Transient receptor potential vanilloid 4 (TRPV4), a calcium-permeable channel, is highly expressed in the apical membrane of choroid plexus epithelial cells (CPECs) in the brain. The function of TRPV4 is unknown. Here, we show physical and functional interaction between TRPV4 and anoctamin 1 (ANO1) in HEK293T cells and CPECs. Chloride currents induced by a TRPV4 activator (GSK1016790A) were markedly increased in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV4 with ANO1, but not with ANO4, ANO6, or ANO10, the mRNAs of which were expressed in the choroid plexus. We also found physical interaction between TRPV4 and ANO1 in both HEK293T cells and choroid plexus. We observed that ANO1 was activated at a warm temperature (37°C) in HEK293T cells and that the heat-evoked chloride currents were markedly enhanced after GSK1016790A application in CPECs. Simultaneous stimulation by warmth and hyposmosis induced chloride current activation in wild-type, but not in TRPV4-deficient, CPECs. Cell volume changes were induced by ANO1-mediated chloride currents in parallel with membrane potential changes, and the cell volume was significantly decreased at negative membrane potentials by TRPV4-induced ANO1 activation. Thus, physical and functional interactions between TRPV4 and ANO1 can modulate water transport in the choroid plexus. PMID:24509911

  5. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  6. Effect of surface potential on epithelial cell adhesion, proliferation and morphology.

    PubMed

    Chang, Hsun-Yun; Kao, Wei-Lun; You, Yun-Wen; Chu, Yi-Hsuan; Chu, Kuo-Jui; Chen, Peng-Jen; Wu, Chen-Yi; Lee, Yu-Hsuan; Shyue, Jing-Jong

    2016-05-01

    Cell adhesion is the basis of individual cell survival, division and motility. Hence, understanding the effects that the surface properties have on cell adhesion, proliferation and morphology are crucial. In particular, surface charge/potential has been identified as an important factor that affects cell behavior. However, how cells respond to incremental changes in surface potential remains unclear. By using binary self-assembled monolayer (SAM) modified Au surfaces that are similar in mechanical/chemical properties and provide a series of surface potentials, the effect of surface potential on the behavior of cells can be studied. In this work, the effect of surface potential on epithelial cells, including human embryonic kidney (HEK293T) and human hepatocellular carcinoma (HepG2), were examined. The results showed that the adhesion density of epithelial cells increased with increasing surface potential, which is similar to but varied more significantly compared with fibroblasts. The proliferation rate is found to be independent of surface potential in both cell types. Furthermore, epithelial cells show no morphological change with respect to surface potential, whereas the morphology of the fibroblasts clearly changed with the surface potential. These differences between the cell types were rationalized by considering the difference in extracellular matrix composition. Laminin-dominant epithelial cells showed higher adhesion density and less morphological change than did fibronectin-dominant fibroblasts because the more significant adsorption of positively charged laminin on the surface enhanced the adhesion of epithelial cells. In contrast, due to the dominance of negatively charged fibronectin that adsorbed weakly on the surface, fibroblasts had to change their morphology to fit the inhomogeneous fibronectin-adsorbed area.

  7. In vitro study of cell death with 5-aminolevulinic acid based photodynamic therapy to improve the efficiency of cancer treatment

    NASA Astrophysics Data System (ADS)

    Firdous, S.; Nawaz, M.; Ikram, M.; Ahmed, M.

    2012-03-01

    Photodynamic therapy (PDT) is a kind of photochemo therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, efforts are underway to optimize PDT protocols for improved efficacy and combination of all three PDT mechanisms involved in the different human carcinomas cell narcosis. Our in vitro cell culture experiments with 5-aminolevulanic acid (ALA) a clinically approved photiosensitizer (PS) and 635 nm laser light have yielded promising results, as follow: (1) (human cervical cancer (HeLa) cell line incubated, for 18 h, with 30 μg/ml of 5-ALA, treated with laser light dose of 50 J/cm2 can produce 85% of cell killing (2) human larynx carcinoma (Hep2c) cell line incubated, for 7 h, with 55 μg/ml of 5-ALA, treated with laser light dose of 85 J/cm2 can produce 75% of cell killing (3) human liver cancer (HepG2) cell line incubated, for 22-48 h, with 262 μg/ml of 5-ALA, treated with laser light dose of 120 J/cm2 can produce 95% of cell killing (4) human muscle cancer (RD) cell line incubated, for 47 h, with 250 μg/ml of 5-ALA, treated with laser light dose of 80 J/cm2 can produce 76% of cell killing (5) Human embryonic kidney (HEK293T) cell line incu-bated, for 18 h, with 400 μg/ml of 5-ALA, treated with laser light dose of 40 J/cm2 can produce 82% of cell killing confirming the efficacy of photodynamic therapy.

  8. Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage

    PubMed Central

    Panieri, Emiliano; Toietta, Gabriele; Mele, Marina; Labate, Valentina; Ranieri, Sofia Chiatamone; Fusco, Salvatore; Tesori, Valentina; Antonini, Annalisa; Maulucci, Giuseppe; Spirito, Marco De; Galeotti, Tommaso; Pani, Giovambattista

    2010-01-01

    Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies. PMID:20739737

  9. Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage.

    PubMed

    Panieri, Emiliano; Toietta, Gabriele; Mele, Marina; Labate, Valentina; Ranieri, Sofia Chiatamone; Fusco, Salvatore; Tesori, Valentina; Antonini, Annalisa; Maulucci, Giuseppe; De Spirito, Marco; Galeotti, Tommaso; Pani, Giovambattista

    2010-08-01

    Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies. PMID:20739737

  10. Vitamin B₆ activates p53 and elevates p21 gene expression in cancer cells and the mouse colon.

    PubMed

    Zhang, Peipei; Suidasari, Sofya; Hasegawa, Tomomi; Yanaka, Noriyuki; Kato, Norihisa

    2014-05-01

    Increasing evidence indicates vitamin B6 acts as a protective factor against colon cancer. However, the mechanisms of the effect of vitamin B6 are poorly understood. The present preliminary study using DNA microarray and real-time PCR indicates p21 mRNA is upregulated in human colon carcinoma (HT29) cells exposed to pyridoxal (PL, 500 µM). A similar effect was observed in human epithelial colorectal adenocarcinoma (Caco2) cells, human colon adenocarcinoma (LoVo) cells, human embryonic kidney (HEK293T) cells, and human hepatoma (HepG2) cells. Adding other B6-vitamers such as pyridoxal 5'-phosphate (PLP), pyridoxine (PN), and pyridoxamine (PM) caused no such effect. In order to understand the mechanism of higher mRNA expression of p21 by PL, effect of PL on the p53 activation was examined (the upstream factor for p21 mRNA transcription) in HT29 cells, LoVo cells, and HepG2 cells. PL increased the phosphorylated p53 protein levels (active form) in whole-cell lysates and the nuclei of the cells. Noteworthy, the consumption of a vitamin B6-deficient diet for 5 weeks significantly reduced p21 mRNA levels and tended to reduce phosphorylated p53 protein levels (P=0.053) in the colons of mice compared to a diet with adequate vitamin B6. Thus, these results suggest vitamin B6 plays a role in increasing p21 gene expression via p53 activation in several cancer cells and the mouse colon.

  11. Characterization of the replication timing program of 6 human model cell lines.

    PubMed

    Hadjadj, Djihad; Denecker, Thomas; Maric, Chrystelle; Fauchereau, Fabien; Baldacci, Giuseppe; Cadoret, Jean-Charles

    2016-09-01

    During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14), Cayrou et al. (2011 Sep), Picard et al. (2014 May 1) [1], [2], [3]), and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9), Pope et al. (2014 Nov 20) [5], [6]). On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb) [7], [8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16) [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308), RKO (GSM2111309), HEK 293T (GSM2111310), HeLa (GSM2111311), MRC5-SV (GSM2111312) and K562 (GSM2111313). A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines. PMID:27508120

  12. MiR-9 downregulates CDX2 expression in gastric cancer cells.

    PubMed

    Rotkrua, Pichayanoot; Akiyama, Yoshimitsu; Hashimoto, Yutaka; Otsubo, Takeshi; Yuasa, Yasuhito

    2011-12-01

    Ectopic expression of CDX2, a caudal-related homeobox protein, is known to be associated with the development of intestinal metaplasia in the stomach and gastric carcinogenesis. Previously, we reported that DNA methylation was partly responsible for CDX2 silencing in gastric cancer (GC). However, the mechanism underlying the aberrant expression of CDX2 during malignant transformation remained unclear. MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators. To elucidate the role of miRNAs in CDX2 downregulation in GC cells, putative miRNAs, such as miR-9, were computationally predicted. After exogenous pre-miR-9 precursor transfection, the luciferase activity of a reporter vector containing a part of the 3'-UTR of CDX2 was downregulated in HEK-293T cells. The inverse correlation between the miR-9 and CDX2 protein levels was demonstrated in GC cell lines. By means of miR-9 overexpression and knockdown techniques, the expression levels of the CDX2 protein and downstream target genes (p21, MUC2 and TFF3) were responsively altered in MKN45 and NUGC-3 cells. Transfection of an anti-miR-9 molecule significantly inhibited cell growth by promoting G(1) cell cycle arrest in MKN45 cells similarly to the effect of CDX2 overexpression. Moreover, examination of the miR-9 levels in primary GC tissues revealed that the amounts of miR-9 in the CDX2-negative group were significantly higher than those in the CDX2-positive group (p = 0.004). Therefore, miR-9 might repress CDX2 expression via the binding site in the 3'-UTR, resulting in the promotion of cell proliferation in GCs.

  13. Mass Spectrometric Analysis of the Cell Surface N-Glycoproteome by Combining Metabolic Labeling and Click Chemistry

    NASA Astrophysics Data System (ADS)

    Smeekens, Johanna M.; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide- N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  14. Mass spectrometric analysis of the cell surface N-glycoproteome by combining metabolic labeling and click chemistry.

    PubMed

    Smeekens, Johanna M; Chen, Weixuan; Wu, Ronghu

    2015-04-01

    Cell surface N-glycoproteins play extraordinarily important roles in cell-cell communication, cell-matrix interactions, and cellular response to environmental cues. Global analysis is exceptionally challenging because many N-glycoproteins are present at low abundances and effective separation is difficult to achieve. Here, we have developed a novel strategy integrating metabolic labeling, copper-free click chemistry, and mass spectrometry (MS)-based proteomics methods to analyze cell surface N-glycoproteins comprehensively and site-specifically. A sugar analog containing an azido group, N-azidoacetylgalactosamine, was fed to cells to label glycoproteins. Glycoproteins with the functional group on the cell surface were then bound to dibenzocyclooctyne-sulfo-biotin via copper-free click chemistry under physiological conditions. After protein extraction and digestion, glycopeptides with the biotin tag were enriched by NeutrAvidin conjugated beads. Enriched glycopeptides were deglycosylated with peptide-N-glycosidase F in heavy-oxygen water, and in the process of glycan removal, asparagine was converted to aspartic acid and tagged with 18O for MS analysis. With this strategy, 144 unique N-glycopeptides containing 152 N-glycosylation sites were identified in 110 proteins in HEK293T cells. As expected, 95% of identified glycoproteins were membrane proteins, which were highly enriched. Many sites were located on important receptors, transporters, and cluster of differentiation proteins. The experimental results demonstrated that the current method is very effective for the comprehensive and site-specific identification of the cell surface N-glycoproteome and can be extensively applied to other cell surface protein studies.

  15. Comparison of Cell Expression Formats for the Characterization of GABAA Channels Using a Microfluidic Patch Clamp System

    PubMed Central

    Chen, Qin; Yim, Peter D.; Yuan, Nina; Johnson, Juliette; Cook, James M.; Smith, Steve; Ionescu-Zanetti, Cristian; Wang, Zhi-Jian; Arnold, Leggy A.

    2012-01-01

    Abstract Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABAA channels. A variety of cell types and methods of GABAA channel expression were successfully studied (defined as IGABA>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α1β3γ2 GABAA channels, frozen ready-to-assay (RTA) HEK cells expressing α1β3γ2 or α3β3γ2 GABAA channels, transiently transfected HEK293T cells expressing α1β3γ2 GABAA channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABAA channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABAA channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABAA channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABAA channels in a wide variety of cell formats can be performed using this automated patch clamp system. PMID:22574655

  16. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection

    NASA Astrophysics Data System (ADS)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-04-01

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  17. Visualization of the pH-dependent dynamic distribution of G2A in living cells.

    PubMed

    Lan, Wanjun; Yamaguchi, Satoshi; Yamamoto, Teruyasu; Yamahira, Shinya; Tan, Modong; Murakami, Naoka; Zhang, Jingyan; Nakamura, Motonao; Nagamune, Teruyuki

    2014-09-01

    G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one-tenth in response to the extracellular acidic pH, and this acidic pH-induced G2A accumulation on cell surfaces may be explained by proton-induced dissociation of G2A from endocytic machinery. PMID:24891524

  18. Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells.

    PubMed

    Rodrigues, A F; Formas-Oliveira, A S; Guerreiro, M R; Tomás, H A; Alves, P M; Coroadinha, A S

    2015-09-01

    This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.

  19. Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

    PubMed

    Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

    2016-01-01

    Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes. PMID:27516189

  20. HER2 confers drug resistance of human breast cancer cells through activation of NRF2 by direct interaction

    PubMed Central

    Kang, Hyo Jin; Yi, Yong Weon; Hong, Young Bin; Kim, Hee Jeong; Jang, Young-Joo; Seong, Yeon-Sun; Bae, Insoo

    2014-01-01

    Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells. PMID:25467193

  1. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection.

    PubMed

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang

    2015-05-14

    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL(-1). Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers. PMID:25893559

  2. Extracellular Tau Paired Helical Filaments Differentially Affect Tau Pathogenic Mechanisms in Mitotic and Post-Mitotic Cells: Implications for Mechanisms of Tau Propagation in the Brain.

    PubMed

    Varghese, Merina; Santa-Maria, Ismael; Ho, Lap; Ward, Libby; Yemul, Shrishailam; Dubner, Lauren; Księżak-Reding, Hanna; Pasinetti, Giulio Maria

    2016-09-01

    The release of paired helical filaments (PHFs) from neurons into the extracellular space may contribute to the propagation of tau pathology across brain regions in Alzheimer's disease (AD) and other tauopathies. The majority of available mechanistic studies exploring the pathologic role of extracellular PHFs are conducted in proliferating cell lines. Here, we compare how extracellular PHFs induce tauopathy in mitotic cells and in post-mitotic brain neurons. In a mitotic cell line (HEK 293T), extracellular exposure to AD PHFs leads to an intracellular "aggresomal" type deposition of tau, coincidental with redistribution of dynein, a retrograde motor protein. We also observed that PHFs impaired proteasome degradation, but not autophagy. Exposure of cells to proteasome inhibitors was sufficient to induce intracellular tau aggregate formation as well as reorganization of dynein and the intermediate filament protein, vimentin. Thus, in mitotic cells, extracellular PHFs promote cellular tau aggregation, in part, by interfering with cellular proteasome degradation processes. In contrast with our observations with proliferating cells, exposure of post-mitotic primary neuronal cultures to AD PHFs did not promote "aggresomal" tau deposition, but instead resulted in a widespread accumulation of phosphorylated tau-immunoreactive swellings in neuritic processes, characterized by disturbed cytoskeletal organization of dynein and vimentin. Collectively, our observations suggest that extracellular PHFs may contribute to the propagation of tau pathology by independent mechanisms in post-mitotic and mitotic brain cells. These outcomes indicate that in addition to post-mitotic brain neurons, mitotic brain cells should also be considered as targets for therapeutic interventions to attenuate propagation of tauopathy.

  3. Neurotransmitter transporter family including SLC6A6 and SLC6A13 contributes to the 5-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin IX and photodamage, through uptake of ALA by cancerous cells.

    PubMed

    Tran, Tai Tien; Mu, Anfeng; Adachi, Yuka; Adachi, Yasushi; Taketani, Shigeru

    2014-01-01

    δ-Aminolevulinic acid (ALA)-induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA-induced accumulation of protoporphyrin by the treatment of colon cancer DLD-1 and epithelial cancer HeLa cells with γ-aminobutyric acid (GABA)-related compounds. When the cells were treated with GABA, taurine and β-alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA- and ALA methylester-dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA-treated cells were exposed to white light, the extent of photodamage increased in SLC6A6- and SLC6A13-expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD-1 and HeLa cells decreased the ALA-induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA-induced accumulation of protoporphyrin in cancerous cells.

  4. Endocytosis of Chikungunya Virus into Mammalian Cells: Role of Clathrin and Early Endosomal Compartments

    PubMed Central

    Gay, Bernard; Chazal, Nathalie; Higgs, Stephen; Devaux, Christian; Briant, Laurence

    2010-01-01

    Background The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells. Methodology/Principal Findings Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl β-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island. Conclusions Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms. PMID:20628602

  5. Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains.

    PubMed

    Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

    2015-01-01

    Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176-284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and-B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules. PMID:26258424

  6. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division.

    PubMed

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-09-01

    N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs). A proximity ligation assay (PLA) for NAGK and DYNLRB1 revealed NAGK-dynein complex on nuclear envelopes in prophase cells and on chromosomes in metaphase cells. NAGK-DYNLRB1 PLA followed by Lis1/NudE1 immunostaining showed NAGK-dynein complexes were colocalized with Lis1 and NudE1 signals, and PLA for NAGK-Lis1 showed similar signal patterns, suggesting a functional link between NAGK and dynein-Lis1 complex. Subsequently, NAGK-dynein complexes were found in KTs and on nuclear membranes where KTs were marked with CENP-B ICC and nuclear membrane with lamin ICC. Furthermore, knockdown of NAGK by small hairpin (sh) RNA was found to delay cell division. These results indicate that the NAGK-dynein interaction with the involvements of Lis1 and NudE1 plays an important role in prophase nuclear envelope breakdown (NEB) and metaphase MT-KT attachment during eukaryotic cell division. PMID:27646688

  7. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division

    PubMed Central

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-01-01

    N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs). A proximity ligation assay (PLA) for NAGK and DYNLRB1 revealed NAGK-dynein complex on nuclear envelopes in prophase cells and on chromosomes in metaphase cells. NAGK-DYNLRB1 PLA followed by Lis1/NudE1 immunostaining showed NAGK-dynein complexes were colocalized with Lis1 and NudE1 signals, and PLA for NAGK-Lis1 showed similar signal patterns, suggesting a functional link between NAGK and dynein-Lis1 complex. Subsequently, NAGK-dynein complexes were found in KTs and on nuclear membranes where KTs were marked with CENP-B ICC and nuclear membrane with lamin ICC. Furthermore, knockdown of NAGK by small hairpin (sh) RNA was found to delay cell division. These results indicate that the NAGK-dynein interaction with the involvements of Lis1 and NudE1 plays an important role in prophase nuclear envelope breakdown (NEB) and metaphase MT-KT attachment during eukaryotic cell division. PMID:27646688

  8. N-Acetyl-D-Glucosamine Kinase Interacts with Dynein-Lis1-NudE1 Complex and Regulates Cell Division.

    PubMed

    Sharif, Syeda Ridita; Islam, Ariful; Moon, Il Soo

    2016-09-01

    N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs). A proximity ligation assay (PLA) for NAGK and DYNLRB1 revealed NAGK-dynein complex on nuclear envelopes in prophase cells and on chromosomes in metaphase cells. NAGK-DYNLRB1 PLA followed by Lis1/NudE1 immunostaining showed NAGK-dynein complexes were colocalized with Lis1 and NudE1 signals, and PLA for NAGK-Lis1 showed similar signal patterns, suggesting a functional link between NAGK and dynein-Lis1 complex. Subsequently, NAGK-dynein complexes were found in KTs and on nuclear membranes where KTs were marked with CENP-B ICC and nuclear membrane with lamin ICC. Furthermore, knockdown of NAGK by small hairpin (sh) RNA was found to delay cell division. These results indicate that the NAGK-dynein interaction with the involvements of Lis1 and NudE1 plays an important role in prophase nuclear envelope breakdown (NEB) and metaphase MT-KT attachment during eukaryotic cell division.

  9. Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

    SciTech Connect

    Amand, Helene L.

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Reversible CPP dimerisation is a simple yet efficient strategy to improve delivery. Black-Right-Pointing-Pointer Dimer formation enhances peptiplex stability, resulting in increased transfection. Black-Right-Pointing-Pointer By dimerisation, the CPP EB1 even gain endosomal escape properties while lowering cytotoxicity. -- Abstract: Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ('peptiplexes') enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the 'chelate effect' and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from

  10. Identification of a functional nuclear export signal in the green fluorescent protein asFP499

    SciTech Connect

    Mustafa, Huseyin . E-mail: huseyinm@hotmail.com; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

    2006-04-21

    The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified {sub 194}LRMEKLNI{sub 201} as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES.

  11. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells

    PubMed Central

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  12. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

    PubMed

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

    2014-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

  13. DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

    PubMed Central

    Bose, Arindam; Pande, Paritosh; Jasti, Vijay P.; Millsap, Amy D.; Hawkins, Edward K.; Rizzo, Carmelo J.; Basu, Ashis K.

    2015-01-01

    The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5′-G1G2CG3CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass. PMID:26220181

  14. Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

    PubMed

    Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

    2012-02-01

    The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.

  15. Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    PubMed Central

    Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H.; Sosinsky, Gina E.

    2015-01-01

    Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system

  16. Inhibition of Myo6 gene expression by co‑expression of a mutant of transcription factor POU4F3 (BRN‑3C) in hair cells.

    PubMed

    Ma, Deng-Bin; Chen, Jie; Xia, Yang; Zhu, Guang-Jie; Ma, Xiao-Feng; Zhou, Han; Gu, Ya-Jun; Yu, Chen-Jie; Zhu, Min-Sheng; Qian, Xiao-Yun; Gao, Xia

    2014-04-01

    An eight‑base pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3‑Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferase‑thymidine kinase vectors expressing Renilla luciferase and the Myo6 promoter‑driven firefly luciferase expressing vectors along with pIRES2‑enhanced green fluorescent protein (EGFP)‑Pou4f3 (expressing wild‑type Pou4f3) or pIRES2‑EGFP‑Pou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by co‑expression of wild‑type Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2‑EGFP‑Pou4f3 and the empty control vectors. However, co‑expression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P<0.001). The data suggests that mutated Pou4f3 has a negative role in the promoter activity of Myo6, and by extension, the expression of myosin VI, and this may be an underlying mechanism of DFNA15 hearing loss.

  17. Heme oxygenase-1 regulates matrix metalloproteinase MMP-1 secretion and chondrocyte cell death via Nox4 NADPH oxidase activity in chondrocytes.

    PubMed

    Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

    2013-01-01

    Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22(phox) heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

  18. Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

    PubMed Central

    Rousset, Francis; Nguyen, Minh Vu Chuong; Grange, Laurent; Morel, Françoise; Lardy, Bernard

    2013-01-01

    Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis. PMID:23840483

  19. Lipoplexes of dicationic gemini surfactants with DNA: Structural features of DNA compaction and transfection efficiency.

    PubMed

    Faizullin, D A; Zuev, Yu F; Zakharova, L Ya; Pokrovsky, A G; Korobeinikov, V A; Mukhametzyanov, T A; Konovalov, A I

    2015-01-01

    The internal structure of DNA lipoplexes with hydroxyethylated alkylammonium gemini surfactants (GS) with high transfection activity was studied by circular dichroism. It was shown that the efficiency of transfection of HEK293T cells with the pEGFP-N1 circular plasmid was different from zero only in the region of existence of chiral supramolecular DNA-GS complexes and reaches a maximum at concentrations at which the spontaneous aggregation of components is observed.

  20. Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1.

    PubMed

    Shakeri, Raheleh; Hosseinkhani, Saman; Los, Marek J; Davoodi, Jamshid; Jain, Mayur V; Cieślar-Pobuda, Artur; Rafat, Mehrdad; Ardestani, Sussan Kaboudanian

    2015-11-01

    Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form; however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1. E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike - HL-60 cancer cell line. In vitro apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP.

  1. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  2. Integrated nanoscale tools for interrogating living cells

    NASA Astrophysics Data System (ADS)

    Jorgolli, Marsela

    and fabricated a new hybrid chip that combines a front-side nanowire-based interface for neuronal recording with backside complementary metal oxide semiconductor (CMOS) circuits for on-chip multiplexing, voltage control for stimulation, signal amplification, and signal processing. Individual chips contain 1024 stimulation/recording sites enabling large-scale interfacing of neuronal networks with single cell resolution. Through electrical and electrochemical characterization of the devices, we demonstrated their enhanced functionality at a massively parallel scale. In our initial cell experiments, we achieved intracellular stimulations and recordings of changes in the membrane potential in a variety of cells including: HEK293T, cardiomyocytes, and rat cortical neurons. This demonstrated the device capability for single-cell-resolution recording/stimulation which when extended to a large number of neurons in a massively parallel fashion will enable the functional mapping of a complex neuronal network.

  3. Peroxidase-like activity of Fe3O4@carbon nanoparticles enhances ascorbic acid-induced oxidative stress and selective damage to PC-3 prostate cancer cells.

    PubMed

    An, Qiao; Sun, Chuanyu; Li, Dian; Xu, Ke; Guo, Jia; Wang, Changchun

    2013-12-26

    Ascorbic acid (AA) is capable of inhibiting cancer cell growth by perturbing the normal redox state of cells and causing toxic effects through the generation of abundant reactive-oxygen species (ROS). However, the clinical utility of AA at a tolerable dosage is plagued by a relatively low in vivo efficacy. This study describes the development of a peroxidase-like composite nanoparticle for use in an AA-mediated therapeutic strategy. On the basis of a high-throughput, one-pot solvothermal approach, Fe3O4@C nanoparticles (NPs) were synthesized and then modified with folic acid (FA) on the surface. Particular focus is concentrated on the assessment of peroxidase-like catalytic activity by a chromogenic reaction in the presence of H2O2. The carbon shell of Fe3O4@C NPs contains partially graphitized carbon and thus facilitates electron transfer in the catalytic decomposition of H2O2, leading to the production of highly reactive hydroxyl radicals. Along with magnetic responsiveness and receptor-binding specificity, the intrinsic peroxidase-like catalytic activity of Fe3O4@C-FA NPs pronouncedly promotes AA-induced oxidative stress in cancer cells and optimizes the ROS-mediated antineoplastic efficacy of exogenous AA. In vitro experiments using human prostate cancer PC-3 cells demonstrate that Fe3O4@C-FA NPs serve as a peroxidase mimic to create hydroxyl radicals from endogenous H2O2 that is yielded in response to exogenous AA via an oxidative stress process. The usage of a dual agent leads to the enhanced cytotoxicity of PC-3 cells, and, because of the synergistic effect of NPs, the administrated dosage of AA is reduced markedly. However, because normal cells (HEK 293T cells) appear to have a higher capacity to cope with additionally generated ROS than cancer cells, the NP-AA combination shows little damage in this case, proving that selective killing of cancer cells could be achieved owing to preferential accumulation of ROS in cancer cells. A possible ROS

  4. Peroxidase-like activity of Fe3O4@carbon nanoparticles enhances ascorbic acid-induced oxidative stress and selective damage to PC-3 prostate cancer cells.

    PubMed

    An, Qiao; Sun, Chuanyu; Li, Dian; Xu, Ke; Guo, Jia; Wang, Changchun

    2013-12-26

    Ascorbic acid (AA) is capable of inhibiting cancer cell growth by perturbing the normal redox state of cells and causing toxic effects through the generation of abundant reactive-oxygen species (ROS). However, the clinical utility of AA at a tolerable dosage is plagued by a relatively low in vivo efficacy. This study describes the development of a peroxidase-like composite nanoparticle for use in an AA-mediated therapeutic strategy. On the basis of a high-throughput, one-pot solvothermal approach, Fe3O4@C nanoparticles (NPs) were synthesized and then modified with folic acid (FA) on the surface. Particular focus is concentrated on the assessment of peroxidase-like catalytic activity by a chromogenic reaction in the presence of H2O2. The carbon shell of Fe3O4@C NPs contains partially graphitized carbon and thus facilitates electron transfer in the catalytic decomposition of H2O2, leading to the production of highly reactive hydroxyl radicals. Along with magnetic responsiveness and receptor-binding specificity, the intrinsic peroxidase-like catalytic activity of Fe3O4@C-FA NPs pronouncedly promotes AA-induced oxidative stress in cancer cells and optimizes the ROS-mediated antineoplastic efficacy of exogenous AA. In vitro experiments using human prostate cancer PC-3 cells demonstrate that Fe3O4@C-FA NPs serve as a peroxidase mimic to create hydroxyl radicals from endogenous H2O2 that is yielded in response to exogenous AA via an oxidative stress process. The usage of a dual agent leads to the enhanced cytotoxicity of PC-3 cells, and, because of the synergistic effect of NPs, the administrated dosage of AA is reduced markedly. However, because normal cells (HEK 293T cells) appear to have a higher capacity to cope with additionally generated ROS than cancer cells, the NP-AA combination shows little damage in this case, proving that selective killing of cancer cells could be achieved owing to preferential accumulation of ROS in cancer cells. A possible ROS

  5. Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo‡

    PubMed Central

    Sadhukha, Tanmoy; Prabha, Swayam; Sanaú, Mercedes; Rotenberg, Susan A.

    2016-01-01

    Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO−) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds. PMID

  6. Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives

    PubMed Central

    Brito, Hugo; Martins, Ana Cláudia; Lavrado, João; Mendes, Eduarda; Francisco, Ana Paula; Santos, Sofia A.; Ohnmacht, Stephan A.; Kim, Nam-Soon; Rodrigues, Cecília M. P.; Moreira, Rui; Neidle, Stephen; Borralho, Pedro M.; Paulo, Alexandra

    2015-01-01

    Background A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. Methodology/Principal Findings IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 μM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). Conclusions Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer. PMID:26024321

  7. A synthetic cGMP-sensitive gene switch providing Viagra(®)-controlled gene expression in mammalian cells and mice.

    PubMed

    Kim, Taeuk; Folcher, Marc; Charpin-El Hamri, Ghislaine; Fussenegger, Martin

    2015-05-01

    Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell

  8. Induction of apoptosis through ER stress and TP53 in MCF-7 cells by the nanoparticle [Gd@C82(OH)22]n: A systems biology study.

    PubMed

    Wang, Lin; Meng, Jie; Cao, Weipeng; Li, Qizhai; Qiu, Yuqing; Sun, Baoyun; Li, Lei M

    2014-06-01

    those of HEK293T and MCF-7 cells induced by the miR-23a∼27a∼24-2 cluster. Furthermore, one of the inferred regulatory mechanisms comprises the apoptosis network centered around TP53, whose effective regulation of apoptosis is somehow reestablished after [Gd@C82(OH)22]n treatment. These results elucidate the application and development of [Gd@C82(OH)22]n and other fullerene derivates.

  9. NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

    SciTech Connect

    Miyazaki, Masaya; Nishihara, Hiroshi; Hasegawa, Hideki; Tashiro, Masato; Wang, Lei; Kimura, Taichi; Tanino, Mishie; Tsuda, Masumi; Tanaka, Shinya

    2013-11-29

    Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while the physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.

  10. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

    PubMed

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays

  11. Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS.

    PubMed

    Gopal, Angelica A; Rappaz, Benjamin; Rouger, Vincent; Martyn, Iain B; Dahlberg, Peter D; Meland, Rachel J; Beamish, Ian V; Kennedy, Timothy E; Wiseman, Paul W

    2016-02-01

    Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or

  12. Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

    PubMed Central

    Pritchard, Laura K.; Harvey, David J.; Bonomelli, Camille

    2015-01-01

    ABSTRACT The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. IMPORTANCE Development of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as

  13. β2-Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function.

    PubMed

    Kim, Donghwa; Pauer, Susan H; Yong, Hwan M; An, Steven S; Liggett, Stephen B

    2016-08-19

    Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of β2-adrenergic receptors (β2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when β2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected β2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca(2+)]i with β2AR co-transfection, ∼53% decrease in [Ca(2+)]i signaling with shRNA knockdown of β2AR in H292 cells, and ∼60% loss of [Ca(2+)]i responsiveness in βAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with β2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of β2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged β-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with β-agonist co-treatment. Thus the β2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by β-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function. PMID:27342779

  14. β2-Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function*

    PubMed Central

    Kim, Donghwa; Pauer, Susan H.; Yong, Hwan M.; An, Steven S.; Liggett, Stephen B.

    2016-01-01

    Bitter taste receptors (TAS2Rs) are G-protein-coupled receptors now recognized to be expressed on extraoral cells, including airway smooth muscle (ASM) where they evoke relaxation. TAS2Rs are difficult to express in heterologous systems, with most receptors being trapped intracellularly. We find, however, that co-expression of β2-adrenergic receptors (β2AR) in HEK-293T routes TAS2R14 to the cell surface by forming receptor heterodimers. Cell surface TAS2R14 expression was increased by ∼5-fold when β2AR was co-expressed. Heterodimer formation was shown by co-immunoprecipitation with tagged receptors, biomolecular fluorescence complementation, and merged confocal images. The dynamic nature of this interaction was shown by: a gene-dose relationship between transfected β2AR and TAS2R14 expression, enhanced (up to 3-fold) TAS2R14 agonist stimulation of [Ca2+]i with β2AR co-transfection, ∼53% decrease in [Ca2+]i signaling with shRNA knockdown of β2AR in H292 cells, and ∼60% loss of [Ca2+]i responsiveness in βAR knock-out mouse ASM. Once expressed on the surface, we detected unidirectional, conformation-dependent, interaction within the heterodimer, with β2AR activation rapidly uncoupling TAS2R14 function (∼65% desensitization). Cross-talk was independent of β2AR internalization and cAMP/PKA, and not accompanied by TAS2R14 internalization. With prolonged β-agonist exposure, TAS2R14 internalized, consistent with slow recycling of naked TAS2R14 in the absence of the heterodimeric milieu. In studies of ASM mechanics, rapid cross-talk was confirmed at the physiologic level, where relaxation from TAS2R14 agonist was decreased by ∼50% with β-agonist co-treatment. Thus the β2AR acts as a double-edged sword: increasing TAS2R14 cell surface expression, but when activated by β-agonist, partially offsetting the expression phenotype by direct receptor:receptor desensitization of TAS2R14 function. PMID:27342779

  15. Target sequencing, cell experiments, and a population study establish endothelial nitric oxide synthase (eNOS) gene as hypertension susceptibility gene.

    PubMed

    Salvi, Erika; Kuznetsova, Tatiana; Thijs, Lutgarde; Lupoli, Sara; Stolarz-Skrzypek, Katarzyna; D'Avila, Francesca; Tikhonoff, Valerie; De Astis, Silvia; Barcella, Matteo; Seidlerová, Jitka; Benaglio, Paola; Malyutina, Sofia; Frau, Francesca; Velayutham, Dinesh; Benfante, Roberta; Zagato, Laura; Title, Alexandra; Braga, Daniele; Marek, Diana; Kawecka-Jaszcz, Kalina; Casiglia, Edoardo; Filipovsky, Jan; Nikitin, Yuri; Rivolta, Carlo; Manunta, Paolo; Beckmann, Jacques S; Barlassina, Cristina; Cusi, Daniele; Staessen, Jan A

    2013-11-01

    A case-control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase (eNOS) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% (P<0.01) lower. In the population, systolic/diastolic blood pressure increased over 7.6 years (median) by 9.7/6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6-11.1; P=0.028) and 4.8 mm Hg diastolic (CI, 1.5-8.2; P=0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes (P≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24-3.37; P=0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension.

  16. Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

    PubMed Central

    MaCaulay, S Lance; Stoichevska, Violet; Grusovin, Julian; Gough, Keith H; Castelli, Laura A; Ward, Colin W

    2003-01-01

    SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested. PMID:12917015

  17. Target sequencing, cell experiments, and a population study establish endothelial nitric oxide synthase (eNOS) gene as hypertension susceptibility gene.

    PubMed

    Salvi, Erika; Kuznetsova, Tatiana; Thijs, Lutgarde; Lupoli, Sara; Stolarz-Skrzypek, Katarzyna; D'Avila, Francesca; Tikhonoff, Valerie; De Astis, Silvia; Barcella, Matteo; Seidlerová, Jitka; Benaglio, Paola; Malyutina, Sofia; Frau, Francesca; Velayutham, Dinesh; Benfante, Roberta; Zagato, Laura; Title, Alexandra; Braga, Daniele; Marek, Diana; Kawecka-Jaszcz, Kalina; Casiglia, Edoardo; Filipovsky, Jan; Nikitin, Yuri; Rivolta, Carlo; Manunta, Paolo; Beckmann, Jacques S; Barlassina, Cristina; Cusi, Daniele; Staessen, Jan A

    2013-11-01

    A case-control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase (eNOS) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% (P<0.01) lower. In the population, systolic/diastolic blood pressure increased over 7.6 years (median) by 9.7/6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6-11.1; P=0.028) and 4.8 mm Hg diastolic (CI, 1.5-8.2; P=0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes (P≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24-3.37; P=0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension. PMID:24019403

  18. The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6.

    PubMed

    Küffer, Alexander; Lakkaraju, Asvin K K; Mogha, Amit; Petersen, Sarah C; Airich, Kristina; Doucerain, Cédric; Marpakwar, Rajlakshmi; Bakirci, Pamela; Senatore, Assunta; Monnard, Arnaud; Schiavi, Carmen; Nuvolone, Mario; Grosshans, Bianka; Hornemann, Simone; Bassilana, Frederic; Monk, Kelly R; Aguzzi, Adriano

    2016-08-25

    Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options. PMID:27501152

  19. Sodium butyrate down-regulates tristetraprolin-mediated cyclin B1 expression independent of the formation of processing bodies.

    PubMed

    Zheng, Xiang-Tao; Xiao, Xiao-Qiang; Dai, Ju-Ji

    2015-12-01

    Butyrate regulates multiple host cellular events including the cell cycle; however, little is known about the molecular mechanism by which butyrate induces a global down-regulation of the expression of genes associated with the cell cycle. Here, we demonstrate that treating HEK293T cells and the non-small-cell lung cancer cell line A549 with a high concentration of sodium butyrate reduces cyclin B1 expression. The underlying mechanism is related to the destabilization of its mRNA by tristetraprolin, which is up-regulated in response to sodium butyrate. Specifically, the sodium butyrate stimulation reduces the mRNA and protein expression of cyclin B1 and, conversely, upregulates tristetraprolin expression. Importantly, the overexpression of tristetraprolin in HEK293T decreases the mRNA and protein expression of cyclin B1; in contrast, knockdown of tristetraprolin mediated by small interfering RNA increases its expression in response to sodium butyrate treatment for both HEK293T and A549 cells. Furthermore, results from luciferase reporter assays and RNA immunoprecipitation indicate that sodium butyrate accelerates 3' UTR-dependent cyclin B1 decay by enhancing the binding of tristetraprolin to the 3' untranslated region of cyclin B1. Surprisingly, the overexpression of tristetraprolin prevents the formation of processing bodies, and the siRNA-mediated silencing of EDC4 does not restore the sodium butyrate-induced reduction of cyclin B1 expression. Thus, we confirm that NaBu regulates ZFP36-mediated cyclin B1 expression in a manner that is independent of the formation of P-bodies. The above findings disclose a novel mechanism of sodium butyrate-mediated gene expression regulation and might benefit its application in tumor treatment.

  20. A cationic fluorescent polymeric thermometer for the ratiometric sensing of intracellular temperature.

    PubMed

    Uchiyama, Seiichi; Tsuji, Toshikazu; Ikado, Kumiko; Yoshida, Aruto; Kawamoto, Kyoko; Hayashi, Teruyuki; Inada, Noriko

    2015-07-01

    We developed new cationic fluorescent polymeric thermometers containing both benzothiadiazole and BODIPY units as an environment-sensitive fluorophore and as a reference fluorophore, respectively. The temperature-dependent fluorescence spectra of the thermometers enabled us to perform highly sensitive and practical ratiometric temperature sensing inside living mammalian cells. Intracellular temperatures of non-adherent MOLT-4 (human acute lymphoblastic leukaemia) and adherent HEK293T (human embryonic kidney) cells could be monitored with high temperature resolutions (0.01-1.0 °C) using the new cationic fluorescent polymeric thermometer.

  1. The protective effect of magnesium lithospermate B against glucose-induced intracellular oxidative damage

    SciTech Connect

    Qu, Jian; Ren, Xian; Hou, Rui-ying; Dai, Xing-ping; Zhao, Ying-chun; Xu, Xiao-jing; Zhang, Wei; Zhou, Gan; Zhou, Hong-hao; Liu, Zhao-qian

    2011-07-22

    Highlights: {yields} LAB reduced the ROS production in HEK293T cells cultured under oxidative stress. High dose of glucose enhanced the expression of HO-1 mRNA and HO-1 protein in a time-dependent manner. {yields} LAB enhanced the expression of HO-1 mRNA and HO-1 protein in a dose-dependent manner treated with high dose of glucose. {yields} LAB plays an important role against glucose-induced intracellular oxidative damage. {yields} The enhanced expression of HO-1 mRNA and HO-1 protein caused by LAB is regulated via Nrf2 signal pathway. -- Abstract: Objectives: To investigate the effects of magnesium lithospermate B (LAB) on intracellular reactive oxygen species (ROS) production induced by high dose of glucose or H{sub 2}O{sub 2}, we explored the influences of LAB on the expression of heme oxygenase-1 (HO-1) and nuclear factor E2-related factor-2 (Nrf2) in HEK293T cells after treatment with high dose of glucose. Materials and methods: The total nuclear proteins in HEK293T cells were extracted with Cytoplasmic Protein Extraction Kit. The ROS level was determined by flow cytometry. The mRNA and protein expression of HO-1 and Nrf2 were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Results: LAB reduced the ROS production in HEK293T cells cultured under oxidative stress. High dose of glucose enhanced the expression of HO-1 mRNA and HO-1 protein in a time-dependent manner. LAB enhanced the expression of HO-1 mRNA and HO-1 protein in a dose-dependent manner treated with high dose of glucose. The amount of Nrf2 translocation was enhanced after cells were pretreated with 50 {mu}mol/L or 100 {mu}mol/L LAB. Silencing of Nrf2 gene eliminated the enhanced expression of HO-1 protein induced by high dose of glucose plus LAB. Conclusions: LAB plays an important role against glucose-induced intracellular oxidative damage. The enhanced expression of HO-1 mRNA and HO-1 protein caused by LAB is regulated via Nrf2 signal pathway.

  2. POSH stimulates the ubiquitination and the clathrin-independent endocytosis of ROMK1 channels.

    PubMed

    Lin, Dao-Hong; Yue, Peng; Pan, Chu-Yang; Sun, Peng; Zhang, Xin; Han, Zeguang; Roos, Marcel; Caplan, Michael; Giebisch, Gerhard; Wang, Wen-Hui

    2009-10-23

    POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-alpha, beta, and gamma subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Delta373-378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHDeltaRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHDeltaRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK

  3. POSH Stimulates the Ubiquitination and the Clathrin-independent Endocytosis of ROMK1 Channels*

    PubMed Central

    Lin, Dao-Hong; Yue, Peng; Pan, Chu-Yang; Sun, Peng; Zhang, Xin; Han, Zeguang; Roos, Marcel; Caplan, Michael; Giebisch, Gerhard; Wang, Wen-Hui

    2009-01-01

    POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-α, β, and γ subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Δ373–378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHΔRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHΔRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We

  4. CyHV-2 ORF104 activates the p38 MAPK pathway.

    PubMed

    Du, Mi; Chen, Mingliang; Shen, Haifeng; Wang, Wei; Li, Zengpeng; Wang, Weiyi; Huang, Jianhui; Chen, Jianming

    2015-10-01

    Cyprinid herpesvirus 2 (CyHV-2) is the pathogen responsible for herpesviral hematopoietic necrosis disease, which causes huge losses on aquaculture. So far the studies of CyHV-2 mainly focus on the identification and detection of this virus, but little is known about the role of specific CyHV-2 genes in the infection process. Based on the genomic information, CyHV-2 ORF104 encodes a kinase-like protein, which is highly conserved among the three CyHVs. Our study was initiated to investigate the role of kinase-like protein ORF104 during virus infection. Subcellular localization study showed that ORF104 was mainly expressed in the nucleus in both human HEK293T and fish EPC cells. However, deletion of the putative nuclear localization signal of ORF104 (ORF104M) resulted in the cytoplasmic distribution in HEK293T. We then examined whether MAPKs were involved in the ORF104-mediated signaling pathway by overexpressing ORF104 and ORF104M in HEK293T. Overexpression of ORF104 and ORF104M resulted in the up-regulation of p38 phosphorylation, but not JNK or ERK, indicating that ORF104 specifically activates p38 signaling pathway. In vivo study showed that CyHV-2 infection enhanced p38 phosphorylation in gibel carp (Carassius auratus gibelio). Interestingly, p38 inhibitor SB203580 strongly reduced fish death caused by CyHV-2 infection. Therefore, our study for the first time reveals the function of ORF104 during CyHV-2 infection, indicating that ORF104 is a potential vaccine candidate for CyHV-2.

  5. A Novel Class of Metal-Directed Supramolecular DNA-Delivery Systems

    SciTech Connect

    Cruz-Campa, I.; Arzola, A.; Santiago, L.; Parsons, J.G.; Varela-Ramirez, A.; Aguilera, R.J.; Noveron, J.C.

    2009-06-03

    The bis-complexes [Cu(L{sub dt}){sub 2}](OTf){sub 2} ( 1) and [Cu(L{sub ot}){sub 2}](OTf){sub 2} ( 2), where L{sub dt} = 1-dodecyl-1,4,7-triazacyclononane, L{sup ot} = 1-octadecyl-1,4,7-triazacyclononane and OTf = trifluoromethanesulfonate, formed a novel class of metallo-liposomes in water that transfect pEGFP-N1 plasmids into HEK 293-T cells at 38% and 4% efficiency, respectively.

  6. Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin*

    PubMed Central

    Berezniuk, Iryna; Vu, Hang T.; Lyons, Peter J.; Sironi, Juan J.; Xiao, Hui; Burd, Berta; Setou, Mitsutoshi; Angeletti, Ruth H.; Ikegami, Koji; Fricker, Lloyd D.

    2012-01-01

    The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2–3-fold) or knockdown (80–90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo. PMID:22170066

  7. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.

    PubMed

    Lin, Steven; Staahl, Brett T; Alla, Ravi K; Doudna, Jennifer A

    2014-12-15

    The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

  8. Unconventional actin conformations localize on intermediate filaments in mitosis

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-03-04

    Research highlights: {yields} Unconventional actin conformations colocalize with vimentin on a cage-like structure in metaphase HEK 293T cells. {yields} These conformations are detected with the anti-actin antibodies 1C7 ('lower dimer') and 2G2 ('nuclear actin'), but not C4 (monomeric actin). {yields} Mitotic unconventional actin cables are independent of filamentous actin or microtubules. {yields} Unconventional actin colocalizes with vimentin on a nocodazole-induced perinuclear dense mass of cables. -- Abstract: Different structural conformations of actin have been identified in cells and shown to reside in distinct subcellular locations of cells. In this report, we describe the localization of actin on a cage-like structure in metaphase HEK 293T cells. Actin was detected with the anti-actin antibodies 1C7 and 2G2, but not with the anti-actin antibody C4. Actin contained in this structure is independent of microtubules and actin filaments, and colocalizes with vimentin. Taking advantage of intermediate filament collapse into a perinuclear dense mass of cables when microtubules are depolymerized, we were able to relocalize actin to such structures. We hypothesize that phosphorylation of intermediate filaments at mitosis entry triggers the recruitment of different actin conformations to mitotic intermediate filaments. Storage and partition of the nuclear actin and antiparallel 'lower dimer' actin conformations between daughter cells possibly contribute to gene transcription and transient actin filament dynamics at G1 entry.

  9. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6.

    PubMed

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-07-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

  10. Bioinformatics analysis and expression of a novel protein ROP48 in Toxoplasma gondii.

    PubMed

    Zhou, Jian; Wang, Lin; Zhou, Aihua; Lu, Gang; Li, Qihang; Wang, Zhilin; Zhu, Meiyan; Zhou, Huaiyu; Cong, Hua; He, Shenyi

    2016-03-01

    Toxoplasma gondii is an obligate intracellular apicomplexan parasite, and can infect warmblooded animals and humans all over the world. In the past years, ROP family genes encoding particular proteins of T. gondii had made a great contribution to toxoplasmosis. In this study, we used multiple bioinformatics approaches to predict the physical and chemical characteristics, transmembrane domain, epitope, and topological structure of the rhoptry protein 48 (ROP48). The results indicated that ROP48 protein was mainly located in the membrane and had several positive linear-B cell epitopes and Th-cell epitopes, which suggested that ROP48 is a potential DNA vaccine candidate against toxoplasmosis. Then the PCR product amplified from the ROP48 cDNA was inserted into a pEASY-T1 vector to build a recombinant cloning plasmid. After sequencing, ROP48 was subcloned into a eukaryotic expression plasmid pEGFP-C1 to obtain pEGFP-C1-ROP48 (pROP48). After identification by PCR and restriction enzyme digestion, the recombinant plasmid pROP48 was transfected into HEK 293-T cell and identified by RT-PCR. The results showed that the eukaryotic expression plasmid pROP48 was constructed and transfected to the cells of HEK 293-T successfully. Western blotting showed that the expressed proteins can be recognized by anti-STAg mouse sera. PMID:27078655

  11. Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies

    PubMed Central

    Chen, Jing; Shinde, Sudhirkumar; Koch, Markus-Hermann; Eisenacher, Martin; Galozzi, Sara; Lerari, Thilo; Barkovits, Katalin; Subedi, Prabal; Krüger, Rejko; Kuhlmann, Katja; Sellergren, Börje; Helling, Stefan; Marcus, Katrin

    2015-01-01

    Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, “plastic antibodies”) featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites. PMID:26126808

  12. Micro-optical coherence tomography tracking of magnetic gene transfection via Au-Fe3O4 dumbbell nanoparticles

    NASA Astrophysics Data System (ADS)

    Shi, Wei; Liu, Xinyu; Wei, Chao; Xu, Zhichuan J.; Sim, Stanley Siong Wei; Liu, Linbo; Xu, Chenjie

    2015-10-01

    Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT.Heterogeneous Au-Fe3O4 dumbbell nanoparticles (NPs) are composed of Au NPs and Fe3O4 NPs that bring in optical and magnetic properties respectively. This article reports the engineering of Au-Fe3O4 NPs as gene carriers for magnetic gene transfection as well as contrast agents for micro-optical coherence tomography (μOCT). As a proof-of-concept, Au-Fe3O4 NPs are used to deliver the green fluorescent protein to HEK 293T cells and their entrance into the cells is monitored through μOCT. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05459a

  13. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

    PubMed Central

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-01-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer. PMID:27364926

  14. Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies.

    PubMed

    Chen, Jing; Shinde, Sudhirkumar; Koch, Markus-Hermann; Eisenacher, Martin; Galozzi, Sara; Lerari, Thilo; Barkovits, Katalin; Subedi, Prabal; Krüger, Rejko; Kuhlmann, Katja; Sellergren, Börje; Helling, Stefan; Marcus, Katrin

    2015-01-01

    Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here, we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs, "plastic antibodies") featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells, human neuroblastoma SH-SY5Y cells, mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography, pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate, exceeding the number identified by TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites. PMID:26126808

  15. A Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK)-dependent Transcriptional Program Controls Activation of the Early Growth Response 1 (EGR1) Gene during Amino Acid Limitation*

    PubMed Central

    Shan, Jixiu; Balasubramanian, Mukundh N.; Donelan, William; Fu, Lingchen; Hayner, Jaclyn; Lopez, Maria-Cecilia; Baker, Henry V.; Kilberg, Michael S.

    2014-01-01

    Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism. PMID:25028509

  16. Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata.

    PubMed

    Wei, Ning-Ning; Lv, Hai-Ning; Wu, Yang; Yang, Shi-Long; Sun, Xiao-Ying; Lai, Ren; Jiang, Yong; Wang, KeWei

    2016-01-01

    Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy. PMID:26515068

  17. Nanodiamond-Mediated Intercellular Transport of Proteins through Membrane Tunneling Nanotubes.

    PubMed

    Epperla, Chandra Prakash; Mohan, Nitin; Chang, Che-Wei; Chen, Chia-Chun; Chang, Huan-Cheng

    2015-12-01

    Recently discovered tunneling nanotubes (TNTs) are capable of creating intercellular communication pathways through which transport of proteins and other cytoplasmic components occurs. Intercellular transport is related to many diseases and nanotubes are potentially useful as drug-delivery channels for cancer therapy. Here, we apply fluorescent nanodiamond (FND) as a photostable tracker, as well as a protein carrier, to illustrate the transport events in TNTs of human cells. Proteins, including bovine serum albumin and green fluorescent protein, are first coated on 100-nm FNDs by physical adsorption and then single-particle tracking of the bioconjugates in the transient membrane connections is carried out by fluorescence microscopy. Stop-and-go and to-and-fro motions mediated by molecular motors are found for the active transport of protein-loaded FNDs trapped in the endosomal vehicles of human embryonic kidney cells (HEK293T). Quantitative analysis of the heterotypical transport between HEK293T and SH-SY5Y neuroblastoma cells by flow cytometry confirm the formation of open-ended nanotubes between them, despite that their TNTs differ in structural components. Our results demonstrate the promising applications of this novel carbon-based nanomaterial for intercellular delivery of biomolecular cargo down to the single-particle level.

  18. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  19. Selective Activation of Nociceptor TRPV1 Channel and Reversal of Inflammatory Pain in Mice by a Novel Coumarin Derivative Muralatin L from Murraya alata.

    PubMed

    Wei, Ning-Ning; Lv, Hai-Ning; Wu, Yang; Yang, Shi-Long; Sun, Xiao-Ying; Lai, Ren; Jiang, Yong; Wang, KeWei

    2016-01-01

    Coumarin and its derivatives are fragrant natural compounds isolated from the genus Murraya that are flowering plants widely distributed in East Asia, Australia, and the Pacific Islands. Murraya plants have been widely used as medicinal herbs for relief of pain, such as headache, rheumatic pain, toothache, and snake bites. However, little is known about their analgesic components and the molecular mechanism underlying pain relief. Here, we report the bioassay-guided fractionation and identification of a novel coumarin derivative, named muralatin L, that can specifically activate the nociceptor transient receptor potential vanilloid 1 (TRPV1) channel and reverse the inflammatory pain in mice through channel desensitization. Muralatin L was identified from the active extract of Murraya alata against TRPV1 transiently expressed in HEK-293T cells in fluorescent calcium FlexStation assay. Activation of TRPV1 current by muralatin L and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV1-expressing HEK-293T cells and dorsal root ganglion neurons isolated from mice. Furthermore, muralatin L could reverse inflammatory pain induced by formalin and acetic acid in mice but not in TRPV1 knock-out mice. Taken together, our findings show that muralatin L specifically activates TRPV1 and reverses inflammatory pain, thus highlighting the potential of coumarin derivatives from Murraya plants for pharmaceutical and medicinal applications such as pain therapy.

  20. Soluble form of canine transferrin receptor inhibits canine parvovirus infection in vitro and in vivo.

    PubMed

    Wen, Jiexia; Pan, Sumin; Liang, Shuang; Zhong, Zhenyu; He, Ying; Lin, Hongyu; Li, Wenyan; Wang, Liyue; Li, Xiujin; Zhong, Fei

    2013-01-01

    Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo.

  1. ChBax/Bak as key regulators of the mitochondrial apoptotic pathway: cloned and characterized in Crassostrea hongkongensis.

    PubMed

    Xiang, Zhiming; Qu, Fufa; Wang, Fuxuan; Xiao, Shu; Jun, Li; Zhang, Yang; Yu, Ziniu

    2015-02-01

    Apoptosis has been primarily investigated in mammals, and little is known about apoptosis in mollusks. The proteins Bax and Bak play critical roles in the mitochondrial apoptosis pathway and in determining cell fate. In this study, ChBax and ChBak, homologs of the well-known Bax and Bak proteins, were identified from the oyster Crassostrea hongkongensis. The ChBax/Bak proteins consist of 207/232 amino acids with the typical domains found in BCL-2 family members. ChBax and ChBak mRNA expression were detected in all 8 of the selected oyster tissues and at the different stages of development. Fluorescence microscopy revealed that the full-length proteins of ChBax/Bak were located in the cytoplasm and mitochondrial outer membrane, of HEK293T cells, respectively. Furthermore, both of the genes' expression levels were found to increase in the hemocytes of oysters challenged with pathogens. The over-expression of ChBax or ChBak activates the p53-Luc reporter gene in HEK293T cells in a dose-dependent manner. These results indicate that ChBax and ChBak may play important roles in the mitochondrial apoptotic pathway in oysters.

  2. Isolation of koala retroviruses from koalas in Japan.

    PubMed

    Miyazawa, Takayuki; Shojima, Takayuki; Yoshikawa, Rokusuke; Ohata, Takuji

    2011-01-01

    Koala retrovirus (KoRV) is considered to be associated with leukemia, lymphoma and immunodeficiency-like diseases in koalas. We therefore conducted a pilot study of KoRV infection in five Queensland koalas in Kobe Municipal Oji Zoo. By polymerase chain reaction to detect partial env and pol genes of KoRV in genomic DNA isolated from whole blood and feces, all five koalas were found to be positive for KoRV proviruses. We succeeded in culturing koala lymphocytes from less than 1 ml blood for over 14 days in the presence of recombinant human interleukin-2. By coculturing the lymphocytes with human embryonic kidney (HEK) 293T cells, we isolated KoRVs from all five koalas. We designated these isolates as strains OJ-1 to OJ-5. By electron microscopy, we observed C-type retroviral particles in HEK 293T cells chronically infected with KoRV strain OJ-4. This is the first report on the isolation of KoRV from koalas in a Japanese zoo.

  3. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel.

    PubMed

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-09-14

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, OPEN ACCESS Toxins 2015, 7 3672 BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation.

  4. Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol.

    PubMed

    Kudo, Yuma; Abe, Akihiro; Ito, Kumiko; Cho, Yuko; Yotsu-Yamashita, Mari; Konoki, Keiichi

    2014-01-01

    Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the (26)Asp, (359)Arg, and (361)Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.

  5. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

    PubMed Central

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-01-01

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation. PMID:26389953

  6. Characterization of two mutations in the SPTLC1 subunit of serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathy type I.

    PubMed

    Rotthier, Annelies; Penno, Anke; Rautenstrauss, Bernd; Auer-Grumbach, Michaela; Stettner, Georg M; Asselbergh, Bob; Van Hoof, Kim; Sticht, Heinrich; Lévy, Nicolas; Timmerman, Vincent; Hornemann, Thorsten; Janssens, Katrien

    2011-06-01

    Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy leading to progressive distal sensory loss and severe ulcerations. Mutations in SPTLC1 and SPTLC2, encoding the two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the first and rate-limiting step in the de novo synthesis of sphingolipids, have been reported to cause HSAN-I. Here, we demonstrate that the SPTLC1 mutations p.S331F and p.A352V result in a reduction of SPT activity in vitro and are associated with increased levels of the deoxysphingoid bases 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine in patients' plasma samples. Stably expressing p.S331F-SPTLC1 HEK293T cell lines likewise show accumulation of deoxysphingoid bases, but this accumulation is not observed in HEK293T cells overexpressing p.A352V-SPTLC1. These results confirm that the increased formation of deoxysphingoid bases is a key feature for HSAN-I as it is associated with all pathogenic SPTLC1 and SPTLC2 mutations reported so far, but also warrant for caution in the interpretation of in vitro data. PMID:21618344

  7. Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel.

    PubMed

    Hakim, Md Abdul; Jiang, Wenbin; Luo, Lei; Li, Bowen; Yang, Shilong; Song, Yuzhu; Lai, Ren

    2015-09-01

    The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, OPEN ACCESS Toxins 2015, 7 3672 BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation. PMID:26389953

  8. Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio.

    PubMed

    Qi, Lin; Xiang, Zhiming

    2015-07-01

    The death domain-associated protein Daxx exerts many functions including the induction and inhibition of apoptosis, regulation of chromatin remodeling and gene transcription. In this report, we have cloned and characterized a Daxx ortholog from the zebrafish, Danio rerio. The bioinformatics analysis results indicated that the open reading frame (ORF) of zebrafish Daxx is 2,151bp long and encodes a putative protein of 716 amino acids containing Daxx domain. Though quantitative PCR analyses, Daxx mRNA was detected in embryonic development from 6 h to 120 h and in all 11 selected zebrafish tissues, and the expression of Daxx was increased first and then decreased during megalocytivirus infectious spleen and kidney necrosis virus (ISKNV) infection. Fluorescence microscopy indicated that the full-length protein was located in the nuclei of the tested Hela cells uniformly but punctiform distribution in HEK293T. In the luciferase report assays, the GAL4-Daxx fusion protein inhibited the transcriptional activity of L8G5-Luc reporter gene showed that Daxx might act as a transcriptional repressor, following the over-expression in HEK293T, the activation of NF-κB-Luc and p53/p21-Luc reporter genes were repressed by the protein. These results suggested that Daxx might play definite role in apoptosis and innate immunity in zebrafish.

  9. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  10. GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses.

    PubMed

    Fang, Cheng; Deng, Lunbin; Keller, Cheryl A; Fukata, Masaki; Fukata, Yuko; Chen, Gong; Lüscher, Bernhard

    2006-12-01

    Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma2 subunit of GABA(A) receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABA(A) receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

  11. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  12. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  13. Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

    PubMed Central

    2012-01-01

    Background Probiotic bacteria are increasingly used as immunomodulatory agents. Yet detailed molecular knowledge on the immunomodulatory molecules of these bacteria is lagging behind. Lipoteichoic acid (LTA) is considered a major microbe-associated molecular pattern (MAMP) of Gram-positive bacteria. However, many details and quantitative data on its immune signalling capacity are still unknown, especially in beneficial bacteria. Recently, we have demonstrated that a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having modified LTA molecules, has an enhanced probiotic efficacy in a DSS-induced colitis model as compared to wild-type. Results In this study, the importance of D-alanylated and acylated LTA for the pro-inflammatory activity of LGG was studied in vitro. Purified native LTA of LGG wild-type exhibited a concentration-dependent activation of NF-κB signalling in HEK293T cells after interaction with TLR2/6, but not with TLR2 alone. Chemical deacylation of LTA interfered with the TLR2/6 interaction, while a moderate effect was observed with chemical dealanylation. Similarly, the dltD mutant of LGG exhibited a significantly reduced capacity to activate TLR2/6-dependent NF-κB signalling in a HEK293T reporter cell line compared to wild-type. In addition, the dltD mutant of LGG showed a reduced induction of mRNA of the chemokine IL-8 in the Caco-2 epithelial cell line compared to wild-type. Experiments with highly purified LTA of LGG confirmed that LTA is a crucial factor for IL-8 mRNA induction in Caco-2 epithelial cells. Chemical dealanylation and deacylation reduced IL-8 mRNA expression. Conclusions Taken together, our results indicate that LTA of LGG is a crucial MAMP with pro-inflammatory activities such as IL-8 induction in intestinal epithelial cells and NF-κB induction in HEK293T cells via TLR2/6 interaction. The lipid chains of LGG LTA are needed for these activities, while also the D-alanine substituents are important, especially

  14. A Novel Nonsense Mutation in the MIP Gene Linked to Congenital Posterior Polar Cataracts in a Chinese Family

    PubMed Central

    Song, Zixun; Wang, Lianqing; Liu, Yaping; Xiao, Wei

    2015-01-01

    Purpose To detect the causative mutation for congenital posterior polar cataracts in a five-generation Chinese family and further explore the potential pathogenesis of this disease. Methods Coding exons, with flanking sequences of five candidate genes, were screened using direct DNA sequencing. The identified mutations were confirmed by restriction fragment length polymorphism (RFLP) analysis. A full-length wild-type or an Y219* mutant aquaporin0 (AQP0) fused with an N-terminal FLAG tag, was transfected into HEK293T cells. For co-localization studies, FLAG-WT-AQP0 and Myc-Y219*-AQP0 constructs were co-transfected. Quantitative real-time RT-PCR, western blotting and immunofluorescence studies were performed to determine protein expression levels and sub-cellular localization, respectively. Results We identified a novel nonsense mutation in MIP (c.657 C>G; p.Y219*) (major intrinsic protein gene) that segregates with congenital posterior polar cataract in a Chinese family. This mutation altered a highly conserved tyrosine to a stop codon (Y219*) within AQP0.When FLAG-WT-AQP0 and FLAG-Y219*-AQP0 expression constructs were singly transfected into HEK 293T cells, mRNA expression showed no significant difference between the wild-type and the mutant, while Y219*-AQP0 protein expression was significantly lower than that of wild-type AQP0. Wild-type AQP0 predominantly localized to the plasma membrane, while the mutated protein was abundant within the cytoplasm of HEK293T cells. However, when FLAG-WT-AQP0 andMyc-MU-AQP0were co-expressed, both proteins showed high fluorescence in the cytoplasm. Conclusions The novel nonsense mutation in the MIP gene (c.657 C>G) identified in a Chinese family may cause posterior polar cataracts. The dominant negative effect of the mutated protein on the wild-type protein interfered with the trafficking of wild-type protein to the cell membrane and both the mutant and wild-type protein were trapped in the cytoplasm. Consequently, both wild

  15. [OATP1B1 in drug-drug interactions between traditional Chinese medicine Danshensu and rosuvastatin].

    PubMed

    Wen, Jin-hua; Wei, Xiao-hua; Cheng, Xiao-hua; Zuo, Rong; Peng, Hong-wei; Lü, Yan-ni; Zhou, Jian; Zheng, Xue-lian; Cai, Jun; Xiong, Yu-qing; Cao, Li

    2016-01-01

    The study was designed to explore the drug-drug interactions mechanisms mediated by OATP1B1 between traditional Chinese medicine Danshensu and rosuvastatin. First, the changes of rosuvastatin pharmacokinetics were investigated in presence of Danshensu in rats. Then, the primary rat hepatocytes model was established to explore the effects of Danshensu on the uptake of rosuvastatin by hepatocytes. Finally, HEK293T cells with overexpression of OATP1B1*a and OATP1B1*5 were established using a lentiviral delivery system to explore the effects of Danshensu on the uptake of rosuvastatin. Rosuvastatin pharmacokinetic parameters of C(max0, AUCO(0-t), AUC(0-∞) were increased about 123%, 194% and 195%, by Danshensu in rats, while the CL z/F value was decreased by 60%. Uptake of rosuvastatin in the primary rat hepatocytes was decreased by 3.13%, 41.15% and 74.62%, respectively in the presence of 20, 40 and 80 μmol x L(-1) Danshensu. The IC50 parameters was (53.04 ± 2.43) μmol x L(-1). The inhibitory effect of Danshensu on OATP1B1 mediated transport of rosuvastatin was related to the OATP1B1 gene type. In OATP1B1*5-HEK293T mutant cells, transport of rosuvastatin were reduced by (39.11 ± 4.94)% and (63.61 ± 3.94)%, respectively, by Danshensu at 1 and 10 μmol x L(-1). While transport of rosuvastatin was reduced by (8.22 ± 2.40)% and (11.56 ± 3.04)% and in OATP1B1*1a cells, respectively. Danshensu significantly altered the pharmacokinetics of rosuvastatin in rats, which was related to competitive inhibition of transport by OATPJBI. Danshensu exhibited a significant activity in the inhibition of rosuvastatin transport by OATP1B1*5-HEK293T, but not by OATP1B1*1a, suggesting a dependence on OATP1B1 sequence. PMID:27405165

  16. α1-Syntrophin Variant Identified in Drug-Induced Long QT Syndrome Increases Late Sodium Current

    PubMed Central

    Choi, Jong-Il; Wang, Chaojian; Thomas, Matthew J.; Pitt, Geoffrey S.

    2016-01-01

    Drug-induced long-QT syndrome (diLQTS) is often due to drug block of IKr, especially in genetically susceptible patients with subclinical mutations in the IKr-encoding KCHN2. Few variants in the cardiac NaV1.5 Na+ channel complex have been associated with diLQTS. We tested whether a novel SNTA1 (α1-syntrophin) variant (p.E409Q) found in a patient with diLQTS increases late sodium current (INa-L), thereby providing a disease mechanism. Electrophysiological studies were performed in HEK293T cells co-expressing human NaV1.5/nNOS/PMCA4b with either wild type (WT) or SNTA1 variants (A390V-previously reported in congenital LQTS; and E409Q); and in adult rat ventricular cardiomyocytes infected with SNTA1 expressing adenoviruses (WT or one of the two SNTA1 variants). In HEK293T cells and in cardiomyocytes, there was no significant difference in the peak INa densities among the SNTA1 WT and variants. However, both variants increased INa-L (% of peak current) in HEK293T cells (0.58±0.10 in WT vs. 0.90±0.11 in A390V, p = 0.048; vs. 0.88±0.07 in E409Q, p = 0.023). In cardiomyocytes, INa-L was significantly increased by E409Q, but not by A390V compared to WT (0.49±0.14 in WT vs.0.94±0.23 in A390V, p = 0.099; vs. 1.12±0.24 in E409Q, p = 0.019). We demonstrated that a novel SNTA1 variant is likely causative for diLQTS by augmenting INa-L. These data suggest that variants within the NaV1.5-interacting α1-syntrophin are a potential mechanism for diLQTS, thereby expanding the concept that variants within congenital LQTS loci can cause diLQTS. PMID:27028743

  17. Effect of Thermodiffusion Nitriding on Cytocompatibility of Ti-6Al-4V Titanium Alloy

    NASA Astrophysics Data System (ADS)

    Pohrelyuk, I. M.; Tkachuk, O. V.; Proskurnyak, R. V.; Boiko, N. M.; Kluchivska, O. Yu.; Stoika, R. S.

    2016-04-01

    The nitrided layer was formed on the surface of Ti-6Al-4V titanium alloy by the thermodiffusion saturation in nitrogen at the atmospheric pressure. The study of the vitality of pseudonormal human embryo kidney cells of the HEK293T line showed that their cultivation in the presence of the untreated alloy sample is accompanied by a statistically significant reduction in the number of living cells compared with the control sample (untreated cells), whereas their cultivation in the presence of the nitrided alloy sample does not change the cell number considerably. In addition, it was shown that cell behavior in the presence of the nitrided sample differs only slightly from the control sample, whereas the growth of cells in the presence of the untreated alloy differed significantly from that in the control sample, demonstrating small groups of cells instead of their big clusters.

  18. Surface expression of the Anoctamin-1 (ANO1) channel is suppressed by protein-protein interactions with β-COP.

    PubMed

    Lee, Young-Sun; Bae, Yeonju; Park, Nammi; Yoo, Jae Cheal; Cho, Chang-Hoon; Ryoo, Kanghyun; Hwang, Eun Mi; Park, Jae-Yong

    2016-06-24

    Anoctamin-1 (ANO1) is a Ca(2+)-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with β-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of β-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with β-COP in U251 glioblastoma cells, and silencing of β-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that β-COP negatively regulates ANO1 surface expression. PMID:27207835

  19. Surface expression of the Anoctamin-1 (ANO1) channel is suppressed by protein-protein interactions with β-COP.

    PubMed

    Lee, Young-Sun; Bae, Yeonju; Park, Nammi; Yoo, Jae Cheal; Cho, Chang-Hoon; Ryoo, Kanghyun; Hwang, Eun Mi; Park, Jae-Yong

    2016-06-24

    Anoctamin-1 (ANO1) is a Ca(2+)-activated chloride channel (CaCC) that plays important physiological roles in normal and cancerous tissues. However, the plasma membrane trafficking mechanisms of ANO1 remain poorly characterized. In yeast two-hybrid screening experiments, we observed direct interactions of ANO1 with β-COP, which is a subunit of Coat Protein Complex I (COPI). This interaction was then confirmed using several in vitro and in vivo binding assays. Moreover, the cotransfection of β-COP with ANO1 into HEK293T cells led to decreased the surface expression and the channel activity of ANO1. Accordingly, endogenous ANO1 was associated with β-COP in U251 glioblastoma cells, and silencing of β-COP enhanced surface expression and whole-cell currents of ANO1 in these cells. Taken together, these data suggest that β-COP negatively regulates ANO1 surface expression.

  20. Cellular Activity of New Small Molecule Protein Arginine Deiminase 3 (PAD3) Inhibitors.

    PubMed

    Jamali, Haya; Khan, Hasan A; Tjin, Caroline C; Ellman, Jonathan A

    2016-09-01

    The protein arginine deiminases (PADs) catalyze the post-translational deimination of arginine side chains. Multiple PAD isozymes have been characterized, and abnormal PAD activity has been associated with several human disease states. PAD3 has been characterized as a modulator of cell growth via apoptosis inducing factor and has been implicated in the neurodegenerative response to spinal cord injury. Here, we describe the design, synthesis, and evaluation of conformationally constrained versions of the potent and selective PAD3 inhibitor 2. The cell activity of representative inhibitors in this series was also demonstrated for the first time by rescue of thapsigargin-induced cell death in PAD3-expressing HEK293T cells. PMID:27660689

  1. Lipocalin 2 regulation by thermal stresses: Protective role of Lcn2/NGAL against cold and heat stresses

    SciTech Connect

    Roudkenar, Mehryar Habibi; Halabian, Raheleh; Roushandeh, Amaneh Mohammadi; Nourani, Mohammad Reza; Masroori, Nasser; Ebrahimi, Majid; Nikogoftar, Mahin; Rouhbakhsh, Mehdi; Bahmani, Parisa; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2009-11-01

    Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.

  2. Defoliation and plasmid delivery with layer-by-layer coated colloids.

    PubMed

    Reibetanz, Uta; Claus, Claudia; Typlt, Elke; Hofmann, Jörg; Donath, Edwin

    2006-02-10

    The uptake of polyelectrolyte multilayer coated colloids into cells, subsequent defoliation and plasmid delivery was studied by means of confocal microscopy and flow cytometry. Silica particles coated layer-wise with protamine and dextran sulfate were given to HEK 293T cells. Optimum uptake was found with protamine as the top layer. The particle uptake likely follows an non-receptor-mediated endocytotic pathway. Defoliation of polyelectrolyte multilayer coated particles within cells was demonstrated by the release of incorporated plasmids as indicated by the expression of plasmid encoded proteins using the enhanced green fluorescence proteine (pEGFP-C1) plasmid and a red fluorescence protein (pDsRed1-N1) plasmid. This proves, together with the direct observation of fluorescent layer debris, the defoliation of coated particles and the release of layer components into the cytoplasm. Particle uptake and GFP expression.

  3. Synthesis and Antiproliferative Activity of Steroidal Thiosemicarbazone Platinum (Pt(II)) Complexes

    PubMed Central

    Huang, Yanmin; Kong, Erbin; Gan, Chunfang; Liu, Zhiping; Lin, Qifu; Cui, Jianguo

    2015-01-01

    Steroidal compounds exhibit particular physiological activities. In this paper, some steroidal thiosemicarbazones platinum (Pt(II)) complexes were synthesized by the condensation of steroidal ketones with thiosemicarbazide using estrone, chenodeoxycholic acid, and 7-deoxycholic acid as starting materials and complexation of steroidal thiosesemicarbazones with Pt(II). The complexes were characterized by IR, NMR, and MS, and their antiproliferative activities were evaluated. The results showed that some steroidal thiosemicarbazones platinum (Pt(II)) complexes displayed moderate cytotoxicity to HeLa and Bel-7404 cells. Thereinto, complex 6 showed an excellent inhibited selectivity to HeLa cells with an IC50 value of 9.2 μM and SI value of 21.7. At the same time, all compounds were almost inactive to HEK293T (normal kidney epithelial cells). The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs. PMID:26635511

  4. Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

    PubMed

    Loughran, H Marie; Han, Ziying; Wrobel, Jay E; Decker, Sarah E; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N; Reitz, Allen B

    2016-08-01

    We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4.

  5. Attenuation of fibroblast growth factor signaling by poly-N-acatyllactosamine type glycans

    PubMed Central

    Sugihara, Kazuhiro; Shibata, Toshiaki K.; Takata, Kayoko; Kimura, Takako; Kanayama, Naohiro; Williams, Roy; Hatakeyama, Shingo; Akama, Tomoya O.; Kuo, Chu-Wei; Khoo, Kay-Hooi; Fukuda, Michiko N.

    2014-01-01

    Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells. PMID:23968720

  6. Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress.

    PubMed

    Loughran, H Marie; Han, Ziying; Wrobel, Jay E; Decker, Sarah E; Ruthel, Gordon; Freedman, Bruce D; Harty, Ronald N; Reitz, Allen B

    2016-08-01

    We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4. PMID:27377328

  7. Transcriptional regulation of the c-Myc promoter by NFAT1 involves negative and positive NFAT-responsive elements.

    PubMed

    Mognol, Giuliana P; de Araujo-Souza, Patricia S; Robbs, Bruno K; Teixeira, Leonardo K; Viola, Joao P B

    2012-03-01

    A number of physiological processes in both normal and cancer cells are regulated by the proto-oncogene c-Myc. Among them, processes such as cell cycle regulation, apoptosis, angiogenesis and metastasis are also controlled by the nuclear factor of activated T cells (NFAT) family of transcription factors. It is already known that NFAT upregulates c-Myc expression by binding to an element located in the minimal c-Myc promoter. However, the importance of other NFAT sites in the context of the full promoter has not been evaluated. In this work, we demonstrate that the regulation of c-Myc by NFAT1 is more complex than previously conceived. In addition to the proximal site, NFAT1 directly binds to distal sites in the c-Myc promoter with different affinities. Promoter deletions and site-directed mutagenesis of NFAT binding sites in HEK293T cells suggest that in NFAT1-mediated transactivation, some NFAT elements are negative and dominant and others are positive and recessive. Furthermore, we demonstrate that cooperation with partner proteins, such as p300, enhances NFAT1-mediated transactivation of the c-Myc promoter. At last, the newly identified sites are also responsive to NFAT2 in HEK293T cells. However, in NIH3T3 cells, the regulation mediated by NFAT proteins is not dependent on the known NFAT sites, including the site previously described. Thus, our data suggest that the contribution of NFAT to the regulation of c-Myc expression may depend on a balance between the binding to positive and negative NFAT-responsive elements and cooperation with transcriptional cofactors, which may differ according to the context and/or cell type.

  8. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

  9. MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1.

    PubMed

    Lin, Dao-Hong; Yue, Peng; Zhang, Chengbiao; Wang, Wen-Hui

    2014-01-01

    The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1-3'UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3'UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3'UTR but not with 3'UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity. PMID:24197061

  10. CDCP1 cleavage is necessary for homodimerization-induced migration of triple-negative breast cancer

    PubMed Central

    Wright, HJ; Arulmoli, J; Motazedi, M; Nelson, LJ; Heinemann, FS; Flanagan, LA; Razorenova, OV

    2016-01-01

    Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic form of breast cancer that lacks the estrogen, progesterone and HER2 receptors and is resistant to targeted and hormone therapies. TNBCs express high levels of the transmembrane glycoprotein, complement C1r/C1s, Uegf, Bmp1 (CUB)-domain containing protein 1 (CDCP1), which has been correlated with the aggressiveness and poor prognosis of multiple carcinomas. Full-length CDCP1 (flCDCP1) can be proteolytically cleaved, resulting in a cleaved membrane-bound isoform (cCDCP1). CDCP1 is phosphorylated by Src family kinases in its full-length and cleaved states, which is important for its pro-metastatic signaling. We observed that cCDCP1, compared with flCDCP1, induced a dramatic increase in phosphorylation of the migration-associated proteins: PKCδ, ERK1/2 and p38 mitogen-activated protein kinase in HEK 293T. In addition, only cCDCP1 induced migration of HEK 293T cells and rescued migration of the TNBC cell lines expressing short hairpin RNA against CDCP1. Importantly, we found that only cCDCP1 is capable of dimerization, which can be blocked by expression of the extracellular portion of cCDCP1 (ECC), indicating that dimerization occurs through CDCP1’s ectodomain. We found that ECC inhibited phosphorylation of PKCδ and migration of TNBC cells in two-dimensional culture. Furthermore, ECC decreased cell invasiveness, inhibited proliferation and stimulated apoptosis of TNBC cells in three-dimensional culture, indicating that the cCDCP1 dimer is an important contributor to TNBC aggressiveness. These studies have important implications for the development of a therapeutic to block CDCP1 activity and TNBC metastasis. PMID:26876198

  11. Synthesis, Biological Evaluation and Molecular Modeling of Substituted Indeno[1,2-b]indoles as Inhibitors of Human Protein Kinase CK2

    PubMed Central

    Alchab, Faten; Ettouati, Laurent; Bouaziz, Zouhair; Bollacke, Andre; Delcros, Jean-Guy; Gertzen, Christoph G.W.; Gohlke, Holger; Pinaud, Noël; Marchivie, Mathieu; Guillon, Jean; Fenet, Bernard; Jose, Joachim; Le Borgne, Marc

    2015-01-01

    Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM). Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF. PMID:26061121

  12. Measurement of antibodies to varicella-zoster virus using a virus-free fluorescent-antibody-to-membrane-antigen (FAMA) test.

    PubMed

    Park, Rackhyun; Hwang, Ji Young; Lee, Kang Il; Namkoong, Sim; Choi, Seuk-Keun; Park, Songyong; Park, Hosun; Park, Junsoo

    2015-02-01

    The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

  13. Polyamide Nanogels from Generally Recognized as Safe Components and Their Toxicity in Mouse Preimplantation Embryos.

    PubMed

    Prasad, Priyaa; Molla, Mijanur Rahaman; Cui, Wei; Canakci, Mine; Osborne, Barbara; Mager, Jesse; Thayumanavan, S

    2015-11-01

    Safe delivery systems that can not only encapsulate hydrophobic drug molecules, but also release them in response to specific triggers are important in several therapeutic and biomedical applications. In this paper, we have designed a nanogel based on molecules that are generally recognized as safe (GRAS). We have shown that the resultant polymeric nanogels exhibit responsive molecular release and also show high in vitro cellular viability on HEK 293T, HeLa, MCF 7, and A549 cell lines. The toxicity of these nanogels was further evaluated with a highly sensitive assay using mouse preimplantation embryo development, where blastocysts were formed after 4 days of in vitro culture, and live pups were born when morulae/early blastocysts were transferred to the uteri of surrogate recipients. Our results indicate that these nanogels are nontoxic during mammalian development and do not alter normal growth or early embryo success rate. PMID:26367020

  14. Nitrooxyacyl derivatives of salicylic acid: aspirin-like molecules that covalently inactivate cyclooxygenase-1.

    PubMed

    Cena, Clara; Tosco, Paolo; Marini, Elisabetta; Lazzarato, Loretta; Piccinini, Marco; Ramondetti, Cristina; Lupino, Elisa; Fruttero, Roberta; Gasco, Alberto

    2011-03-01

    A recently described series of nitrooxyacyl derivatives of salicylic acid, displaying aspirin-like anti-inflammatory and platelet anti-aggregatory properties, were evaluated for their abilities to inhibit cyclooxygenase (COX). A number of these compounds irreversibly inhibited both COX-1 and COX-2 isoforms when tested in isolated human platelets and monocytes. Further studies using COX-1 expressed in human HEK293T cells showed that this inhibition mechanism is similar to that of aspirin; namely, the products are able to covalently bind to the Ser 530 residue present in the active cleft of the enzyme. Molecular modeling enabled us to rationalize this behavior. Because these products were previously found to display NO-dependent properties in rat animal models, particularly as they decreased in vivo gastrotoxicity and induced in vitro vasodilation, they represent a new and interesting class of potential aspirin-like antithrombotic agents worthy of further study.

  15. NME2 associates with PTPσ to transduce signals from chondroitin sulfate proteoglycans.

    PubMed

    Hamasaki, Hajime; Fujitani, Masashi; Yamashita, Toshihide

    2016-03-18

    Chondroitin sulfate proteoglycans (CSPGs) are a major component of glial scars, inhibiting axonal growth in the central nervous system. Protein tyrosine phosphatase, receptor type S (PTPσ) has been identified as a receptor for CSPGs, whereas its downstream signaling pathway remains to be fully understood. Here, we report that nucleoside diphosphate kinase 2 (NME2) interacts with PTPσ. We screened proteins associated with PTPσ by mass spectrometry, and obtained NME2. Immunoprecipitation analysis revealed that NME2 associated with the PTPσ intracellular domain in HEK-293T cells. NME2 was expressed in the cytoplasm and nucleus of cortical neurons, and knockdown of NME2 in the cortical neurons completely rescued neurite outgrowth inhibition induced by CSPGs. These results demonstrate that NME2 associates with PTPσ to elicit neurite outgrowth inhibition in response to CSPGs. PMID:26896769

  16. Theophylline-7-acetic acid derivatives with amino acids as anti-tuberculosis agents.

    PubMed

    Voynikov, Yulian; Valcheva, Violeta; Momekov, Georgi; Peikov, Plamen; Stavrakov, Georgi

    2014-07-15

    A series of amides were synthesized by condensation of theophylline-7-acetic acid and eight commercially available amino acid methyl ester hydrochlorides. Consecutive hydrolysis of six of the amido-esters resulted in the formation of corresponding amido-acids. The newly synthesized compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. The activity varied depending on the amino acid fragments and in seven cases exerted excellent values with MICs 0.46-0.26 μM. Assessment of the cytotoxicity revealed that the compounds were not cytotoxic against the human embryonal kidney cell line HEK-293T. The theophylline-7-acetamides containing amino acid moieties appear to be promising lead compounds for the development of antimycobacterial agents.

  17. Proteomics Analysis of Cellular Proteins Co-Immunoprecipitated with Nucleoprotein of Influenza A Virus (H7N9).

    PubMed

    Sun, Ningning; Sun, Wanchun; Li, Shuiming; Yang, Jingbo; Yang, Longfei; Quan, Guihua; Gao, Xiang; Wang, Zijian; Cheng, Xin; Li, Zehui; Peng, Qisheng; Liu, Ning

    2015-01-01

    Avian influenza A viruses are serious veterinary pathogens that normally circulate among avian populations, causing substantial economic impacts. Some strains of avian influenza A viruses, such as H5N1, H9N2, and recently reported H7N9, have been occasionally found to adapt to humans from other species. In order to replicate efficiently in the new host, influenza viruses have to interact with a variety of host factors. In the present study, H7N9 nucleoprotein was transfected into human HEK293T cells, followed by immunoprecipitated and analyzed by proteomics approaches. A series of host proteins co-immunoprecipitated were identified with high confidence, some of which were found to be acetylated at their lysine residues. Bioinformatics analysis revealed that spliceosome might be the most relevant pathway involved in host response to nucleoprotein expression, increasing our emerging knowledge of host proteins that might be involved in influenza virus replication activities. PMID:26528969

  18. Maleimidyl magnetic nanoplatform for facile molecular MRI

    NASA Astrophysics Data System (ADS)

    Heo, Dan; Lee, Eugene; Ku, Minhee; Hwang, Seungyeon; Kim, Bongjune; Park, Yeonji; Lee, Young Han; Huh, Yong-Min; Haam, Seungjoo; Cheong, Jae-Ho; Yang, Jaemoon; Suh, Jin-Suck

    2014-07-01

    In this study, we developed the maleimidyl magnetic nanoplatform, which enables functional targeting of a biomarker-specific moiety for molecular imaging via MRI. The maleimide group of the maleimidyl magnetic nanoplatform is conjugated with a thiol group without additional crosslinkers and side products. A physicochemical analysis was conducted to verify the effectiveness of the maleimidyl magnetic nanoplatform, and the existence of the maleimidyl group was investigated using the platform. To prepare biomarker-specific MRI probes, a thiolated aptamer and peptide were immobilized onto the maleimidyl group of the maleimidyl magnetic nanoplatform. The fabricated MRI probes were applied to four cancer cell lines: HT1080, MCF7, MKN45, and HEK293T. To investigate the potential of the molecular MRI probe, the target-biomarker specificity was confirmed without serious cytotoxicity, and in vivo MRI analysis using a xenograft mouse model was demonstrated. We believe these results will be useful for fabricating molecular MRI probes for the diagnosis of cancer.

  19. Polyamide Nanogels from GRAS Components and Their Toxicity in Mouse Pre-implantation Embryos

    PubMed Central

    Prasad, Priyaa; Molla, Mijanur Rahaman; Cui, Wei; Canakci, Mine; Osborne, Barbara; Mager, Jesse; Thayumanavan, S.

    2016-01-01

    Safe delivery systems that can not only encapsulate hydrophobic drug molecules, but also release them in response to specific triggers, are important in several therapeutic and biomedical applications. In this paper, we have designed a nanogel based on molecules that are generally recognized as safe (GRAS). We have shown that the resultant polymeric nanogels exhibit responsive molecular release, and also show high in vitro cellular viability on HEK 293T, HeLa, MCF 7 and A549 cell lines. The toxicity of these nanogels was further evaluated with a highly sensitive assay using mouse preimplantation embryo development, where blastocysts were formed after four days of in vitro culture and live pups were born when morulae/early blastocysts were transferred to the uteri of surrogate recipients. Our results indicate that these nanogels are non-toxic during mammalian development and do not alter normal growth or early embryo success rate. PMID:26367020

  20. The broad spectrum 2-oxoglutarate oxygenase inhibitor N-oxalylglycine is present in rhubarb and spinach leaves.

    PubMed

    Al-Qahtani, Khalid; Jabeen, Bushra; Sekirnik, Rok; Riaz, Naheed; Claridge, Timothy D W; Schofield, Christopher J; McCullagh, James S O

    2015-09-01

    2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases are involved in many biological processes in organisms ranging from humans (where some are therapeutic targets) to plants. These enzymes are of significant biomedicinal interest because of their roles in hypoxic signaling and epigenetic regulation. Synthetic N-oxalylglycine (NOG) has been identified as a broad-spectrum 2OG oxygenase inhibitor and is currently widely used in studies on the hypoxic response and chromatin modifications in animals. We report the identification of NOG as a natural product present in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves; NOG was not observed in Escherchia coli or human embryonic kidney cells (HEK 293T). The finding presents the possibility that NOG plays a natural role in regulating gene expression by inhibiting 2OG dependent oxygenases. This has significance because tricarboxylic acid cycle (TCA) intermediate inhibition of 2OG dependent oxygenases has attracted major interest in cancer research.

  1. An inhibitory receptor of VLRB in the agnathan lamprey

    PubMed Central

    Wu, Fenfang; Chen, Liyong; Ren, Yong; Yang, Xiaojing; Yu, Tongzhou; Feng, Bo; Chen, Shangwu; Xu, Anlong

    2016-01-01

    Lamprey, the primitive jawless vertebrate, uses variable lymphocyte receptor (VLR) as alternative adaptive immune system instead of immunoglobulin (Ig)-based receptors used in jawed vertebrates. In the present study, we characterized a potential inhibitory receptor of VLRB from leucocytes in lamprey. It is a novel ITIM-containing IgSF protein and was therefore named as NICIP. NICIP has two Ig-like domains in extracellular region, a transmembrane domain and two classical ITIM motifs in cytoplasmic domain. It is mainly expressed on the surface of granulocytes and monocytes and can interact with VLRB. In transiently transfected HEK293T cells, it was confirmed again that it could interact with VLRB and the two phosphorylated ITIM motifs could recruit SHP-1 and SHP-2. These results imply that NICIP may play a role as a potential inhibitory receptor of VLRB and involve in negative regulation of immune response mediated by VLRB. PMID:27762335

  2. Reaction between lawsone and aminophenol derivatives: Synthesis, characterization, molecular structures and antiproliferative activity

    NASA Astrophysics Data System (ADS)

    Kathawate, Laxmi; Joshi, Pranya V.; Dash, Tapan Kumar; Pal, Sanjima; Nikalje, Milind; Weyhermüller, Thomas; Puranik, Vedavati G.; Konkimalla, V. Badireenath; Salunke-Gawali, Sunita

    2014-10-01

    Reaction between two bioreductive reactants lawsone (2-hydroxy-1,4-napthoquinone) and derivatives 2-aminophenol without catalyst is reported. The reaction between lawsone and 4-chloro-2-aminophenol leads to formation of red colored major product 1A:[2-[(5-chloro-hydroxyphenyl)amino]naphthalene-1,4-dione] and fluorescent orange colored minor compound 1B:[10-chloro-benzo[α]phenoxazine-5-one]. Molecular structure of 1A and 1B were determined by single crystal X-ray diffraction. Two mechanisms were proposed to the formation of red 1A and 1B. ‘Ortho-para’ tautomeric equilibrium was observed in DMSO-d6 solution in 1A, which was revealed by 1H, 13C NMR and LC-MS studies. Molecules of 1A formed dimers via Nsbnd H⋯O interaction and polymeric chain of dimers was formed by Osbnd H⋯O interactions. Cl⋯Cl interactions were observed between the polymeric chains of dimers in 1A. Molecules of 1B show Cl⋯N interaction. Antiproliferative properties is studied for 1A-5A compounds (obtained by the reaction of lawsone with 2-amino-4-methylphenol;2A, 2-aminophenol;3A, 3-aminophenol;4A and 4-aminophenol;5A) and evaluated against two cancer cell lines, THP1 (human monocytic leukemia cells) and COLO205 (colorectal adenocarcinoma) and one normal cell line, HEK293T (human embryonic kidney). The values of 50% inhibitory concentration (IC50) of compounds 1A-5A was determined using XTT assay. The cytotoxic effects of compounds 2A and 3A were observed against COLO205 and compounds 4A and 5A on THP1 were observed to be higher in comparison to their effect on HEK293T cell lines.

  3. Characterization of a novel sphingosine 1-phosphate receptor, Edg-8.

    PubMed

    Im, D S; Heise, C E; Ancellin, N; O'Dowd, B F; Shei, G J; Heavens, R P; Rigby, M R; Hla, T; Mandala, S; McAllister, G; George, S R; Lynch, K R

    2000-05-12

    Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.

  4. Comparative Processing and Function of Human and Ferret Cystic Fibrosis Transmembrane Conductance Regulator*

    PubMed Central

    Fisher, John T.; Liu, Xiaoming; Yan, Ziying; Luo, Meihui; Zhang, Yulong; Zhou, Weihong; Lee, Ben J.; Song, Yi; Guo, Chenhong; Wang, Yujiong; Lukacs, Gergely L.; Engelhardt, John F.

    2012-01-01

    The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRΔF508/ΔF508 animal model may be useful. PMID:22570484

  5. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.

    PubMed

    Singh, Nidhi; Yadav, Manisha; Singh, Abhishek Kumar; Kumar, Harish; Dwivedi, Shailendra Kumar Dhar; Mishra, Jay Sharan; Gurjar, Anagha; Manhas, Amit; Chandra, Sharat; Yadav, Prem Narayan; Jagavelu, Kumaravelu; Siddiqi, Mohammad Imran; Trivedi, Arun Kumar; Chattopadhyay, Naibedya; Sanyal, Sabyasachi

    2014-05-01

    The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.

  6. [Production and evaluation of immunologic characteristics of mzNLA-3, a non-infectious HIV-1 clone with a large deletion in the pol sequence].

    PubMed

    Aghasadeghi, M R; Zabihollahi, R; Sadat, S M; Salehi, M; Ashtiani, S H; Namazi, R; Kashanizadeh, N; Azadmanesh, K

    2013-01-01

    Inactivation ofintegrase and reverse transcriptase can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive in replication HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNLA-3) was cut at two points in the pol region using the Bal I restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were not able to infect MT2 and HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments to improve the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations. PMID:23808159

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

    PubMed

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

    2016-04-01

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

  8. Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions.

    PubMed

    Atwood, Blake L; Woolnough, Jessica L; Lefevre, Gaelle M; Saint Just Ribeiro, Mariana; Felsenfeld, Gary; Giles, Keith E

    2016-08-19

    The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF. PMID:27288410

  9. A unique feature of Toll/IL-1 receptor domain-containing adaptor protein is partially responsible for lipopolysaccharide insensitivity in zebrafish with a highly conserved function of MyD88.

    PubMed

    Liu, Yanhui; Li, Mengzhen; Fan, Shan; Lin, Yiqun; Lin, Bin; Luo, Fang; Zhang, Chenxu; Chen, Shangwu; Li, Yingqiu; Xu, Anlong

    2010-09-15

    MyD88 and Toll/IL-1R domain-containing adaptor protein (TIRAP) are required for the TLR4 response to LPS stimulation in mammals, but the functions of the two adaptors and their involvement in zebrafish insensitivity to LPS remains unknown. We present a functional analysis of zebrafish Myd88 and Tirap and suggest that Myd88 is more important than Tirap for the activation of Tlr-mediated NF-kappaB, which may be a novel mechanism of Myd88-dependent TLR signaling in teleosts. Zebrafish Tirap lacks the phosphatidylinositol 4,5-bisphosphate binding motif required for human TIRAP location and has leucine at position 233 rather than the conserved proline of human TIRAP, as well as 105 additional aa at the N terminus. Overexpression of zebrafish Tirap in HEK293T cells did not activate NF-kappaB and IFN-beta, but slightly activated NF-kappaB in carp leukocyte cells. Zebrafish Myd88 alone strongly induced the activation of NF-kappaB and IFN-beta both in HEK293T and carp leukocyte cells. The function of Myd88 was dependent on its cellular location and the proline in the Toll/IL-1R domain. Although zebrafish Tirap was distributed throughout the cell rather than localized to the cytoplasmic membrane, its impaired ability to activate downstream Tlr molecules was unlikely to be related to its location because chimera TIRAP with a human TIRAP N terminus and membrane-binding domain also did not activate NF-kappaB. However, the mutation of leucine to proline increased the ability of Tirap to activate NF-kappaB. We suggest that the zebrafish Tirap needs a longer N terminus to perform its function and could be partially responsible for the resistance to LPS in zebrafish.

  10. Transcriptome-wide ribonuclease-mediated protein footprinting to identify RNA-protein interaction sites.

    PubMed

    Silverman, Ian M; Gregory, Brian D

    2015-01-15

    RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, investigating the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions interrogate only individual RBPs. However, there are hundreds of these proteins encoded in the human genome, and each cell type expresses a different repertoire, greatly limiting the ability of current methods to capture the global landscape of RNA-protein interactions. To address this gap, we and others have recently developed methods to globally identify regions of RNAs that are bound by proteins in an unbiased manner. Here, we describe a detailed protocol for performing our ribonuclease-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and unbound regions are digested with ribonucleases (RNases), leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and degraded, then protein-depleted RNAs are subjected to RNase treatment. After high-throughput sequencing of the remaining fragments, peak calling is performed to identify protein-protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line (HEK293T) and perform basic quality control, reproducibility, and benchmarking analyses. Finally, we delineate the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental and disease processes will help to further uncover the role of RBPs in post-transcriptional regulation.

  11. Transcriptome-wide ribonuclease-mediated protein footprinting to identify RNA-protein interaction sites.

    PubMed

    Silverman, Ian M; Gregory, Brian D

    2015-01-15

    RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, investigating the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions interrogate only individual RBPs. However, there are hundreds of these proteins encoded in the human genome, and each cell type expresses a different repertoire, greatly limiting the ability of current methods to capture the global landscape of RNA-protein interactions. To address this gap, we and others have recently developed methods to globally identify regions of RNAs that are bound by proteins in an unbiased manner. Here, we describe a detailed protocol for performing our ribonuclease-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and unbound regions are digested with ribonucleases (RNases), leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and degraded, then protein-depleted RNAs are subjected to RNase treatment. After high-throughput sequencing of the remaining fragments, peak calling is performed to identify protein-protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line (HEK293T) and perform basic quality control, reproducibility, and benchmarking analyses. Finally, we delineate the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental and disease processes will help to further uncover the role of RBPs in post-transcriptional regulation. PMID:25448484

  12. CMP-N-acetylneuraminic acid synthetase interacts with fragile X related protein 1

    PubMed Central

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-01-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. PMID:27357083

  13. The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

    PubMed Central

    Dagil, Yulia A.; Arbatsky, Nikolai P.; Alkhazova, Biana I.; L’vov, Vyacheslav L.; Mazurov, Dmitriy V.; Pashenkov, Mikhail V.

    2016-01-01

    Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants. PMID:27513337

  14. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  15. Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons.

    PubMed

    Ohishi, A; Keno, Y; Marumiya, A; Sudo, Y; Uda, Y; Matsuda, K; Morita, Y; Furuta, T; Nishida, K; Nagasawa, K

    2016-04-01

    Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

  16. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A).

    PubMed

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-Thai

    2010-10-01

    Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells. PMID:20833140

  17. MAP3K1 functionally interacts with Axin1 in the canonical Wnt signalling pathway.

    PubMed

    Sue Ng, Ser; Mahmoudi, Tokameh; Li, Vivian S W; Hatzis, Pantelis; Boersema, Paul J; Mohammed, Shabaz; Heck, Albert J; Clevers, Hans

    2010-01-01

    A central point of regulation in the Wnt/beta-catenin signalling pathway is the formation of the beta-catenin destruction complex. Axin1, an essential negative regulator of Wnt signalling, serves as a scaffold within this complex and is critical for rapid turnover of beta-catenin. To examine the mechanism by which Wnt signalling disables the destruction complex, we used an immunoprecipitation-coupled proteomics approach to identify novel endogenous binding partners of Axin1. We found mitogen-activated protein kinase kinase kinase 1 (MAP3K1) as an Axin1 interactor in Ls174T colorectal cancer (CRC) cells. Importantly, confirmation of this interaction in HEK293T cells indicated that the Axin1-MAP3K1 interaction is induced and modulated by Wnt stimulation. siRNA depletion of MAP3K1 specifically abrogated TCF/LEF-driven transcription and Wnt3A-driven endogenous gene expression in both HEK293T as well as DLD-1 CRC. Expression of ubiquitin ligase mutants of MAP3K1 abrogated TCF/LEF transcription, whereas kinase mutants had no effect in TCF-driven activity, highlighting the essential role of the MAP3K1 E3 ubiquitin ligase activity in regulation of the Wnt/beta-catenin pathway. These results suggest that MAP3K1, previously reported as an Axin1 inter-actor in c-Jun NH(2)-terminal kinase pathway, is also involved in the canonical Wnt signalling pathway and positively regulates expression of Wnt target genes. PMID:20128690

  18. A novel H395R mutation in MKKS/BBS6 causes retinitis pigmentosa and polydactyly without other findings of Bardet-Biedl or McKusick-Kaufman syndrome

    PubMed Central

    Hulleman, John D.; Nguyen, Annie; Ramprasad, V.L.; Murugan, Sakthivel; Gupta, Ravi; Mahindrakar, Avinash; Angara, Ravi; Sankurathri, Chandrasekhar

    2016-01-01

    Purpose To identify the causative mutation in two siblings from a consanguineous family in India with retinitis pigmentosa (RP) and polydactyly without other findings of Bardet-Biedl syndrome (BBS). We also performed functional characterization of the mutant protein to explore its role in this limited form of BBS. Methods The siblings underwent a thorough ophthalmological examination, including retinal optical coherence tomography (OCT) imaging, and an extensive physical examination with abdominal ultrasonography to characterize the disease phenotype. Next-generation sequencing (NGS) using a panel targeting retinal degeneration genes was performed on genomic DNA samples from the siblings and parents. Upon identification of the causative mutation, functional characterization was accomplished by performing protein–protein interaction studies in human embryonic kidney (HEK-293T) and human adult retinal pigmented epithelium (ARPE-19) cells. Results The two siblings showed signs of RP and polydactyly. The patients did not have truncal obesity, renal anomalies, hydrometrocolpos, congenital heart disease, or overt cognitive defects. NGS identified a homozygous c.1184A>G mutation in the MKKS/BBS6 gene in both patients resulting in a p.H395R substitution in the MKKS/BBS6 protein. This mutant protein decreased the interaction of MKKS/BBS6 with BBS12 but did so to a different extent in the HEK-293T versus ARPE-19 cells. Nonetheless, the effect of the H395R variant on disrupting interactions with BBS12 was not as profound as other reported MKKS/BBS6 mutations associated with syndromic RP. Conclusions We identified a novel H395R substitution in MKKS/BBS6 that results in a unique phenotype of only RP and polydactyly. Our observations reaffirm the notion that mutations in MKKS/BBS6 cause phenotypic heterogeneity and do not always result in classic MKKS or BBS findings. PMID:26900326

  19. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    PubMed

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis.

  20. Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.

    PubMed

    Wang, Zhi-Qiang; Wang, Wei; Shi, Lin; Chai, Ji-Tian; Zhang, Xin-Jun; Tao, Ya-Xiong

    2016-04-01

    The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis. PMID:26896843

  1. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  2. Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered enzyme kinetics.

    PubMed

    Tabatabaie, L; de Koning, T J; Geboers, A J J M; van den Berg, I E T; Berger, R; Klomp, L W J

    2009-05-01

    Three-phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare recessive inborn error in the biosynthesis of the amino acid L-serine characterized clinically by congenital microcephaly, psychomotor retardation, and intractable seizures. The biochemical abnormalities associated with this disorder are low concentrations of L-serine, D-serine, and glycine in cerebrospinal fluid (CSF). Only two missense mutations (p.V425M and p.V490M) have been identified in PHGDH, the gene encoding 3-PGDH, but it is currently unclear how these mutations in the carboxy-terminal regulatory domain of the protein affect enzyme function. We now describe five novel mutations in five patients with 3-PGDH deficiency; one frameshift mutation (p.G238fsX), and four missense mutations (p.R135W, p.V261M, p.A373T, and p.G377S). The missense mutations were located in the nucleotide binding and regulatory domains of 3-PGDH and did not affect steady-state expression, protein stability, and protein degradation rates. Patients' fibroblasts displayed a significant, but incomplete, reduction in maximal enzyme activities associated with all missense mutations. In transient overexpression studies in HEK293T cells, the p.A373T, p.V425M, and p.V490M mutations resulted in almost undetectable enzyme activities. Molecular modeling of the p.R135W and p.V261M mutations onto the partial crystal structure of 3-PGDH predicted that these mutations affect substrate and cofactor binding. This prediction was confirmed by the results of kinetic measurements in fibroblasts and transiently transfected HEK293T cells, which revealed a markedly decreased V(max) and an increase in K(m) values, respectively. Taken together, these data suggest that missense mutations associated with 3-PGDH deficiency either primarily affect substrate binding or result in very low residual enzymatic activity. PMID:19235232

  3. PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A*

    PubMed Central

    Matsuda, Shinya; Kominato, Kyohei; Koide-Yoshida, Shizuyo; Miyamoto, Kenji; Isshiki, Kinuka; Tsuji, Akihiko; Yuasa, Keizo

    2014-01-01

    PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser12, Ser66, and Ser109 and that PCTK3 activity significantly increased via phosphorylation at Ser12 by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3. PMID:24831015

  4. Production, purification and titration of a lentivirus-based vector for gene delivery purposes.

    PubMed

    Nasri, Masoud; Karimi, Ali; Allahbakhshian Farsani, Mehdi

    2014-12-01

    Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at -80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 10(8) transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 10(5) TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells.

  5. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  6. Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor

    PubMed Central

    Sarkar, Sanjay; Chelvarajan, Lakshman; Cook, Frank; Artiushin, Sergey; Mondal, Shankar; Anderson, Kelsi; Eberth, John; Timoney, Peter J.; Kalbfleisch, Theodore S.; Bailey, Ernest

    2016-01-01

    ABSTRACT Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from <3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14+ monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus. IMPORTANCE Outbreaks of EVA can be a source of significant economic loss for the equine industry from high rates of abortion in pregnant mares, death in young foals, establishment of the carrier state in stallions, and trade restrictions imposed by various countries. Similar to other arteriviruses, EAV primarily targets cells of the monocyte/macrophage lineage, which, when infected, are believed to play a

  7. Activated STING in a Vascular and Pulmonary Syndrome

    PubMed Central

    Yang, D.; Ramsey, S.E.; Sanchez, G.A. Montealegre; Tenbrock, K.; Wittkowski, H.; Jones, O.Y.; Kuehn, H.S.; Lee, C.-C.R.; DiMattia, M.A.; Cowen, E.W.; Gonzalez, B.; Palmer, I.; DiGiovanna, J.J.; Biancotto, A.; Kim, H.; Tsai, W.L.; Trier, A.M.; Huang, Y.; Stone, D.L.; Hill, S.; Kim, H.J.; Hilaire, C. St.; Gurprasad, S.; Plass, N.; Chapelle, D.; Horkayne-Szakaly, I.; Foell, D.; Barysenka, A.; Candotti, F.; Holland, S.M.; Hughes, J.D.; Mehmet, H.; Issekutz, A.C.; Raffeld, M.; McElwee, J.; Fontana, J.R.; Minniti, C.P.; Moir, S.; Kastner, D.L.; Gadina, M.; Steven, A.C.; Wingfield, P.T.; Brooks, S.R.; Rosenzweig, S.D.; Fleisher, T.A.; Goldbach-Mansky, R.

    2014-01-01

    BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mono-nuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibro-blasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was

  8. E2F1-Mediated FOS Induction in Arsenic Trioxide–Induced Cellular Transformation: Effects of Global H3K9 Hypoacetylation and Promoter-Specific Hyperacetylation in Vitro

    PubMed Central

    Rahman, Sunniyat; Housein, Zjwan; Dabrowska, Aleksandra; Mayán, Maria Dolores; Boobis, Alan R.

    2015-01-01

    Background: Aberrant histone acetylation has been observed in carcinogenesis and cellular transformation associated with arsenic exposure; however, the molecular mechanisms and cellular outcomes of such changes are poorly understood. Objective: We investigated the impact of tolerated and toxic arsenic trioxide (As2O3) exposure in human embryonic kidney (HEK293T) and urothelial (UROtsa) cells to characterize the alterations in histone acetylation and gene expression as well as the implications for cellular transformation. Methods: Tolerated and toxic exposures of As2O3 were identified by measurement of cell death, mitochondrial function, cellular proliferation, and anchorage-independent growth. Histone extraction, the MNase sensitivity assay, and immunoblotting were used to assess global histone acetylation levels, and gene promoter-specific interactions were measured by chromatin immunoprecipitation followed by reverse-transcriptase polymerase chain reaction. Results: Tolerated and toxic dosages, respectively, were defined as 0.5 μM and 2.5 μM As2O3 in HEK293T cells and 1 μM and 5 μM As2O3 in UROtsa cells. Global hypoacetylation of H3K9 at 72 hr was observed in UROtsa cells following tolerated and toxic exposure. In both cell lines, tolerated exposure alone led to H3K9 hyperacetylation and E2F1 binding at the FOS promoter, which remained elevated after 72 hr, contrary to global H3K9 hypoacetylation. Thus, promoter-specific H3K9 acetylation is a better predictor of cellular transformation than are global histone acetylation patterns. Tolerated exposure resulted in an increased expression of the proto-oncogenes FOS and JUN in both cell lines at 72 hr. Conclusion: Global H3K9 hypoacetylation and promoter-specific hyperacetylation facilitate E2F1-mediated FOS induction in As2O3-induced cellular transformation. Citation: Rahman S, Housein Z, Dabrowska A, Mayán MD, Boobis AR, Hajji N. 2015. E2F1-mediated FOS induction in arsenic trioxide–induced cellular

  9. Hepatitis E Virus Inhibits Type I Interferon Induction by ORF1 Products

    PubMed Central

    Nan, Yuchen; Yu, Ying; Ma, Zexu; Khattar, Sunil K.; Fredericksen, Brenda

    2014-01-01

    ABSTRACT Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-β) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. IMPORTANCE Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host

  10. Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

    PubMed

    Powers, Alicia D; Piras, Bryan A; Clark, Robert K; Lockey, Timothy D; Meagher, Michael M

    2016-06-01

    Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products. PMID:27229773

  11. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    SciTech Connect

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  12. [Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity].

    PubMed

    Tian, Wei-Wei; Qiao, Zhen-Hua; Yang, Lin-Hua; Wang, Hong-Wei; Tang, Yan-Hong; Bian, Si-Cheng

    2011-04-01

    This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.

  13. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    NASA Astrophysics Data System (ADS)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  14. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    NASA Astrophysics Data System (ADS)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2015-12-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  15. CMP‑N‑acetylneuraminic acid synthetase interacts with fragile X related protein 1.

    PubMed

    Ma, Yun; Tian, Shuai; Wang, Zongbao; Wang, Changbo; Chen, Xiaowei; Li, Wei; Yang, Yang; He, Shuya

    2016-08-01

    Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P‑interacting proteins and determine the biological effect of the interaction. The current study identified CMP‑N‑acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two‑hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β‑galactosidase assay and growth studies with selective media. Furthermore, co‑immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue‑specific regulator of GM1 levels in SH‑SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS.

  16. A RING finger protein 114 (RNF114) homolog from Chinese sturgeon (Acipenser sinensis) possesses immune-regulation properties via modulating RIG-I signaling pathway-mediated interferon expression.

    PubMed

    Liao, Zhiyong; Chen, Xiaojun; Nie, Dongsong; Wang, Jiajia; Wu, Mingjiang

    2014-12-01

    Ubiquitin ligases play important roles in immune regulation. The human RNF114 (RING finger protein 114), an ubiquitin ligase, was recently reported to be involved in immune response to double-stranded RNA in disease pathogenesis. Here, we identified a RNF114 homolog in Chinese sturgeon (Acipenser sinensis) and investigated its potential role in immune response. The full-length cDNA of Chinese sturgeon RNF114 (csRNF114) contains an open reading frame (ORF) of 681 nucleotides coding a protein of 227 amino acids. csRNF114 shares the highest identity of 76% at amino acid level to other RNF114 homologs, clustering with bony fish RNF114s based on phylogenetic analysis. The main structural features of csRNF114, including a C3HC4 (Cys3-His-Cys4) RING domain, a C2HC (Cys2-His-Cys)-type zinc finger motif, a C2H2 (Cys2-His2)-type zinc finger motif, and a UIM (ubiquitin-interacting motif), take csRNF114 as an ubiquitin ligase. csRNF114 mRNA was widely expressed in various tissues and significantly up-regulated in poly(I:C)-treated Chinese sturgeon. Over-expression of csRNF114 in HEK293T cells significantly promoted both basal and poly(I:C)-induced activation of interferon regulatory transcription factor 3 (IRF3) and nuclear factor-κB (NF-κB) downstream retinoic acid inducible gene I (RIG-I) signaling pathway and expression of target genes type I interferon (IFN), which was nearly abolished by knockdown of RIG-I with specific human siRNA and by mutation of the C3HC4 RING domain (C28A/C31A) in csRNF114 as well. Furthermore, csRNF114 associated with ubiquitinated proteins in HEK293T cells, for which the C3HC4 RING domain was essential. These data suggested that an ubiquitin ligase RNF114 homolog with a potential role in antiviral response possibly through modulating RIG-I signaling pathway was cloned from Chinese sturgeon, which might contribute to our understanding of the immune biology of Chinese sturgeon.

  17. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  18. Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications.

    PubMed

    Farley, Daniel C; Iqball, Sharifah; Smith, Joanne C; Miskin, James E; Kingsman, Susan M; Mitrophanous, Kyriacos A

    2007-05-01

    Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

  19. Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens

    PubMed Central

    Dörlich, René M.; Chen, Qing; Niklas Hedde, Per; Schuster, Vittoria; Hippler, Marc; Wesslowski, Janine; Davidson, Gary; Nienhaus, G. Ulrich

    2015-01-01

    Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues. PMID:25951521

  20. Identification and characterization of IKKε gene from grass carp Ctenopharyngodon idella.

    PubMed

    Li, Jun; Zhou, Man; Peng, Lingzhi; Sun, Wenzheng; Yang, Peilin; Yan, Jun; Feng, Hao

    2015-11-01

    IKKε is an IKK-related kinase implicated in antiviral immune response in higher vertebrates. To elucidate the function of IKKε in teleost fish, grass carp IKKε (gcIKKε) has been cloned and characterized in this paper. The full-length cDNA of gcIKKε is composed of 2529 nucleotides and encodes a polypeptide of 723 amino acids. The mRNA transcription of gcIKKε was constitutively detected in all the selected tissues and the gcIKKε mRNA level increased at 36 h after GCRV infection. Western blot data of both HEK293T cells and EPC cells demonstrated that gcIKKε was around 80 KDa; and immunofluorescence staining data of both NIH3T3 cells and EPC cells determined gcIKKε was a cytosolic protein. The mRNA level of gcIKKε in CIK cells was increased more than 150 times right after poly(I:C) treatment and PMA treatment triggered gcIKKε mRNA transcription in CIK cells more than 100 times. Over-expression of gcIKKε in EPC cells activated the promoter activity of both zebrafish IFN and fathead minnow IFN. gcIKKε mRNA transcription level in CIK cells was increased from 48 h post GCRV infection with different MOIs. All the data support the idea that gcIKKε is a novel teleost IκB kinase recruited in the IFN-mediated antiviral immunity of grass carp. PMID:26370541

  1. Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.

    PubMed

    Bompiani, Kristin M; Tsai, Cheng-Yu; Achatz, Felix P; Liebig, Janika K; Howell, Stephen B

    2016-09-01

    The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

  2. Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I.

    PubMed

    Stroud, David A; Formosa, Luke E; Wijeyeratne, Xiaonan W; Nguyen, Thanh N; Ryan, Michael T

    2013-01-18

    Transcription activator-like effector nucleases (TALENs) represent a promising approach for targeted knock-out of genes in cultured human cells. We used TALEN-technology to knock out the nuclear gene encoding NDUFA9, a subunit of mitochondrial respiratory chain complex I in HEK293T cells. Screening for the knock-out revealed a mixture of NDUFA9 cell clones that harbored partial deletions of the mitochondrial N-terminal targeting signal but were still capable of import. A cell line lacking functional copies of both NDUFA9 alleles resulted in a loss of NDUFA9 protein expression, impaired assembly of complex I, and cells incapable of growth in galactose medium. Cells lacking NDUFA9 contained a complex I subcomplex consisting of membrane arm subunits but not marker subunits of the matrix arm. Re-expression of NDUFA9 restored the defects in complex I assembly. We conclude that NDUFA9 is involved in stabilizing the junction between membrane and matrix arms of complex I, a late assembly step critical for complex I biogenesis and activity.

  3. Identification of BST-2/tetherin-induced hepatitis B virus restriction and hepatocyte-specific BST-2 inactivation

    PubMed Central

    Lv, Mingyu; Zhang, Biao; Shi, Ying; Han, Zhu; Zhang, Yan; Zhou, Yulai; Zhang, Wenyan; Niu, Junqi; Yu, Xiao-Fang

    2015-01-01

    BST-2/tetherin is an interferon-inducible antiviral protein that blocks the release of various enveloped viruses, including HIV-1. Hepatitis B virus (HBV), a major cause of liver disease, belongs to the Hepadnaviridae family of enveloped DNA viruses. Whether BST-2 regulates HBV production is largely unknown. In this report, we have demonstrated that HBV particle release is modulated by BST-2 in a cell type-dependent fashion. In HEK293T cells, ectopically expressed or interferon-induced BST-2 strongly inhibited HBV release. BST-2 co-localized with HBV surface protein at multivesicular bodies (MVBs) and physically interacted with HBV particles. However, exogenous BST-2-induced HBV restriction was weak in Huh-7 hepatoma cells, and the interferon-induced anti-HBV effect was independent of BST-2 induction in hepatic L02 cells. Notably, HBV could promote HIV-1 ΔVpu virus release from BST-2-positive HepG2 hepatoma cells but not HeLa cells, whereas Vpu failed to efficiently inhibit BST-2-induced HBV restriction. HBx exhibited an enhanced interaction and co-localization with BST-2 in hepatocytes. These observations indicate that BST-2 restricts HBV production at intracellular MVBs but is inactivated by HBV through a novel mechanism requiring hepatocyte-specific cellular co-factors or a hepatocyte-specific environment. Further understanding of BST-2-induced HBV restriction may provide new therapeutic targets for future HBV treatments. PMID:26119070

  4. Study of Cytotoxic Effects of Benzonitrile Pesticides

    PubMed Central

    Lovecka, Petra; Thimova, Marketa; Grznarova, Petra; Lipov, Jan; Knejzlik, Zdenek; Stiborova, Hana; Nindhia, Tjokorda Gde Tirta; Demnerova, Katerina; Ruml, Tomas

    2015-01-01

    The benzonitrile herbicides bromoxynil, chloroxynil, dichlobenil, and ioxynil have been used actively worldwide to control weeds in agriculture since 1970s. Even though dichlobenil is prohibited in EU since 2008, studies addressing the fate of benzonitrile herbicides in the environment show that some metabolites of these herbicides are very persistent. We tested the cytotoxic effects of benzonitrile herbicides and their microbial metabolites using two human cell lines, Hep G2 and HEK293T, representing liver and kidneys as potential target organs in humans. The cell viability and proliferation were determined by MTT test and RTCA DP Analyzer system, respectively. The latter allows real-time monitoring of the effect of added substances. As the cytotoxic compounds could compromise cell membrane integrity, the lactate dehydrogenase test was performed as well. We observed high toxic effects of bromoxynil, chloroxynil, and ioxynil on both tested cell lines. In contrast, we determined only low inhibition of cell growth in presence of dichlobenil and microbial metabolites originating from the tested herbicides. PMID:26339609

  5. Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

    SciTech Connect

    Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

    2008-07-18

    Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.

  6. Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B).

    PubMed

    Duangtum, Natapol; Junking, Mutita; Sawasdee, Nunghathai; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2011-09-16

    Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl(-)/HCO(3)(-) exchange and the failure of proton (H(+)) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells. PMID:21871436

  7. IFNa of black carp is an antiviral cytokine modified with N-linked glycosylation.

    PubMed

    Huang, Zhilin; Chen, Song; Liu, Jiachen; Xiao, Jun; Yan, Jun; Feng, Hao

    2015-10-01

    Type I interferons (IFNs) play an important role in the antiviral immune response in teleost fish. In this study, one type I interferon (bcIFNa) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNa gene consists of 783 nucleotides and the predicted bcIFNa protein contains 185 amino acids. Semi-quantitative RT-PCR analysis demonstrated that bcIFNa mRNA transcription level in all the selected tissues of black carp was greatly increased at 33 h post spring viremia of carp virus (SVCV) infection. The protein of bcIFNa could be detected in both the whole cell lysate and the supernatant media of HEK293T cells transfected with plasmids expressing bcIFNa through immunoblot assay. EPC cells showed greatly increased antiviral ability when the cells were treated with the bcIFNa-containing conditioned media for 24 h before SVCV infection. Mass spectrum assay and glycosidase digestion analysis determined that bcIFNa is modified with N-linked glycosylation, which occurs on the Asn (N) of 38 site of this cytokine. The un-glycosylated mutant bcIFNa-N38Q could be secreted out of the cell and showed the similar antiviral ability against SVCV as that of wild type bcIFNa, which suggested that N-linked glycosylation does not contribute directly to the antiviral property of this fish cytokine.

  8. Potential anticancer heterometallic Fe-Au and Fe-Pd agents: initial mechanistic insights.

    PubMed

    Lease, Nicholas; Vasilevski, Vadim; Carreira, Monica; de Almeida, Andreia; Sanaú, Mercedes; Hirva, Pipsa; Casini, Angela; Contel, María

    2013-07-25

    A series of gold(III) and palladium(II) heterometallic complexes with new iminophosphorane ligands derived from ferrocenylphosphanes [{Cp-P(Ph2)═N-Ph}2Fe] (1), [{Cp-P(Ph2)═N-CH2-2-NC5H4}2Fe] (2), and [{Cp-P(Ph2)═N-CH2-2-NC5H4}Fe(Cp)] (3) have been synthesized and structurally characterized. Ligands 2 and 3 afford stable coordination complexes [AuCl2(3)]ClO4, [{AuCl2}2(2)](ClO4)2, [PdCl2(3)], and [{PdCl2}2(2)]. The complexes have been evaluated for their antiproliferative properties in human ovarian cancer cells sensitive and resistant to cisplatin (A2780S/R), in human breast cancer cells (MCF7) and in a nontumorigenic human embryonic kidney cell line (HEK-293T). The highly cytotoxic trimetallic derivatives M2Fe (M = Au, Pd) are more cytotoxic to cancer cells than their corresponding monometallic fragments. Moreover, these complexes were significantly more cytotoxic than cisplatin in the resistant A2780R and the MCF7 cell lines. Studies of the interactions of the trimetallic compounds with DNA and the zinc-finger protein PARP-1 indicate that they exert anticancer effects in vitro based on different mechanisms of actions with respect to cisplatin. PMID:23786413

  9. Caveolin-1-mediated suppression of cyclooxygenase-2 via a beta-catenin-Tcf/Lef-dependent transcriptional mechanism reduced prostaglandin E2 production and survivin expression.

    PubMed

    Rodriguez, Diego A; Tapia, Julio C; Fernandez, Jaime G; Torres, Vicente A; Muñoz, Nicolas; Galleguillos, Daniela; Leyton, Lisette; Quest, Andrew F G

    2009-04-01

    Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E(2) (PGE(2)) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and beta-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE(2) and cell proliferation. Moreover, COX-2 overexpression or PGE(2) supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE(2) to the medium prevented effects attributed to caveolin-1-mediated inhibition of beta-catenin-Tcf/Lef-dependent transcription. Finally, PGE(2) reduced the coimmunoprecipitation of caveolin-1 with beta-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE(2)-induced signaling events linked to beta-catenin/Tcf/Lef-dependent transcription of tumor survival genes including cox-2 itself and survivin. PMID:19244345

  10. Cellular response to empty and palladium-conjugated amino-polystyrene nanospheres uptake: a proteomic study.

    PubMed

    Pietrovito, Laura; Cano-Cortés, Victoria; Gamberi, Tania; Magherini, Francesca; Bianchi, Laura; Bini, Luca; Sánchez-Martín, Rosario M; Fasano, Mauro; Modesti, Alessandra

    2015-01-01

    Amino polystyrene nanospheres are shown to be efficient and controllable delivery devices, capable of transporting several bioactive cargoes. Recently, the design of a new device for prodrug activation, using these nanospheres with palladium encapsulated onto them, has been developed successfully. To study the influence of the cellular uptake of these nanodevices, we investigated the cellular response of human embryonic kidney cells (HEK-293T) and murine fibroblasts (L929) treated with empty or palladium-conjugated amino polystyrene nanospheres. To identify differentially expressed proteins, we performed an exhaustive proteomic analysis. In accordance with genomic data previously obtained, the uptake of the empty nanospheres did not induce significant variation in protein expression levels. Following the treatment with palladium-conjugated nanospheres, some changes in protein profiles in both cell lines were observed; these alterations affect proteins involved in cell metabolism and intracellular transport. No key regulator of the cell cycle result was differentially expressed after the treatment, confirming that these innovative drug delivery systems are harmless and well tolerated by the cells.

  11. Preparation of hierarchical mesoporous CaCO3 by a facile binary solvent approach as anticancer drug carrier for etoposide

    PubMed Central

    2013-01-01

    To develop a nontoxic system for targeting therapy, a new highly ordered hierarchical mesoporous calcium carbonate nanospheres (CCNSs) as small drug carriers has been synthesized by a mild and facile binary solvent approach under the normal temperature and pressure. The hierarchical structure by multistage self-assembled strategy was confirmed by TEM and SEM, and a possible formation process was proposed. Due to the large fraction of voids inside the nanospheres which provides space for physical absorption, the CCNSs can stably encapsulate the anticancer drug etoposide with the drug loading efficiency as high as 39.7 wt.%, and etoposide-loaded CCNS (ECCNS) nanoparticles can dispersed well in the cell culture. Besides, the drug release behavior investigated at three different pH values showed that the release of etoposide from CCNSs was pH-sensitive. MTT assay showed that compared with free etoposide, ECCNSs exhibited a higher cell inhibition ratio against SGC-7901 cells and also decreased the toxicity of etoposide to HEK 293 T cells. The CLSM image showed that ECCNSs exhibited a high efficiency of intracellular delivery, especially in nuclear invasion. The apoptosis test revealed that etoposide entrapped in CCNSs could enhance the delivery efficiencies of drug to achieve an improved inhibition effect on cell growth. These results clearly implied that the CCNSs are a promising drug delivery system for etoposide in cancer therapy. PMID:23849350

  12. Study of Cytotoxic Effects of Benzonitrile Pesticides.

    PubMed

    Lovecka, Petra; Thimova, Marketa; Grznarova, Petra; Lipov, Jan; Knejzlik, Zdenek; Stiborova, Hana; Nindhia, Tjokorda Gde Tirta; Demnerova, Katerina; Ruml, Tomas

    2015-01-01

    The benzonitrile herbicides bromoxynil, chloroxynil, dichlobenil, and ioxynil have been used actively worldwide to control weeds in agriculture since 1970s. Even though dichlobenil is prohibited in EU since 2008, studies addressing the fate of benzonitrile herbicides in the environment show that some metabolites of these herbicides are very persistent. We tested the cytotoxic effects of benzonitrile herbicides and their microbial metabolites using two human cell lines, Hep G2 and HEK293T, representing liver and kidneys as potential target organs in humans. The cell viability and proliferation were determined by MTT test and RTCA DP Analyzer system, respectively. The latter allows real-time monitoring of the effect of added substances. As the cytotoxic compounds could compromise cell membrane integrity, the lactate dehydrogenase test was performed as well. We observed high toxic effects of bromoxynil, chloroxynil, and ioxynil on both tested cell lines. In contrast, we determined only low inhibition of cell growth in presence of dichlobenil and microbial metabolites originating from the tested herbicides. PMID:26339609

  13. Development of a doxycycline-inducible lentiviral plasmid with an instant regulatory feature.

    PubMed

    Yang, Tian; Burrows, Christopher; Park, Jeong Hyeon

    2014-03-01

    Lentiviruses provide highly efficient gene delivery vehicles in both dividing and non-dividing cells. Inducible gene expression systems often employ a specific cell line that constitutively expresses a regulatory protein for transgene expression. As one of such inducible expression systems the Tet-On system uses a cell line expressing reverse tetracycline-responsive transcriptional activator (rtTA). The rtTA protein binds to the tetracycline-responsive element (TRE) in the promoter and activates transcription of a transgene in a doxycycline-dependent manner. To establish a universal and instant regulatory system without generating Tet-On cell lines, the cDNAs of rtTA and a testing target gene (PPM1B) were cloned in the bi-directional TRE-containing promoters. Here, we examined whether a basal leaky expression of rtTA allows instantly inducible expression of both rtTA itself and the target gene, PPM1B in a single plasmid using the two mini-CMV promoters. Transient transfection of the lentiviral plasmids into human embryonic kidney HEK293T cells showed a significant induction of PPM1B expression in response to doxycycline, suggesting that these lentiviral plasmids can be used as an instantly inducible mammalian expression vector. However, the expression of rtTA by lentiviral transduction shows a minimal expression without a consistent response to doxycycline, suggesting that the utility of these lentiviral vectors is limited. A potential solution to overcome lentiviral transgene inactivation is proposed. PMID:24727543

  14. Harnessing functional plasticity of enzymes: a fluorogenic probe for imaging 17beta-HSD10 dehydrogenase, an enzyme involved in Alzheimer's and Parkinson's diseases.

    PubMed

    Froemming, Mary K; Sames, Dalibor

    2007-11-21

    In this paper, we describe the development of a fluorogenic substrate for 17beta-hydroxysteroid-dehydrogenase type 10 (17beta-HSD10), which is a multifunctional metabolic enzyme fulfilling several metabolic roles (beta-oxidation of fatty acids, catabolism of isoleucine, and metabolism of steroids). In recent years, it has emerged as an important stress and pathological marker in neurons and glial cells (expression down-regulation in Parkinson's disease, up-regulation and association with beta-amyloid peptide in Alzheimer's disease). Through the iterative molecular design and chemical synthesis described herein, compound 1 was developed, which possesses all required properties for a selective optical reporter substrate: alcohol-ketone optical switching, the ability to function as a good enzyme substrate (expressed in kinetic parameters), cell permeability, and cell retention. Probe 1 provides a blue-to-green/yellow bright switch and enables non-invasive, real-time imaging of 17beta-HSD10 in live human cells. The selectivity of reporter 1 was established by the quantitative correlation of metabolic activity to protein expression in human kidney cell line HEK-293T. PMID:17958419

  15. Cathepsin D inhibitors as potential therapeutics for breast cancer treatment: Molecular docking and bioevaluation against triple-negative and triple-positive breast cancers.

    PubMed

    Anantaraju, Hasitha Shilpa; Battu, Madhu Babu; Viswanadha, Srikant; Sriram, Dharmarajan; Yogeeswari, Perumal

    2016-05-01

    The main aim of this study was to discover small molecule inhibitors against Cathepsin D (CatD) (EC.3.4.23.5), a clinically proven prognostic marker for breast cancer, and to explore the mechanisms by which CatD could be a useful therapeutic target for triple-positive and triple-negative breast cancers (TPBC & TNBC). The crystal structure of CatD at 2.5 Å resolution (PDB: 1LYB), which was complexed with Pepstatin A, was selected for computer-aided molecular modeling. The methods used in our study were pharmacophore modeling and molecular docking. Virtual screening was performed to identify small molecules from an in-house database and a large commercial chemical library. Cytotoxicity studies were performed on human normal cell line HEK293T and growth inhibition studies on breast adenocarcinoma cell lines, namely MCF-7, MDA-MB-231, SK-BR-3, and MDA-MB-468. Furthermore, RT-PCR analysis, in vitro enzyme assay, and cell cycle analysis ascertained the validity of the selected molecules. A set of 28 molecules was subjected to an in vitro fluorescence-based inhibitory activity assay, and among them six molecules exhibited >50 % inhibition at 25μM. These molecules also exhibited good growth inhibition against TPBC and TNBC cancer types. Among them, molecules 1 and 17 showed single-digit micromolar GI50 values against MCF-7 and MDA-MB-231 cell lines.

  16. Glucose modulates respiratory complex I activity in response to acute mitochondrial dysfunction.

    PubMed

    Cannino, Giuseppe; El-Khoury, Riyad; Pirinen, Marja; Hutz, Bettina; Rustin, Pierre; Jacobs, Howard T; Dufour, Eric

    2012-11-01

    Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an "emergency shutdown" system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.

  17. Auranofin and N-heterocyclic carbene gold-analogs are potent inhibitors of the bacteria Helicobacter pylori.

    PubMed

    Owings, Joshua P; McNair, Nina N; Mui, Yiu Fung; Gustafsson, Tomas N; Holmgren, Arne; Contel, Maria; Goldberg, Joanna B; Mead, Jan R

    2016-07-01

    Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicate that auranofin targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as five auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and five gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro Auranofin completely inhibited bacterial growth at 1.2 μM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ∼88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 μM), while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13-20-fold higher compared to auranofin indicating that they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo Finally, auranofin was synergistic with the antibiotic amoxicillin, suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections. PMID:27279627

  18. Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation

    PubMed Central

    Kapoor, Isha; Kanaujiya, Jitendra; Kumar, Yogesh; Thota, Jagadeshwar Reddy; Bhatt, Madan L.B.; Chattopadhyay, Naibedya; Sanyal, Sabyasachi; Trivedi, Arun Kumar

    2016-01-01

    Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents. PMID:26506232

  19. Photoexcited quantum dots for killing multidrug-resistant bacteria

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen M.; Goodman, Samuel M.; McDaniel, Jessica A.; Madinger, Nancy E.; Chatterjee, Anushree; Nagpal, Prashant

    2016-05-01

    Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

  20. Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

    PubMed Central

    Ronstedt, Katharina; Sternberg, Damien; Detro-Dassen, Silvia; Gramkow, Thomas; Begemann, Birgit; Becher, Toni; Kilian, Petra; Grieschat, Matthias; Machtens, Jan-Philipp; Schmalzing, Günther; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    Mutations in the muscle chloride channel gene (CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function. PMID:26502825

  1. An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Fontaine, Danielle M; Kohrt, Dawn; Talukder, Munya; Michelini, Elisa; Cevenini, Luca; Roda, Aldo; Grossel, Martha J

    2015-09-01

    Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. We previously reported the approximately 2-fold enhanced activity and 1.4-fold greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomole levels of ATP. In addition, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and living cells with 4.4-fold and approximately 3.0-fold greater sensitivity, respectively. PLG2 could be an improved alternative to Promega's luc2 for reporter and imaging applications.

  2. Signaling of chloroquine-induced stress in the yeast Saccharomyces cerevisiae requires the Hog1 and Slt2 mitogen-activated protein kinase pathways.

    PubMed

    Baranwal, Shivani; Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S

    2014-09-01

    Chloroquine (CQ) has been under clinical use for several decades, and yet little is known about CQ sensing and signaling mechanisms or about their impact on various biological pathways. We employed the budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by CQ. Our screening with yeast mutants revealed that it targets histone proteins and histone deacetylases (HDACs). Here, we also describe the novel role of mitogen-activated protein kinases Hog1 and Slt2, which aid in survival in the presence of CQ. Cells deficient in Hog1 or Slt2 are found to be CQ hypersensitive, and both proteins were phosphorylated in response to CQ exposure. CQ-activated Hog1p is translocated to the nucleus and facilitates the expression of GPD1 (glycerol-3-phosphate dehydrogenase), which is required for the synthesis of glycerol (one of the major osmolytes). Moreover, cells treated with CQ exhibited an increase in intracellular reactive oxygen species (ROS) levels and the effects were rescued by addition of reduced glutathione to the medium. The deletion of SOD1, the superoxide dismutase in yeast, resulted in hypersensitivity to CQ. We have also observed P38 as well as P42/44 phosphorylation in HEK293T human cells upon exposure to CQ, indicating that the kinds of responses generated in yeast and human cells are similar. In summary, our findings define the multiple biological pathways targeted by CQ that might be useful for understanding the toxicity modulated by this pharmacologically important molecule.

  3. Assessment of flavaglines as potential chikungunya virus entry inhibitors.

    PubMed

    Wintachai, Phitchayapak; Thuaud, Frédéric; Basmadjian, Christine; Roytrakul, Sittiruk; Ubol, Sukathida; Désaubry, Laurent; Smith, Duncan R

    2015-03-01

    Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that recently caused large epidemics in islands in, and countries around, the Indian Ocean. There is currently no specific drug for therapeutic treatment or for use as a prophylactic agent against infection and no commercially available vaccine. Prohibitin has been identified as a receptor protein used by chikungunya virus to enter mammalian cells. Recently, synthetic sulfonyl amidines and flavaglines (FLs), a class of naturally occurring plant compounds with potent anti-cancer and cytoprotective and neuroprotective activities, have been shown to interact directly with prohibitin. This study therefore sought to determine whether three prohibitin ligands (sulfonyl amidine 1 m and the flavaglines FL3 and FL23) were able to inhibit CHIKV infection of mammalian Hek293T/17 cells. All three compounds inhibited infection and reduced virus production when cells were treated before infection but not when added after infection. Pretreatment of cells for only 15 minutes prior to infection followed by washing out of the compound resulted in significant inhibition of entry and virus production. These results suggest that further investigation of prohibitin ligands as potential Chikungunya virus entry inhibitors is warranted.

  4. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole.

  5. Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/calmodulin.

    PubMed

    Jung, Jinsei; Nam, Joo Hyun; Park, Hyun Woo; Oh, Uhtaek; Yoon, Joo-Heon; Lee, Min Goo

    2013-01-01

    Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO(3)(-) secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca(2+)/calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO(3)(-) at high [Ca(2+)](i). Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca(2+)](i) is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca(2+)](i)-dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P(HCO3)/P(Cl). In addition, the high [Ca(2+)](i)-induced increase in HCO(3)(-) permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO(3)(-) permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO(3)(-) transport, especially in transepithelial HCO(3)(-) secretion.

  6. Early growth response 1 (EGR-1) is a transcriptional regulator of mitochondrial carrier homolog 1 (MTCH 1)/presenilin 1-associated protein (PSAP).

    PubMed

    Nelo-Bazán, María Alejandra; Latorre, Pedro; Bolado-Carrancio, Alfonso; Pérez-Campo, Flor M; Echenique-Robba, Pablo; Rodríguez-Rey, José Carlos; Carrodeguas, José Alberto

    2016-03-01

    Attempts to elucidate the cellular function of MTCH1 (mitochondrial carrier homolog 1) have not yet rendered a clear insight into the function of this outer mitochondrial membrane protein. Classical biochemical and cell biology approaches have not produced the expected outcome. In vitro experiments have indicated a likely role in the regulation of cell death by apoptosis, and its reported interaction with presenilin 1 suggests a role in the cellular pathways in which this membrane protease participates, nevertheless in vivo data are missing. In an attempt to identify cellular pathways in which this protein might participate, we have studied its promoter looking for transcriptional regulators. We have identified several putative binding sites for EGR-1 (Early growth response 1; a protein involved in growth, proliferation and differentiation), in the proximal region of the MTCH1 promoter. Chromatin immunoprecipitation showed an enrichment of these sequences in genomic DNA bound to EGR-1 and transient overexpression of EGR-1 in cultured HEK293T cells induces an increase of endogenous MTCH1 levels. We also show that MTCH1 levels increase in response to treatment of cells with doxorubicin, an apoptosis inducer through DNA damage. The endogenous levels of MTCH1 decrease when EGR-1 levels are lowered by RNA interference. Our results indicate that EGR-1 is a transcriptional regulator of MTCH1 and give some clues about the cellular processes in which MTCH1 might participate. PMID:26692143

  7. An organelle K+ channel is required for osmoregulation in Chlamydomonas reinhardtii.

    PubMed

    Xu, Feifei; Wu, Xiaoan; Jiang, Lin-Hua; Zhao, Hucheng; Pan, Junmin

    2016-08-01

    Fresh water protozoa and algae face hypotonic challenges in their living environment. Many of them employ a contractile vacuole system to uptake excessive water from the cytoplasm and expel it to the environment to achieve cellular homeostasis. K(+), a major osmolyte in contractile vacuole, is predicted to create higher osmolarity for water influx. Molecular mechanisms for K(+) permeation through the plasma membrane have been well studied. However, how K(+) permeates organelles such as the contractile vacuole is not clear. Here, we show that the six-transmembrane K(+) channel KCN11 in Chlamydomonas is exclusively localized to contractile vacuole. Ectopic expression of KCN11 in HEK293T cells results in voltage-gated K(+) channel activity. Disruption of the gene or mutation of key residues for K(+) permeability of the channel leads to dysfunction of cell osmoregulation in very hypotonic conditions. The contractile cycle is inhibited in the mutant cells with a slower rate of contractile vacuole swelling, leading to cell death. These data demonstrate a new role for six-transmembrane K(+) channels in contractile vacuole functioning and provide further insights into osmoregulation mediated by the contractile vacuole. PMID:27311484

  8. Cotton Rat (Sigmodon hispidus) Signaling Lymphocyte Activation Molecule (CD150) Is an Entry Receptor for Measles Virus

    PubMed Central

    Carsillo, Thomas; Huey, Devra; Levinsky, Amy; Obojes, Karola; Schneider-Schaulies, Jürgen; Niewiesk, Stefan

    2014-01-01

    Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection. PMID:25295727

  9. Auranofin and N-heterocyclic carbene gold-analogs are potent inhibitors of the bacteria Helicobacter pylori.

    PubMed

    Owings, Joshua P; McNair, Nina N; Mui, Yiu Fung; Gustafsson, Tomas N; Holmgren, Arne; Contel, Maria; Goldberg, Joanna B; Mead, Jan R

    2016-07-01

    Auranofin is an FDA-approved gold-containing compound used for the treatment of rheumatoid arthritis. Recent reports of antimicrobial activity against protozoa and bacteria indicate that auranofin targets the reductive enzyme thioredoxin reductase (TrxR). We evaluated auranofin as well as five auranofin analogs containing N-heterocyclic carbenes (instead of the triethylphosphane present in auranofin) and five gold-carbene controls for their ability to inhibit or kill Helicobacter pylori in vitro Auranofin completely inhibited bacterial growth at 1.2 μM. Purified H. pylori TrxR was inhibited by auranofin in a cell-free assay (IC50 ∼88 nM). The most active gold(I)-N-heterocyclic carbene compounds exhibited MICs comparable to auranofin against H. pylori (2 μM), while also exhibiting lower toxicities for human embryonic kidney cells (HEK-293T cells). Median toxic concentrations (TC50) were 13-20-fold higher compared to auranofin indicating that they were less cytotoxic. The N-heterocyclic carbene analogs maybe well tolerated, but further evaluation is needed in vivo Finally, auranofin was synergistic with the antibiotic amoxicillin, suggesting that targeting both the reductive enzyme TrxR and cell wall synthesis may be effective against H. pylori infections.

  10. Glucose modulates respiratory complex I activity in response to acute mitochondrial dysfunction.

    PubMed

    Cannino, Giuseppe; El-Khoury, Riyad; Pirinen, Marja; Hutz, Bettina; Rustin, Pierre; Jacobs, Howard T; Dufour, Eric

    2012-11-01

    Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an "emergency shutdown" system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases. PMID:23007390

  11. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-01-01

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

  12. C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43.

    PubMed

    Jensen, Sacha A; Aspinall, Georgia; Handford, Penny A

    2014-07-15

    Fibrillin microfibrils are 10-12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature microfibril assembly. PMID:24982166

  13. A study of substrate specificity for a CTD phosphatase, SCP1, by proteomic screening of binding partners.

    PubMed

    Kim, Young Jun; Bahk, Young Yil

    2014-05-30

    RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are a newly emerging family of phosphatases. Recently a CTD-specific phosphatase, small CTD phosphatase 1 (SCP1), has shown to act as an evolutionarily conserved transcriptional corepressor for inhibiting neuronal gene transcription in non-neuronal cells. In this study, using the established NIH/3T3 and HEK293T cells, which are expressing human SCP1 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for SCP1. Although the present findings provide the possibility for new avenues to provide to a better understanding of cellular physiology of SCP1, now these proteomic and some immunological approaches for SCP1 interactome might not represent the accurate physiological relevance in vivo. In this presentation, we focus the substrate specificity to delineate an appearance of the dephosphorylation reaction catalyzed by SCP1 phosphatase. We compared the phosphorylated sequences of the immunologically confirmed binding partners with SCP1 searched in HPRD. We found the similar sequences from CdcA3 and validated the efficiency of enzymatic catalysis for synthetic phosphopeptides the recombinant SCP1. This approach led to the identification of several interacting partners with SCP1. We suggest that CdcA3 could be an enzymatic substrate for SCP1 and that SCP1 might have the relationship with cell cycle regulation through enzymatic activity against CdcA3. PMID:24769477

  14. Calcium homoeostasis modulator 1 (CALHM1) reduces the calcium content of the endoplasmic reticulum (ER) and triggers ER stress.

    PubMed

    Gallego-Sandín, Sonia; Alonso, María Teresa; García-Sancho, Javier

    2011-08-01

    CALHM1 (calcium homoeostasis modulator 1), a membrane protein with similarity to NMDA (N-methyl-D-aspartate) receptor channels that localizes in the plasma membrane and the ER (endoplasmic reticulum) of neurons, has been shown to generate a plasma-membrane Ca(2+) conductance and has been proposed to influence Alzheimer's disease risk. In the present study we have investigated the effects of CALHM1 on intracellular Ca(2+) handling in HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] cells by using targeted aequorins for selective monitorization of Ca(2+) transport by organelles. We find that CALHM1 increases Ca(2+) leak from the ER and, more importantly, reduces ER Ca(2+) uptake by decreasing both the transport capacity and the Ca(2+) affinity of SERCA (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase). As a result, the Ca(2+) content of the ER is drastically decreased. This reduction in the Ca(2+) content of the ER triggered the UPR (unfolded protein response) with induction of several ER stress markers, such as CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein], ERdj4, GRP78 (glucose-regulated protein of 78 kDa) and XBP1 (X-box-binding protein 1). Thus CALHM1 might provide a relevant link between Ca(2+) homoeostasis disruption, ER stress and cell damage in the pathogenesis of neurodegenerative diseases.

  15. Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing.

    PubMed

    Benakanakere, Manjunatha R; Finoti, Livia S; Tanaka, Urara; Grant, Gregory R; Scarel-Caminaga, Raquel M; Kinane, Denis F

    2016-01-01

    Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility. PMID:27499075

  16. The effects of cytosine methylation on general transcription factors

    NASA Astrophysics Data System (ADS)

    Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

    2016-07-01

    DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

  17. Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

    PubMed Central

    Kim, Jun Seok; Lee, Cheolju

    2015-01-01

    Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

  18. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling

    PubMed Central

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-01-01

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

  19. Transmembrane protein 139 (TMEM139) interacts with human kidney isoform of anion exchanger 1 (kAE1).

    PubMed

    Nuiplot, Nalin-On; Junking, Mutita; Duangtum, Natapol; Khunchai, Sasiprapa; Sawasdee, Nunghathai; Yenchitsomanus, Pa-Thai; Akkarapatumwong, Varaporn

    2015-08-01

    Human kidney anion exchanger 1 (kAE1) mediates Cl(-)/HCO3(-) exchanges at the basolateral membrane of the acid-secreting α-intercalated cells. Mutations in SLC4A1 gene encoding kAE1 are associated with distal renal tubular acidosis (dRTA). Several studies have shown that impaired trafficking of the mutant kAE1 is an important molecular mechanism underlying the pathogenesis of dRTA. Proteins involved in kAE1 trafficking were identified but the mechanism resulting in dRTA remained unclear. Thus, this study attempted to search for additional proteins interacting with C-terminal of kAE1 (Ct-kAE1) and involved in kAE1 trafficking to cell membrane. Transmembrane protein 139 (TMEM139) was identified as a protein interacting with Ct-kAE1 by yeast two-hybrid screening. The interaction between kAE1 and TMEM139 was confirmed by affinity co-purification, co-immunoprecipitation (co-IP) and yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA). In addition, flow cytometry results showed that suppression of endogenous TMEM139 by small interfering RNA (siRNA) and over-expression of TMEM139 in HEK293T cells could reduce and increase membrane localization of kAE1, respectively. The presented data demonstrate that TMEM139 interacts with kAE1 and promotes its intracellular trafficking. PMID:26049106

  20. The effects of cytosine methylation on general transcription factors

    PubMed Central

    Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

    2016-01-01

    DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs. PMID:27385050

  1. 2-(3'-Indolyl)-N-arylthiazole-4-carboxamides: Synthesis and evaluation of antibacterial and anticancer activities.

    PubMed

    Tantak, Mukund P; Wang, Jing; Singh, Rajnish Prakash; Kumar, Anil; Shah, Kavita; Kumar, Dalip

    2015-10-01

    A new series of 2-(3'-indolyl)-N-arylthiazole-4-carboxamides 17a-p has been designed and synthesized. Initial reaction of readily available thioamides 15 with bromopyruvic acid under refluxing conditions produced different thiazole carboxylic acids 16 which upon coupling with arylamines by using EDCI·HCl and HOBt afforded diverse arylthiazole-4-carboxamides 17a-p in 78-87% yields. Antibacterial activity evaluation against Gram-positive and Gram-negative bacterial strains led to compounds 17i-k and 17o as potent and selectively (Gram-negative) antibacterial agents. The cytotoxicity of thiazole carboxamides 17a-p was also evaluated on a panel of human cancer cell lines. Among the tested derivatives, compounds 17i (IC50=8.64μM; HEK293T) and 17l (IC50=3.41μM; HeLa) were identified as the most potent analogues of the series. Preliminary mechanism of action studies of thiazole carboxamide 17i suggested that its cytotoxicity against HeLa cells involves the induction of cell death by apoptosis.

  2. Fidgetin-like 1 is a ciliogenesis-inhibitory centrosome protein.

    PubMed

    Zhao, Xiaoyu; Jin, Miaomiao; Wang, Mengzhu; Sun, Lili; Hong, Xuejiao; Cao, Ying; Wang, Chunguang

    2016-09-01

    Fidgetin-like 1 (FIGL-1) is a homolog of fidgetin, an AAA protein that was identified as the protein encoded by the gene mutated in fidget mice. Because the phenotypes of fidget mice are reminiscent of the phenotypes of ciliopathy diseases, and because fidgetin has microtubule-severing activity, we hypothesize that these proteins participate in cilia-related processes. Indeed, overexpression of FIGL-1 interfered with ciliogenesis in cultured cells. In particular, overexpressed FIGL-1 strongly accumulated at the centrosome, and, when highly expressed, perturbed the localization of centrosomal proteins such as pericentrin, CP110, and centrin. Using a polyclonal antibody against human FIGL-1, we found that endogenous FIGL-1 localized preferentially around the mother centriole. Consistently, depletion of FIGL-1 by shRNA treatment enhanced ciliogenesis in HEK293T cells. By checking the integrity of the cytoplasmic microtubule network in FIGL-1-overexpressing cells, we found that FIGL-1 probably has microtubule-severing activity, as suggested by its sequence homology with other microtubule-severing proteins. Furthermore, we showed that overexpression of FIGL-1 in zebrafish embryo decreased the length of cilia and perturbed the heart laterality. Taken together, these results demonstrate that FIGL-1 is a new centrosomal protein and inhibits ciliogenesis. These results extend the already long list of centrosomal proteins and provide new insights into the regulation of ciliogenesis. PMID:27384458

  3. Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.

    PubMed

    Hawke, Lindsey; Bowman, Mackenzie L; Poon, Man-Chiu; Scully, Mary-Frances; Rivard, Georges-Etienne; James, Paula D

    2016-07-28

    Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells. PMID:27317792

  4. Identification of miR-34a-target interactions by a combined network based and experimental approach

    PubMed Central

    Hart, Martin; Rheinheimer, Stefanie; Leidinger, Petra; Backes, Christina; Menegatti, Jennifer; Fehlmann, Tobias; Grässer, Friedrich; Keller, Andreas; Meese, Eckart

    2016-01-01

    Circulating miRNAs have been associated with numerous human diseases. The lack of understanding the functional roles of blood-born miRNAs limits, however, largely their value as disease marker. In a systems biology analysis we identified miR-34a as strongly associated with pathogenesis. Genome-wide analysis of miRNAs in blood cell fractions highlighted miR-34a as most significantly up-regulated in CD3+ cells of lung cancer patients. By our in silico analysis members of the protein kinase C family (PKC) were indicated as miR-34a target genes. Using a luciferase assay, we confirmed binding of miR-34a-5p to target sequences within the 3′UTRs of five PKC family members. To verify the biological effect, we transfected HEK 293T and Jurkat cells with miR-34a-5p causing reduced endogenous protein levels of PKC isozymes. By combining bioinformatics approaches with experimental validation, we demonstrate that one of the most relevant disease associated miRNAs has the ability to control the expression of a gene family. PMID:27144431

  5. Human UBL5 protein interacts with coilin and meets the Cajal bodies

    SciTech Connect

    Švéda, Martin; Častorálová, Markéta; Lipov, Jan; Ruml, Tomáš; Knejzlík, Zdeněk

    2013-06-28

    Highlights: •Localization of the UBL5 protein in Hela cells was determined by fluorescence microscopy and biochemical fractionation. •Colocalization of UBL5 with Cajal bodies was observed. •Interaction of UBL5 with coilin was proven by pull-down. -- Abstract: UBL5 protein, a structural homologue of ubiquitin, was shown to be involved in pre-mRNA splicing and transcription regulation in yeast and Caenorhabditis elegans, respectively. However, role of the UBL5 human orthologue is still elusive. In our study, we observed that endogenous human UBL5 that was localized in the nucleus, partially associates with Cajal bodies (CBs), nuclear domains where spliceosomal components are assembled. Simultaneous expression of exogenous UBL5 and coilin resulted in their nuclear colocalization in HeLa cells. The ability of UBL5 to interact with coilin was proved by GST pull-down assay using coilin that was either in vitro translated or extracted from HEK293T cells. Further, our results showed that the UBL5–coilin interaction was not influenced by coilin phosphorylation. These results suggest that UBL5 could be targeted to CBs via its interaction with coilin. Relation between human UBL5 protein and CBs is in the agreement with current observations about yeast orthologue Hub1 playing important role in alternative splicing.

  6. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  7. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone.

    PubMed

    Kao, Yu-Han; Chern, Yijuang; Yang, Hui-Ting; Chen, Hui-Mei; Lin, Chun-Jung

    2016-08-01

    Huntington's disease (HD) is a neurodegenerative disease marked by an expanded polyglutamine (polyQ) tract on the huntingtin (HTT) protein that may cause transcriptional dysfunction. This study aimed to investigate the regulation and function of P-glycoprotein, an important efflux transporter, in brain capillaries in HD. The results showed that, compared with the littermate controls, R6/2 HD transgenic mice with the human mutant HTT gene had higher levels of P-glycoprotein mRNA and protein and enhanced NF-κB activity in their brain capillaries. Higher P-glycoprotein expression was also observed in the brain capillaries of human HD patients. Consistent with this enhanced P-glycoprotein expression, brain extracellular levels and brain-to-plasma ratios of the antipsychotic agents risperidone and paliperidone were significantly lower in R6/2 mice than in their littermate controls. Exogenous expression of human mutant HTT protein with expanded polyQ (mHTT-109Q) in HEK293T cells enhanced the levels of P-glycoprotein transcripts and NF-κB activity compared with cells expressing normal HTT-25Q. Treatment with the IKK inhibitor, BMS-345541, decreased P-glycoprotein mRNA level in cells transfected with mHTT-109Q or normal HTT-25Q In conclusion, mutant HTT altered the expression of P-glycoprotein through the NF-κB pathway in brain capillaries in HD and markedly affected the availability of P-glycoprotein substrates in the brain.

  8. Development of luminescent coelenterazine derivatives activatable by β-galactosidase for monitoring dual gene expression.

    PubMed

    Lindberg, Eric; Mizukami, Shin; Ibata, Keiji; Miyawaki, Atsushi; Kikuchi, Kazuya

    2013-10-25

    Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain β-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect β-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.

  9. Mycobacterium tuberculosis Peptidyl-Prolyl Isomerases Also Exhibit Chaperone like Activity In-Vitro and In-Vivo

    PubMed Central

    Pandey, Saurabh; Sharma, Ashish; Tripathi, Deeksha; Kumar, Ashutosh; Khubaib, Mohd; Bhuwan, Manish; Chaudhuri, Tapan Kumar; Hasnain, Seyed Ehtesham; Ehtesham, Nasreen Zafar

    2016-01-01

    Peptidyl-prolyl cis-trans isomerases (Ppiases), also known as cyclophilins, are ubiquitously expressed enzymes that assist in protein folding by isomerization of peptide bonds preceding prolyl residues. Mycobacterium tuberculosis (M.tb) is known to possess two Ppiases, PpiA and PpiB. However, our understanding about the biological significance of mycobacterial Ppiases with respect to their pleiotropic roles in responding to stress conditions inside the macrophages is restricted. This study describes chaperone-like activity of mycobacterial Ppiases. We show that recombinant rPpiA and rPpiB can bind to non-native proteins in vitro and can prevent their aggregation. Purified rPpiA and rPpiB exist in oligomeric form as evident from gel filtration chromatography.E. coli cells overexpressing PpiA and PpiB of M.tb could survive thermal stress as compared to plasmid vector control. HEK293T cells transiently expressing M.tb PpiA and PpiB proteins show increased survival as compared to control cells in response to oxidative stress and hypoxic conditions generated after treatment with H2O2 and CoCl2 thereby pointing to their likely role in adaption under host generated oxidative stress and conditions of hypoxia. The chaperone-like function of these M.tuberculosis cyclophilins may possibly function as a stress responder and consequently contribute to virulence. PMID:26981873

  10. Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing.

    PubMed

    Benakanakere, Manjunatha R; Finoti, Livia S; Tanaka, Urara; Grant, Gregory R; Scarel-Caminaga, Raquel M; Kinane, Denis F

    2016-01-01

    Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.

  11. The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

    PubMed Central

    Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

    2014-01-01

    HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

  12. Gadd45a promotes DNA demethylation through TDG

    PubMed Central

    Li, Zheng; Gu, Tian-Peng; Weber, Alain R.; Shen, Jia-Zhen; Li, Bin-Zhong; Xie, Zhi-Guo; Yin, Ruichuan; Guo, Fan; Liu, Xiaomeng; Tang, Fuchou; Wang, Hailin; Schär, Primo; Xu, Guo-Liang

    2015-01-01

    Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)—initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway. PMID:25845601

  13. The effects of cytosine methylation on general transcription factors.

    PubMed

    Jin, Jianshi; Lian, Tengfei; Gu, Chan; Yu, Kai; Gao, Yi Qin; Su, Xiao-Dong

    2016-07-07

    DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

  14. Fidgetin-like 1 is a ciliogenesis-inhibitory centrosome protein.

    PubMed

    Zhao, Xiaoyu; Jin, Miaomiao; Wang, Mengzhu; Sun, Lili; Hong, Xuejiao; Cao, Ying; Wang, Chunguang

    2016-09-01

    Fidgetin-like 1 (FIGL-1) is a homolog of fidgetin, an AAA protein that was identified as the protein encoded by the gene mutated in fidget mice. Because the phenotypes of fidget mice are reminiscent of the phenotypes of ciliopathy diseases, and because fidgetin has microtubule-severing activity, we hypothesize that these proteins participate in cilia-related processes. Indeed, overexpression of FIGL-1 interfered with ciliogenesis in cultured cells. In particular, overexpressed FIGL-1 strongly accumulated at the centrosome, and, when highly expressed, perturbed the localization of centrosomal proteins such as pericentrin, CP110, and centrin. Using a polyclonal antibody against human FIGL-1, we found that endogenous FIGL-1 localized preferentially around the mother centriole. Consistently, depletion of FIGL-1 by shRNA treatment enhanced ciliogenesis in HEK293T cells. By checking the integrity of the cytoplasmic microtubule network in FIGL-1-overexpressing cells, we found that FIGL-1 probably has microtubule-severing activity, as suggested by its sequence homology with other microtubule-severing proteins. Furthermore, we showed that overexpression of FIGL-1 in zebrafish embryo decreased the length of cilia and perturbed the heart laterality. Taken together, these results demonstrate that FIGL-1 is a new centrosomal protein and inhibits ciliogenesis. These results extend the already long list of centrosomal proteins and provide new insights into the regulation of ciliogenesis.

  15. Further progress in cytosolic cellular delivery of quantum dots

    NASA Astrophysics Data System (ADS)

    Boeneman Gemmill, Kelly; Delehanty, James B.; Stewart, Michael H.; Susumu, Kimihiro; Blanco-Canosa, Juan B.; Dawson, Philip E.; Huston, Alan; Medintz, Igor

    2012-03-01

    Currently there is considerable interest in using bioconjugated nanoparticles for in vivo imaging, biosensing and theranostics. Luminescent CdSe/ZnS core shell semiconductor quantum dots (QDs) have unique optical properties and bioconjugation capabilities that make them ideal prototypes for these purposes. We have previously described the metal-affinity association between the imidazole groups of terminal hexahistidine residues of peptides and proteins and the ZnS shell of quantum dots as a useful bioconjugation technique. We have also demonstrated that QDs labeled with an oligohistidine-tagged cell penetrating peptide (CPP) derived from the HIV TAT-protein could undergo specific endocytosis-mediated cellular uptake in both HEK293T/17 and COS-1 cells. However, the QDs were predominantly sequestered in the endosomes. This remains a significant hindrance to future potential cellular imaging applications which require the QDs to access other subcellular organelles. Here we describe the testing of several cytosolic QD delivery modalities including microinjection, the commercial cytosolic delivery agent PULSin, and the cytosolic delivery peptide Palm-1. Palm-1, a palmitylated peptide that is capable of both cellular uptake and rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for cytosolic delivery of QDs. Potential intracellular applications for this peptide are discussed.

  16. Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions.

    PubMed

    Holic, Nathalie; Fenard, David

    2016-01-01

    Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications. PMID:27317171

  17. Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression

    PubMed Central

    Fath, Stephan; Bauer, Asli Petra; Liss, Michael; Spriestersbach, Anne; Maertens, Barbara; Hahn, Peter; Ludwig, Christine; Schäfer, Frank; Graf, Marcus; Wagner, Ralf

    2011-01-01

    Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins – transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators – all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression. PMID:21408612

  18. Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing

    PubMed Central

    Benakanakere, Manjunatha R.; Finoti, Livia S.; Tanaka, Urara; Grant, Gregory R.; Scarel-Caminaga, Raquel M.; Kinane, Denis F.

    2016-01-01

    Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility. PMID:27499075

  19. Buffalo (Bubalus bubalis) interleukin-12: analysis of expression profiles and functional cross-reactivity with bovine system.

    PubMed

    Premraj, Avinash; Sreekumar, E; Jain, Mamta; Rasool, T J

    2006-03-01

    Interleukin-12, a heterodimeric pro-inflammatory cytokine, from water buffaloes (Bubalus bubalis) was analyzed for its for its tissue specific expression and functionality. Concanavalin A stimulated splenocytes displayed an up-regulation of the IL-12 p40 subunit 8-24h post-stimulation, whereas the p35 subunit did not show any quantitative variation at different time intervals. Basal level expressions of both the subunits were observed by RT-PCR in spleen. In addition p40 transcripts could be detected in liver and p35 in brain and muscle tissues as well in very low levels. Functional recombinant buffalo IL-12 was expressed in HEK 293T cells as a heterodimer using foot-and-mouth disease virus 2A polypeptide as a linker. Culture supernatants from transfected cells contained a hetero-dimeric p70 subunit as revealed in western blot of the proteins separated by native polyacrylamide gel electrophoresis (PAGE) using a monoclonal antibody against bovine IL-12 p40. IL-12 containing culture supernatant induced production of nitric oxide in cultured splenocytes of both buffalo and bovine origin. Our study reveals that buffalo IL-12, which shares a high-level sequence identity with bovine IL-12, also has functional cross-reactivity with the bovine immune cells.

  20. Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability.

    PubMed

    Stray-Pedersen, Asbjørg; Cobben, Jan-Maarten; Prescott, Trine E; Lee, Sora; Cang, Chunlei; Aranda, Kimberly; Ahmed, Sohnee; Alders, Marielle; Gerstner, Thorsten; Aslaksen, Kathinka; Tétreault, Martine; Qin, Wen; Hartley, Taila; Jhangiani, Shalini N; Muzny, Donna M; Tarailo-Graovac, Maja; van Karnebeek, Clara D M; Lupski, James R; Ren, Dejian; Yoon, Grace

    2016-01-01

    Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health. PMID:26708751

  1. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex.

    PubMed

    Todd, Matthew A M; Picketts, David J

    2012-08-01

    Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.

  2. Epimedium koreanum Nakai displays broad spectrum of antiviral activity in vitro and in vivo by inducing cellular antiviral state.

    PubMed

    Cho, Won-Kyung; Weeratunga, Prasanna; Lee, Byeong-Hoon; Park, Jun-Seol; Kim, Chul-Joong; Ma, Jin Yeul; Lee, Jong-Soo

    2015-01-20

    Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant's known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakai markedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans.

  3. Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.

    PubMed

    Berger, Adeline; Lorain, Stéphanie; Joséphine, Charlène; Desrosiers, Melissa; Peccate, Cécile; Voit, Thomas; Garcia, Luis; Sahel, José-Alain; Bemelmans, Alexis-Pierre

    2015-05-01

    The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

  4. Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo

    PubMed Central

    Kamezaki, Aosa; Sato, Fuminori; Aoki, Kazuhiro; Asakawa, Kazuhide; Kawakami, Koichi; Matsuzaki, Fumio; Sehara-Fujisawa, Atsuko

    2016-01-01

    Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as ‘ectodomain shedding’, is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo. PMID:27364328

  5. Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea hongkongensis.

    PubMed

    Xiang, Zhiming; Qu, Fufa; Qi, Lin; Ying, Tong; Li, Jun; Shu, Xiao; Yu, Ziniu

    2014-05-01

    Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis.

  6. Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability.

    PubMed

    Stray-Pedersen, Asbjørg; Cobben, Jan-Maarten; Prescott, Trine E; Lee, Sora; Cang, Chunlei; Aranda, Kimberly; Ahmed, Sohnee; Alders, Marielle; Gerstner, Thorsten; Aslaksen, Kathinka; Tétreault, Martine; Qin, Wen; Hartley, Taila; Jhangiani, Shalini N; Muzny, Donna M; Tarailo-Graovac, Maja; van Karnebeek, Clara D M; Lupski, James R; Ren, Dejian; Yoon, Grace

    2016-01-01

    Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.

  7. Multidrug and toxic compound extrusion protein-1 (MATE1/SLC47A1) is a novel flavonoid transporter.

    PubMed

    Lee, Ji Hae; Lee, Jung Eun; Kim, Yeojin; Lee, Hojoung; Jun, Hee-Jin; Lee, Sung-Joon

    2014-10-01

    Dietary flavonoids have various biological functions. However, their cellular transport mechanisms are largely unknown. We have determined that the multidrug and toxic compound extrusion transporter-1 (MATE1) is a membrane transporter for flavonoids and has a high affinity for quercetin. HEK293T cells overexpressing MATE1 exhibited increased intracellular quercetin accumulation. This effect disappeared in the presence of a MATE1 inhibitor and after MATE1 gene knockdown. HepG2 cells expressed MATE1 significantly, with the uptake quercetin of which was dramatically reduced with MATE1 inhibition. On the basis of immunofluorescence analysis, MATE1 was highly expressed in peroxisomes and the endoplasmic reticulum (ER) as well as in plasma membranes in the liver and intestine, which suggests potential accumulation of quercetin in peroxisomes and the ER in these tissues. Fluorescent microscopic analysis confirmed selective accumulation of qurcetin in peroxisome. The effects of quercetin on cellular lipid reduction and glucose uptake were exaggerated with MATE1 overexpression. In conclusion, MATE1 is a membrane transporter for quercetin; its overexpression enhances the hypolipidemic activity of quercetin and cellular glucose transport. Considering the low bioavailability of quercetin, appropriate regulation of MATE1 expression may optimize cellular quercetin concentrations and promote health benefits.

  8. Curcumin inhibits HIV-1 by promoting Tat protein degradation

    PubMed Central

    Ali, Amjad; Banerjea, Akhil C.

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  9. Regulatory role of neuron-restrictive silencing factor in expression of TRPC1

    SciTech Connect

    Ohba, Takayoshi; Watanabe, Hiroyuki; Takahashi, Yoichiro; Suzuki, Takashi; Miyoshi, Ichiro; Nakayama, Shinnsuke; Satoh, Eisaku; Iino, Kenji; Sasano, Hironobu; Mori, Yasuo; Kuromitsu, Sadao; Imagawa, Keiichi; Saito, Yoshihiko; Iijima, Toshihiko; Ito, Hiroshi; Murakami, Manabu . E-mail: mmura0123@hotmail.co.jp

    2006-12-22

    Neuron-restrictive silencer factor (NRSF) binds its consensus element to repress the transcription of various genes. The dominant-negative form (dnNRSF) has a hypertrophic effect on cardiogenesis through an unidentified mechanism. We examined the involvement of transient receptor potential (TRP) channel proteins, using transgenic mice overexpressing dnNRSF (dnNRSF mice). Electrophoretic mobility-shift assays revealed an interaction between NRSF and a neuron-restrictive silencer element-like sequence in intron 4 of TRPC1 genomic DNA. According to RT-PCR and Western analyses, TRPC1 was up-regulated in dnNRSF mouse heart. Transient overexpression of TRPC1 in HEK 293T cells increased the activity of the nuclear factor in activated T cells (NFAT) promoter and stimulated store-operated Ca{sup 2+} channel (SOCC)-mediated Ca{sup 2+} entry. Transfection of TRPC1 into primary cardiomyocytes increased NFAT activity, indicating a major role for TRPC1 in NFAT activation. Our findings strongly suggest that NRSF regulates TRP1 gene expression and causes changes in the levels of calcium entry through SOCCs.

  10. BmP02 Atypically Delays Kv4.2 Inactivation: Implication for a Unique Interaction between Scorpion Toxin and Potassium Channel

    PubMed Central

    Wu, Bin; Zhu, Yan; Shi, Jian; Tao, Jie; Ji, Yonghua

    2016-01-01

    BmP02, a short-chain peptide with 28 residues from the venom of Chinese scorpion Buthus martensi Karsch, has been reported to inhibit the transient outward potassium currents (Ito) in rat ventricular muscle cells. However, it remains unclear whether BmP02 modulates the Kv4.2 channel, one of the main contributors to Ito. The present study investigated the effects of BmP02 on Kv4.2 kinetics and its underlying molecular mechanism. The electrophysiological recordings showed that the inactivation of Kv4.2 expressed in HEK293T cells was significantly delayed by BmP02 in a dose-response manner with EC50 of ~850 nM while the peak current, activation and voltage-dependent inactivation of Kv4.2 were not affected. Meanwhile, the recovery from inactivation of Kv4.2 was accelerated and the deactivation was slowed after the application of BmP02. The site-directed mutagenesis combined with computational modelling identified that K347 and K353, located in the turret motif of the Kv4.2, and E4/E5, D20/D21 in BmP02 are key residues to interact with BmP02 through electrostatic force. These findings not only reveal a novel interaction between Kv4.2 channel and its peptidyl modulator, but also provide valuable information for design of highly-selective Kv4.2 modulators. PMID:27690098

  11. Characterization of C-alkyl amidines as bioavailable covalent reversible inhibitors of human DDAH-1.

    PubMed

    Lluis, Matthew; Wang, Yun; Monzingo, Arthur F; Fast, Walter; Robertus, Jon D

    2011-01-01

    C-Alkyl amidine analogues of asymmetric N(ω),N(ω)-dimethyl-L-arginine are dual-targeted inhibitors of both human DDAH-1 and nitric oxide (NO) synthase, and provide a promising scaffold for the development of therapeutics to control NO overproduction in a variety of pathologies including septic shock and some cancers. Using a two-part click-chemistry-mediated activity probe, a homologated series of C-alkyl amidines were ranked for their ability to inhibit DDAH-1 within cultured HEK 293T cells. N⁵-(1-Iminopentyl)-L-ornithine was determined to be the most potent compound in vitro (K(d)=7 μM) as well as in cultured cells, and the binding conformation and covalent reversible mode of inhibition was investigated by comparison of interactions made with DDAH-1 and a catalytically inactive C274S variant, as gauged by X-ray crystallography and isothermal titration calorimetry. By interrupting the ability of the inhibitor to form a covalent bond, the contribution of this interaction could be estimated. These results suggest that further stabilization of the covalent adduct is a promising strategy for lead optimization in the design of effective reagents to block NO synthesis.

  12. Curcumin inhibits HIV-1 by promoting Tat protein degradation.

    PubMed

    Ali, Amjad; Banerjea, Akhil C

    2016-01-01

    HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity. PMID:27283735

  13. A systematic scan of interactions with tyrosine motifs in the erythropoietin receptor using a mammalian 2-hybrid approach.

    PubMed

    Montoye, Tony; Lemmens, Irma; Catteeuw, Dominiek; Eyckerman, Sven; Tavernier, Jan

    2005-06-01

    Signaling via the erythropoietin receptor (EpoR) depends on the interaction of several proteins with phosphorylated tyrosine-containing motifs in its cytosolic domain. Detailed mapping of these interactions is required for an accurate insight into Epo signaling. We recently developed a mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based 2-hybrid method that operates in intact Hek293-T mammalian cells. As baits, we used intracellular segments of the EpoR containing 1 or 2 tyrosines. Several known signaling molecules, including cytokine-inducible SH2-containing protein (CIS), suppressor of cytokine signaling-2 (SOCS2), phosphatidylinositol 3'-kinase (PI3-K), phospholipase C-gamma (PLC-gamma), and signal transducer and activator of transcription 5 (STAT5) were used as prey. We also extended the MAPPIT method to enable interaction analysis with wild-type EpoR. In this relay MAPPIT approach, instead of using isolated EpoR fragments as bait, we used the full-length EpoR itself as a "receptor bait." Finally, we introduced MAPPIT in the erythroleukemic TF-1 cell line, which is a more natural setting of the EpoR. With these strategies several known interactions with the EpoR were analyzed and evidence for new interactions was obtained. PMID:15644415

  14. Intragenic duplication: a novel mutational mechanism in hereditary pancreatitis

    PubMed Central

    Joergensen, Maiken T.; Geisz, Andrea; Brusgaard, Klaus; de Muckadell, Ove B. Schaffalitzky; Hegyi, Péter; Gerdes, Anne-Marie; Sahin-Tóth, Miklós

    2011-01-01

    Objectives In a hereditary pancreatitis family from Denmark we identified a novel intragenic duplication of 9 nucleotides in exon-2 of the human cationic trypsinogen (PRSS1) gene (c.63_71dup) which at the amino-acid level resulted in the insertion of three amino acids within the activation peptide of cationic trypsinogen (p.K23_I24insIDK). The aim of the present study was to characterize the effect of this unique genetic alteration on the function of human cationic trypsinogen. Methods Wild-type and mutant cationic trypsinogens were produced recombinantly and purified to homogeneity. Trypsinogen activation was followed by enzymatic assays and SDS-PAGE. Trypsinogen secretion was measured from transfected HEK 293T cells. Results Recombinant cationic trypsinogen carrying the p.K23_I24insIDK mutation exhibited >10-fold increased autoactivation. Activation by human cathepsin B was also accelerated by 10-fold. Secretion of the p.K23_I24insIDK mutant from transfected cells was diminished, consistent with intracellular autoactivation. Conclusions This is the first report of an intragenic duplication within the PRSS1 gene causing hereditary pancreatitis. The accelerated activation of p.K23_I24insIDK by cathepsin B is a unique biochemical property not found in any other pancreatitis-associated trypsinogen mutant. In contrast, the robust autoactivation of the novel mutant confirms the notion that increased autoactivation is a disease-relevant mechanism in hereditary pancreatitis. PMID:21499207

  15. TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity

    PubMed Central

    2014-01-01

    Background Recent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission. Results TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents. Conclusions Our data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation. PMID:25139109

  16. Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-dilution Method

    PubMed Central

    Fu, Lijuan; Amato, Nicolas J.; Wang, Pengcheng; McGowan, Sara J.; Niedernhofer, Laura J.; Wang, Yinsheng

    2016-01-01

    The rising interest in understanding the functions, regulation and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Mono-methylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m5C), 2′-O-methylcytidine (Cm), N6-methyladenosine (m6A) and 2′-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of the four methylated nucleosides are tissue-specific. In addition, the 2′-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m5C and m6A) in total RNA isolated from mouse brain, pancreas and spleen, but not mouse heart. We also found that the levels of m5C, Cm and Am are significantly lower (by 6.5-43 fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m6A was slightly higher (by 1.6 fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications. PMID:26158405

  17. HEPeak: an HMM-based exome peak-finding package for RNA epigenome sequencing data

    PubMed Central

    2015-01-01

    Background Methylated RNA Immunoprecipatation combined with RNA sequencing (MeRIP-seq) is revolutionizing the de novo study of RNA epigenomics at a higher resolution. However, this new technology poses unique bioinformatics problems that call for novel and sophisticated statistical computational solutions, aiming at identifying and characterizing transcriptome-wide methyltranscriptome. Results We developed HEP, a Hidden Markov Model (HMM)-based Exome Peak-finding algorithm for predicting transcriptome methylation sites using MeRIP-seq data. In contrast to exomePeak, our previously developed MeRIP-seq peak calling algorithm, HEPeak models the correlation between continuous bins in an m6A peak region and it is a model-based approach, which admits rigorous statistical inference. HEPeak was evaluated on a simulated MeRIP-seq dataset and achieved higher sensitivity and specificity than exomePeak. HEPeak was also applied to real MeRIP-seq datasets from human HEK293T cell line and mouse midbrain cells and was shown to be able to recapitulate known m6A distribution in transcripts and identify novel m6A sites in long non-coding RNAs. Conclusions In this paper, a novel HMM-based peak calling algorithm, HEPeak, was developed for peak calling for MeRIP-seq data. HEPeak is written in R and is publicly available. PMID:25917296

  18. A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction.

    PubMed

    Shao, Qing; Lindstrom, Kristin; Shi, Ruoyang; Kelly, John; Schroeder, Audrey; Juusola, Jane; Levine, Kara L; Esseltine, Jessica L; Penuela, Silvia; Jackson, Michael F; Laird, Dale W

    2016-06-10

    Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G→A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a "PANX1-related disorder." PMID:27129271

  19. Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability

    PubMed Central

    Stray-Pedersen, Asbjørg; Cobben, Jan-Maarten; Prescott, Trine E.; Lee, Sora; Cang, Chunlei; Aranda, Kimberly; Ahmed, Sohnee; Alders, Marielle; Gerstner, Thorsten; Aslaksen, Kathinka; Tétreault, Martine; Qin, Wen; Hartley, Taila; Jhangiani, Shalini N.; Muzny, Donna M.; Tarailo-Graovac, Maja; van Karnebeek, Clara D.M.; Lupski, James R.; Ren, Dejian; Yoon, Grace

    2016-01-01

    Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent “leak” channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health. PMID:26708751

  20. Characterization of a ligand-gated cation channel based on an inositol receptor in the silkworm, Bombyx mori.

    PubMed

    Kikuta, Shingo; Endo, Haruka; Tomita, Natsuo; Takada, Tomoyuki; Morita, Chiharu; Asaoka, Kiyoshi; Sato, Ryoichi

    2016-07-01

    Insect herbivores recognize non-volatile compounds in plants to direct their feeding behavior. Gustatory receptors (Gr) appear to be required for nutrient recognition by gustatory organs in the mouthparts of insects. Gr10 is expressed in Bombyx mori (BmGr10) mouthparts such as maxillary galea, maxillary palp, and labrum. BmGr10 is predicted to function in sugar recognition; however, the precise biochemical function remains obscure. Larvae of B. mori are monophagous feeders able to find and feed on mulberry leaves. Soluble mulberry leaf extract contains sucrose, glucose, fructose, and myo-inositol. In this study, we identified BmGr10 as an inositol receptor using electrophysiological analysis with the Xenopus oocyte expression system and Ca(2+) imaging techniques using mammalian cells. These results demonstrated that Xenopus oocytes or HEK293T cells expressing BmGr10 specifically respond to myo-inositol and epi-inositol but do not respond to any mono-, di-, or tri-saccharides or to some sugar alcohols. These inositols caused Ca(2+) and Na(+) influxes into the cytoplasm independently of a G protein-mediated signaling cascade, indicating that BmGr10 is a ligand-gated cation channel. Overall, BmGr10 plays an important role in the myo-inositol recognition required for B. mori larval feeding behavior. PMID:27132146

  1. Synphilin-1 binds ATP and regulates intracellular energy status.

    PubMed

    Li, Tianxia; Liu, Jingnan; Smith, Wanli W

    2014-01-01

    Recent studies have suggested that synphilin-1, a cytoplasmic protein, is involved in energy homeostasis. Overexpression of synphilin-1 in neurons results in hyperphagia and obesity in animal models. However, the mechanism by which synphilin-1 alters energy homeostasis is unknown. Here, we used cell models and biochemical approaches to investigate the cellular functions of synphilin-1 that may affect energy balance. Synphilin-1 was pulled down by ATP-agarose beads, and the addition of ATP and ADP reduced this binding, indicating that synphilin-1 bound ADP and ATP. Synphilin-1 also bound GMP, GDP, and GTP but with a lower affinity than it bound ATP. In contrast, synphilin-1 did not bind with CTP. Overexpression of synphilin-1 in HEK293T cells significantly increased cellular ATP levels. Genetic alteration to abolish predicted ATP binding motifs of synphilin-1 or knockdown of synphilin-1 by siRNA reduced cellular ATP levels. Together, these data demonstrate that synphilin-1 binds and regulates the cellular energy molecule, ATP. These findings provide a molecular basis for understanding the actions of synphilin-1 in energy homeostasis.

  2. Interaction of Mycobacterium tuberculosis Virulence Factor RipA with Chaperone MoxR1 Is Required for Transport through the TAT Secretion System

    PubMed Central

    Bhuwan, Manish; Arora, Naresh; Sharma, Ashish; Khubaib, Mohd; Pandey, Saurabh; Chaudhuri, Tapan Kumar

    2016-01-01

    ABSTRACT Mycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing β-lactam antibiotics, opening up new candidates for drug repurposing. PMID:26933057

  3. Amyloid-β Precursor Protein Modulates the Sorting of Testican-1 and Contributes to Its Accumulation in Brain Tissue and Cerebrospinal Fluid from Patients with Alzheimer Disease

    PubMed Central

    Barrera-Ocampo, Alvaro; Arlt, Sönke; Matschke, Jakob; Hartmann, Ursula; Puig, Berta; Ferrer, Isidre; Zürbig, Petra; Glatzel, Markus; Jahn, Holger

    2016-01-01

    The mechanisms leading to amyloid-β (Aβ) accumulation in sporadic Alzheimer disease (AD) are unknown but both increased production or impaired clearance likely contribute to aggregation. To understand the potential roles of the extracellular matrix proteoglycan Testican-1 in the pathophysiology of AD, we used samples from AD patients and controls and an in vitro approach. Protein expression analysis showed increased levels of Testican-1 in frontal and temporal cortex of AD patients; histological analysis showed that Testican-1 accumulates and co-aggregates with Aβ plaques in the frontal, temporal and entorhinal cortices of AD patients. Proteomic analysis identified 10 fragments of Testican-1 in cerebrospinal fluid (CSF) from AD patients. HEK293T cells expressing human wild type or mutant Aβ precursor protein (APP) were transfected with Testican-1. The co-expression of both proteins modified the sorting of Testican-1 into the endocytic pathway leading to its transient accumulation in Golgi, which seemed to affect APP processing, as indicated by reduced Aβ40 and Aβ42 levels in APP mutant cells. In conclusion, patient data reflect a clearance impairment that may favor Aβ accumulation in AD brains and our in vitro model supports the notion that the interaction between APP and Testican-1 may be a key step in the production and aggregation of Aβ species. PMID:27486134

  4. The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling.

    PubMed

    Likhite, Neah; Jackson, Christopher A; Liang, Mao-Shih; Krzyzanowski, Michelle C; Lei, Pedro; Wood, Jordan F; Birkaya, Barbara; Michaels, Kerry L; Andreadis, Stelios T; Clark, Stewart D; Yu, Michael C; Ferkey, Denise M

    2015-11-10

    Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyltransferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5-deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide-binding protein-coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins. PMID:26554819

  5. Construction and characterization of an infectious molecular clone of Koala retrovirus.

    PubMed

    Shojima, Takayuki; Hoshino, Shigeki; Abe, Masumi; Yasuda, Jiro; Shogen, Hiroko; Kobayashi, Takeshi; Miyazawa, Takayuki

    2013-05-01

    Koala retrovirus (KoRV) is a gammaretrovirus that is currently endogenizing into koalas. Studies on KoRV infection have been hampered by the lack of a replication-competent molecular clone. In this study, we constructed an infectious molecular clone, termed plasmid pKoRV522, of a KoRV isolate (strain Aki) from a koala reared in a Japanese zoo. The virus KoRV522, derived from pKoRV522, grew efficiently in human embryonic kidney (HEK293T) cells, attaining 10(6) focus-forming units/ml. Several mutations in the Gag (L domain) and Env regions reported to be involved in reduction in viral infection/production in vitro are found in pKoRV522, yet KoRV522 replicated well, suggesting that any effects of these mutations are limited. Indeed, a reporter virus pseudotyped with pKoRV522 Env was found to infect human, feline, and mink cell lines efficiently. Analyses of KoRV L-domain mutants showed that an additional PPXY sequence, PPPY, in Gag plays a critical role in KoRV budding. Altogether, our results demonstrate the construction and characterization of the first infectious molecular clone of KoRV. The infectious clone reported here will be useful for elucidating the mechanism of endogenization of the virus in koalas and screening for antiretroviral drugs for KoRV-infected koalas.

  6. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice.

    PubMed

    Jafarpour, Nazli; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Kohram, Fatemeh; Aghababa, Haniyeh; Khoramabadi, Nima; Mahdavi, Mehdi

    2014-08-01

    Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine. PMID:24842263

  7. Distinct parts of leukotriene C{sub 4} synthase interact with 5-lipoxygenase and 5-lipoxygenase activating protein

    SciTech Connect

    Strid, Tobias; Svartz, Jesper; Franck, Niclas; Hallin, Elisabeth; Ingelsson, Bjoern; Soederstroem, Mats; Hammarstroem, Sven

    2009-04-17

    Leukotriene C{sub 4} is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C{sub 4} synthase (LTC{sub 4}S) participate in its biosynthesis. We report evidence that LTC{sub 4}S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC{sub 4}S. FLAP bound to the N-terminal part/first hydrophobic region of LTC{sub 4}S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC{sub 4}S. Fluorescent FLAP- and LTC{sub 4}S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC{sub 4}S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC{sub 4}S. Direct interaction of 5-LO and LTC{sub 4}S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC{sub 4}S.

  8. Epimedium koreanum Nakai Displays Broad Spectrum of Antiviral Activity in Vitro and in Vivo by Inducing Cellular Antiviral State

    PubMed Central

    Cho, Won-Kyung; Weeratunga, Prasanna; Lee, Byeong-Hoon; Park, Jun-Seol; Kim, Chul-Joong; Ma, Jin Yeul; Lee, Jong-Soo

    2015-01-01

    Epimedium koreanum Nakai has been extensively used in traditional Korean and Chinese medicine to treat a variety of diseases. Despite the plant’s known immune modulatory potential and chemical make-up, scientific information on its antiviral properties and mode of action have not been completely investigated. In this study, the broad antiviral spectrum and mode of action of an aqueous extract from Epimedium koreanum Nakai was evaluated in vitro, and moreover, the protective effect against divergent influenza A subtypes was determined in BALB/c mice. An effective dose of Epimedium koreanum Nakaimarkedly reduced the replication of Influenza A Virus (PR8), Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV) and Newcastle Disease Virus (NDV) in RAW264.7 and HEK293T cells. Mechanically, we found that an aqueous extract from Epimedium koreanum Nakai induced the secretion of type I IFN and pro-inflammatory cytokines and the subsequent stimulation of the antiviral state in cells. Among various components present in the extract, quercetin was confirmed to have striking antiviral properties. The oral administration of Epimedium koreanum Nakai exhibited preventive effects on BALB/c mice against lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3 and H9N2). Therefore, an extract of Epimedium koreanum Nakai and its components play roles as immunomodulators in the innate immune response, and may be potential candidates for prophylactic or therapeutic treatments against diverse viruses in animal and humans. PMID:25609307

  9. Oligomerization of TAS2R bitter taste receptors.

    PubMed

    Kuhn, Christina; Bufe, Bernd; Batram, Claudia; Meyerhof, Wolfgang

    2010-06-01

    A family of 25 G protein-coupled receptors, TAS2Rs, mediates bitter taste in humans. Many of the members of this family are coexpressed in a subpopulation of taste receptor cells on the tongue, thereby allowing the possibility of receptor-receptor interactions with potential influences on their function. In this study, we used several experimental approaches to investigate whether TAS2Rs can form oligomers and if this has an effect on receptor function. Coimmunoprecipitations clearly demonstrated that TAS2Rs can physically interact in HEK293T cells. Further bioluminescence resonance energy transfer analysis of all 325 possible binary combinations of TAS2Rs established that the vast majority of TAS2R pairs form oligomers, both homomers and heteromers. Subsequent screenings of coexpressed bitter receptors with 104 different tastants did not reveal any heteromer-specific agonists. Additional studies also showed no obvious influence of TAS2R hetero-oligomerization on plasma membrane localization or pharmacological properties of the receptors. Thus, our results show that receptor oligomerization occurs between TAS2R bitter taste receptors; however, functional consequences of hetero-oligomerization were not obvious.

  10. The broad pattern recognition spectrum of the Toll-like receptor in mollusk Zhikong scallop Chlamys farreri.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Sun, Rui; Yue, Feng; Yi, Qilin; Song, Linsheng

    2015-10-01

    Toll-like receptors (TLRs) are among the most studied pattern recognition receptors (PRRs) playing essential roles in innate immune defenses. In the present study, the basic features of CfTLR in mollusk Zhikong scallop Chlamys farreri, including sequence homology, tissue distribution, subcellular localization and ligands spectrum, were investigated to elucidate its pattern recognition. The elements of extracellular domains (ECD) in CfTLR displayed high homology to the corresponding parts of the ECDs in TLRs from Homo sapiens. CfTLR protein was detected in hemocytes, mantle, gills, hepatopancreas, kidney and gonad of the scallops, and it was localized in both the plasma membranes and the lysosomes in HEK293T cells. CfTLR could activate NFκB in response to multiple HsTLR ligands including Pam3CSK4, glucan (GLU), peptidoglycan (PGN), polyriboinosinic:polyribocytidylic acid (poly I:C), Imiquimod and three types of CpG. Additionally, the scallop serum could enhance the induction of NFκB in the CfTLR expressing cells elicited by most PAMPs, including GLU, PGN, Imiquimod and four types of CpG. It could be concluded that this primitive mollusk TLR shared a hybrid function in pattern recognition and could recognize broader ligands than mammalian TLRs, and its mosaic capability of pathogen associated molecular pattern (PAMP) recognition might be based on the basic features of its structure, ligand properties and the assistance of some components in scallop serum.

  11. Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins

    PubMed Central

    Kralt, Annemarie; Jagalur, Noorjahan B.; van den Boom, Vincent; Lokareddy, Ravi K.; Steen, Anton; Cingolani, Gino; Fornerod, Maarten; Veenhoff, Liesbeth M.

    2015-01-01

    Endoplasmic reticulum–synthesized membrane proteins traffic through the nuclear pore complex (NPC) en route to the inner nuclear membrane (INM). Although many membrane proteins pass the NPC by simple diffusion, two yeast proteins, ScSrc1/ScHeh1 and ScHeh2, are actively imported. In these proteins, a nuclear localization signal (NLS) and an intrinsically disordered linker encode the sorting signal for recruiting the transport factors for FG-Nup and RanGTP-dependent transport through the NPC. Here we address whether a similar import mechanism applies in metazoans. We show that the (putative) NLSs of metazoan HsSun2, MmLem2, HsLBR, and HsLap2β are not sufficient to drive nuclear accumulation of a membrane protein in yeast, but the NLS from RnPom121 is. This NLS of Pom121 adapts a similar fold as the NLS of Heh2 when transport factor bound and rescues the subcellular localization and synthetic sickness of Heh2ΔNLS mutants. Consistent with the conservation of these NLSs, the NLS and linker of Heh2 support INM localization in HEK293T cells. The conserved features of the NLSs of ScHeh1, ScHeh2, and RnPom121 and the effective sorting of Heh2-derived reporters in human cells suggest that active import is conserved but confined to a small subset of INM proteins. PMID:26179916

  12. Molecular cloning of chicken IL-7 and characterization of its antiviral activity against IBDV in vivo

    PubMed Central

    Huo, Shanshan; Wang, Liyue; Zhang, Yonghong; Zhang, Jianlou; Zuo, Yuzhu; Xu, Jian; Cui, Dan; Li, Xiujin; Zhong, Fei

    2016-01-01

    Mammalian interleukin-7 (IL-7) is able to stimulate lymphocyte proliferation and maturation, and reverse immunosuppression. However, whether poultry IL-7 has similar functions remains unclear. Chicken infectious bursal disease virus (IBDV) causes serious immunosuppression in chicken due to virus-induced immune disorder. Whether chicken IL-7 (chIL-7) has the ability to restore the immunity during IBDV-induced immunosuppression is not clear. To test this, we amplified chIL-7 gene by RT-PCR, prepared recombinant chIL-7 using HEK293T cells and treated the chicken with the chIL-7 prior to IBDV infection. Our results indicate that chIL-7 promoted mouse B cell proliferation in vitro, and significantly reduced virus titer in bursal tissue and chicken morbidity of IBDV-infected chicken. Mechanically, chIL-7 induced chicken lymphocyte proliferation and interferon-γ production, but down-regulated TGF-β expression, suggesting that chIL-7 has the ability to reverse IBDV-induced immunosuppression and might be a potential therapeutic agent for prevention and treatment of infectious bursal disease. PMID:27466431

  13. Amyloid-β Precursor Protein Modulates the Sorting of Testican-1 and Contributes to Its Accumulation in Brain Tissue and Cerebrospinal Fluid from Patients with Alzheimer Disease.

    PubMed

    Barrera-Ocampo, Alvaro; Arlt, Sönke; Matschke, Jakob; Hartmann, Ursula; Puig, Berta; Ferrer, Isidre; Zürbig, Petra; Glatzel, Markus; Sepulveda-Falla, Diego; Jahn, Holger

    2016-09-01

    The mechanisms leading to amyloid-β (Aβ) accumulation in sporadic Alzheimer disease (AD) are unknown but both increased production or impaired clearance likely contribute to aggregation. To understand the potential roles of the extracellular matrix proteoglycan Testican-1 in the pathophysiology of AD, we used samples from AD patients and controls and an in vitro approach. Protein expression analysis showed increased levels of Testican-1 in frontal and temporal cortex of AD patients; histological analysis showed that Testican-1 accumulates and co-aggregates with Aβ plaques in the frontal, temporal and entorhinal cortices of AD patients. Proteomic analysis identified 10 fragments of Testican-1 in cerebrospinal fluid (CSF) from AD patients. HEK293T cells expressing human wild type or mutant Aβ precursor protein (APP) were transfected with Testican-1. The co-expression of both proteins modified the sorting of Testican-1 into the endocytic pathway leading to its transient accumulation in Golgi, which seemed to affect APP processing, as indicated by reduced Aβ40 and Aβ42 levels in APP mutant cells. In conclusion, patient data reflect a clearance impairment that may favor Aβ accumulation in AD brains and our in vitro model supports the notion that the interaction between APP and Testican-1 may be a key step in the production and aggregation of Aβ species. PMID:27486134

  14. A590T mutation in KCNQ1 C-terminal helix D decreases IKs channel trafficking and function but not Yotiao interaction.

    PubMed

    Kinoshita, Koshi; Komatsu, Takuto; Nishide, Kohki; Hata, Yukiko; Hisajima, Nozomi; Takahashi, Hiroyuki; Kimoto, Katsuya; Aonuma, Kei; Tsushima, Eikichi; Tabata, Toshihide; Yoshida, Tomoyuki; Mori, Hisashi; Nishida, Kunihiro; Yamaguchi, Yoshiaki; Ichida, Fukiko; Fukurotani, Kenkichi; Inoue, Hiroshi; Nishida, Naoki

    2014-07-01

    KCNQ1 encodes the α subunit of the voltage-gated channel that mediates the cardiac slow delayed rectifier K(+) current (IKs). Here, we report a KCNQ1 allele encoding an A590T mutation [KCNQ1(A590T)] found in a 39-year-old female with a mild QT prolongation. A590 is located in the C-terminal α helical region of KCNQ1 that mediates subunit tetramerization, membrane trafficking, and interaction with Yotiao. This interaction is known to be required for the proper modulation of IKs by cAMP. Since previous studies reported that mutations in the vicinity of A590 impair IKs channel surface expression and function, we examined whether and how the A590T mutation affects the IKs channel. Electrophysiological measurements in HEK-293T cells showed that the A590T mutation caused a reduction in IKs density and a right-shift of the current-voltage relation of channel activation. Immunocytochemical and immunoblot analyses showed the reduced cell surface expression of KCNQ1(A590T) subunit and its rescue by coexpression of the wild-type KCNQ1 [KCNQ1(WT)] subunit. Moreover, KCNQ1(A590T) subunit interacted with Yotiao and had a cAMP-responsiveness comparable to that of KCNQ1(WT) subunit. These findings indicate that the A590 of KCNQ1 subunit plays important roles in the maintenance of channel surface expression and function via a novel mechanism independent of interaction with Yotiao.

  15. NAC1, a POZ/BTB protein that functions as a corepressor.

    PubMed

    Korutla, Laxminarayana; Wang, Peijie; Jackson, Trevor G; Mackler, Scott A

    2009-01-01

    We now demonstrate that NAC1 acts as a corepressor for other POZ/BTB proteins. NAC1 is a POZ/BTB motif containing transcriptional repressor protein. In a mammalian two hybrid assay in neuronal (N2A) cells and non-neuronal (HEK 293T) cells, VP16 activation domain tagged NAC1 resulted in significant reversal of transcriptional inhibition with the Gal4-ZID, Gal4-BCL6, Gal4-ZF5, and kelch proteins Gal4-MAYVEN and Gal4-NRP/B fusion proteins. We also observed similar results with another corepressor, BCoR Gal4 fusion protein. NAC1 potentiated ZF5 mediated repression in Gal4-DBD fusion transient assays. GST pulldown assays further confirmed protein-protein interactions between these proteins and NAC1. Both the NAC1 isoforms demonstrated selective interaction through the POZ/BTB domain but not with the non-POZ/BTB region. Endogenous NAC1 and BCL6 physically associated in CNS regions. Strikingly, NAC1 did not interact with the pro-myelocytic leukemia zinc finger protein (PLZF), another POZ/BTB protein that is not found in the adult brain. Therefore, we conclude that NAC1 functions as a corepressor for POZ/BTB proteins expressed in the mature CNS.

  16. Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

    PubMed Central

    Seo, Kinya; Rainer, Peter P.; Shalkey Hahn, Virginia; Lee, Dong-ik; Jo, Su-Hyun; Andersen, Asger; Liu, Ting; Xu, Xiaoping; Willette, Robert N.; Lepore, John J.; Marino, Joseph P.; Birnbaumer, Lutz; Schnackenberg, Christine G.; Kass, David A.

    2014-01-01

    Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3–21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management. PMID:24453217

  17. Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy.

    PubMed

    Seo, Kinya; Rainer, Peter P; Shalkey Hahn, Virginia; Lee, Dong-Ik; Jo, Su-Hyun; Andersen, Asger; Liu, Ting; Xu, Xiaoping; Willette, Robert N; Lepore, John J; Marino, Joseph P; Birnbaumer, Lutz; Schnackenberg, Christine G; Kass, David A

    2014-01-28

    Chronic neurohormonal and mechanical stresses are central features of heart disease. Increasing evidence supports a role for the transient receptor potential canonical channels TRPC3 and TRPC6 in this pathophysiology. Channel expression for both is normally very low but is increased by cardiac disease, and genetic gain- or loss-of-function studies support contributions to hypertrophy and dysfunction. Selective small-molecule inhibitors remain scarce, and none target both channels, which may be useful given the high homology among them and evidence of redundant signaling. Here we tested selective TRPC3/6 antagonists (GSK2332255B and GSK2833503A; IC50, 3-21 nM against TRPC3 and TRPC6) and found dose-dependent blockade of cell hypertrophy signaling triggered by angiotensin II or endothelin-1 in HEK293T cells as well as in neonatal and adult cardiac myocytes. In vivo efficacy in mice and rats was greatly limited by rapid metabolism and high protein binding, although antifibrotic effects with pressure overload were observed. Intriguingly, although gene deletion of TRPC3 or TRPC6 alone did not protect against hypertrophy or dysfunction from pressure overload, combined deletion was protective, supporting the value of dual inhibition. Further development of this pharmaceutical class may yield a useful therapeutic agent for heart disease management.

  18. Single-Tailed Lipidoids Enhance the Transfection Activity of Their Double-Tailed Counterparts.

    PubMed

    Wu, Yihang; Li, Linxian; Chen, Qing; Su, Yi; Levkin, Pavel A; Davidson, Gary

    2016-01-11

    Cationic lipid-like molecules (lipidoids) are widely used for in vitro and in vivo gene delivery. Nearly all lipidoids developed to date employ double-tail or multiple-tail structures for transfection. Single-tail lipidoids are seldom considered for transfection as they have low efficiency in gene delivery. So far, there is no detailed study on the contribution to transfection efficiency of single-tail lipidoids when combined with standard double-tail lipidoids. Here, we use combinatorial chemistry to synthesize 17 double-tail and 17 single-tail lipidoids using thiol-yne and thiol-ene click chemistry, respectively. HEK 293T cells were used to analyze transfection efficiency by fluorescence microscopy and calculated based on the percentage of cells transfected. The size and zeta potential of liposomes and lipoplexes were characterized by dynamic light scattering (DLS). Intracellular DNA delivery and trafficking was further examined using confocal microscopy. Our study shows that combining single with double-tail lipidoids increases uptake of lipoplexes, as well as cellular transfection efficiency.

  19. Synthesis of an arrayed sgRNA library targeting the human genome

    PubMed Central

    Schmidt, Tobias; Schmid-Burgk, Jonathan L.; Hornung, Veit

    2015-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested. PMID:26446710

  20. A role for the CaM Kinase II related anchoring protein (αkap) in maintaining the stability of nicotinic Acetylcholine Receptors

    PubMed Central

    Mouslim, Chakib; Aittaleb, Mohamed; Hume, Richard I.; Akaaboune, Mohammed

    2012-01-01

    αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function. PMID:22496563

  1. Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity.

    PubMed

    Zingue, Stéphane; Magne Nde, Chantal Beatrice; Clyne, Colin; Njamen, Dieudonné

    2014-01-01

    Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs. PMID:27433539

  2. Elucidation of Underlying Mechanisms by Which Millettia macrophylla Benth Induces Its Estrogenic Activity

    PubMed Central

    Zingue, Stéphane; Magne Nde, Chantal Beatrice; Njamen, Dieudonné

    2014-01-01

    Millettia macrophylla is used traditionally to treat menopause related symptoms. This plant was shown to exhibit estrogenic effects in vitro on human embryonic kidney cells and in vivo on ovariectomized rats. The present study aimed at elucidating underlying mechanisms by which M. macrophylla induced its estrogenic effects. To accomplish our goal, kidney Hek293T cells transiently transfected with estrogen alpha or beta receptor expression plasmids were cotreated with a pure antiestrogen ICI 182,780 and the dichloromethane or methanol soluble fractions of M. macrophylla. To follow up, we cotreated ovariectomized rats with both extracts and ICI 182,780 for 3 days in the classical uterotrophic assay. Animals were then sacrificed and the uterine wet weight, total protein levels in uteri, uterine, and vaginal epithelial heights, and mammary gland were assessed. In vitro, the results suggested that the induction of the estrogenic activity by M. macrophylla is due to the binding of its secondary metabolites to ERα and ERβ. In vivo, the cotreatment of extracts and ICI 182,780 significantly abrogated the biological responses induced by the extracts alone. Taken together, these results indicate that the active principles of M. macrophylla induce their beneficial effects on menopausal symptoms by activating the ERs. PMID:27433539

  3. Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

    PubMed

    Martins, S A M; Moulas, G; Trabuco, J R C; Monteiro, G A; Chu, V; Conde, J P; Prazeres, D M F

    2014-02-15

    G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 μM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery. PMID:24055937

  4. Cloning, characterization and expression analysis of a caspase-8 like gene from the Hong Kong oyster, Crassostrea hongkongensis.

    PubMed

    Xiang, Zhiming; Qu, Fufa; Qi, Lin; Zhang, Yang; Tong, Ying; Yu, Ziniu

    2013-12-01

    Apoptosis plays a key role in many biological processes, including homeostasis within the immune system. A family of cysteine proteases, the caspases, constitutes the core of the apoptotic machinery. We have characterized the first bivalve caspase-8 ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChCaspase-8). The full-length cDNA is 1945 bp in length encoding a putative protein of 557 amino acids that contains two N-terminal DED domains, and a CASc domain at the C-terminus. ChCaspase-8 is ubiquitously expressed in oysters, with highest expression levels in the gonad and labial palps. Following microbial infection, the expression of ChCaspase-8 increased in hemocytes from 12 to 72 h post-challenge. When expressed in HeLa cells, ChCaspase-8 is located in the cytoplasm, while over-expression of ChCaspase-8 in HEK293T cells activates the transcriptional activities of NF-κB. These results indicate that ChCaspase-8 might play an important role in the immune and apoptotic responses of oysters.

  5. Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase.

    PubMed

    Xiao, Xunjun; Ross, Leah E; Sevilla, Wednesday A; Wang, Yan; Lowe, Mark E

    2013-09-01

    Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy.

  6. Optogenetic Inhibitor of the Transcription Factor CREB.

    PubMed

    Ali, Ahmed M; Reis, Jakeb M; Xia, Yan; Rashid, Asim J; Mercaldo, Valentina; Walters, Brandon J; Brechun, Katherine E; Borisenko, Vitali; Josselyn, Sheena A; Karanicolas, John; Woolley, G Andrew

    2015-11-19

    Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events. PMID:26590638

  7. Regulating the effects of GPR21, a novel target for type 2 diabetes

    NASA Astrophysics Data System (ADS)

    Leonard, Siobhán; Kinsella, Gemma K.; Benetti, Elisa; Findlay, John B. C.

    2016-05-01

    Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.

  8. X-ray fluorescence microscopy artefacts in elemental maps of topologically complex samples: Analytical observations, simulation and a map correction method

    NASA Astrophysics Data System (ADS)

    Billè, Fulvio; Kourousias, George; Luchinat, Enrico; Kiskinova, Maya; Gianoncelli, Alessandra

    2016-08-01

    XRF spectroscopy is among the most widely used non-destructive techniques for elemental analysis. Despite the known angular dependence of X-ray fluorescence (XRF), topological artefacts remain an unresolved issue when using X-ray micro- or nano-probes. In this work we investigate the origin of the artefacts in XRF imaging of topologically complex samples, which are unresolved problems in studies of organic matter due to the limited travel distances of low energy XRF emission from the light elements. In particular we mapped Human Embryonic Kidney (HEK293T) cells. The exemplary results with biological samples, obtained with a soft X-ray scanning microscope installed at a synchrotron facility were used for testing a mathematical model based on detector response simulations, and for proposing an artefact correction method based on directional derivatives. Despite the peculiar and specific application, the methodology can be easily extended to hard X-rays and to set-ups with multi-array detector systems when the dimensions of surface reliefs are in the order of the probing beam size.

  9. The activation effect of hainantoxin-I, a peptide toxin from the Chinese spider, Ornithoctonus hainana, on intermediate-conductance Ca2+-activated K+ channels.

    PubMed

    Huang, Pengfei; Zhang, Yiya; Chen, Xinyi; Zhu, Li; Yin, Dazhong; Zeng, Xiongzhi; Liang, Songping

    2014-08-21

    Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

  10. Light-dependent activation of G proteins by two isoforms of chicken melanopsins.

    PubMed

    Torii, Masaki; Kojima, Daisuke; Nishimura, Akiyuki; Itoh, Hiroshi; Fukada, Yoshitaka

    2015-11-01

    In the chicken pineal gland, light stimuli trigger signaling pathways mediated by two different subtypes, Gt and G11. These G proteins may be activated by any of the three major pineal opsins, pinopsin, OPN4-1 and OPN4-2, but biochemical evidence for the coupling has been missing except for functional coupling between pinopsin and Gt. Here we investigated the relative expression levels and the functional difference among the three pineal opsins. In the chicken pineal gland, the pinopsin mRNA level was significantly more abundant than the others, of which the OPN4-2 mRNA level was higher than that of OPN4-1. In G protein activation assays, Gt was strongly activated by pinopsin in a light-dependent manner, being consistent with previous studies, and weakly activated by OPN4-2. Unexpectedly, illuminated OPN4-2 more efficiently activated G protein(s) that was endogenously expressed in HEK293T cells in culture. On the other hand, Gq, the closest analogue of G11, was activated only by OPN4-1 although the activity was relatively weak under these conditions. These results suggest that OPN4-1 and OPN4-2 couple with Gq and Gt, respectively. Two melanopsins, OPN4-1 and OPN4-2, appear to have acquired mutually different functions through the evolution.

  11. The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression.

    PubMed

    Taylor-Wells, Jennina; Meredith, David

    2014-01-01

    The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

  12. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  13. SNORD116 and SNORD115 change expression of multiple genes and modify each other's activity.

    PubMed

    Falaleeva, Marina; Surface, Justin; Shen, Manli; de la Grange, Pierre; Stamm, Stefan

    2015-11-10

    The loss of two gene clusters encoding small nucleolar RNAs, SNORD115 and SNORD116 contribute to Prader-Willi syndrome (PWS), the most common syndromic form of obesity in humans. SNORD115 and SNORD116 are considered to be orphan C/D box snoRNAs (SNORDs) as they do not target rRNAs or snRNAs. SNORD115 exhibits sequence complementarity towards the serotonin receptor 2C, but SNORD116 shows no extended complementarities to known RNAs. To identify molecular targets, we performed genome-wide array analysis after overexpressing SNORD115 and SNORD116 in HEK 293T cells, either alone or together. We found that SNORD116 changes the expression of over 200 genes. SNORD116 mainly changed mRNA expression levels. Surprisingly, we found that SNORD115 changes SNORD116's influence on gene expression. In similar experiments, we compared gene expression in post-mortem hypothalamus between individuals with PWS and aged-matched controls. The synopsis of these experiments resulted in 23 genes whose expression levels were influenced by SNORD116. Together our results indicate that SNORD115 and SNORD116 influence expression levels of multiple genes and modify each other activity. PMID:26220404

  14. Identification and primary immune characteristics of an amphioxus akirin homolog.

    PubMed

    Yan, Jie; Dong, Xuan; Kong, Yu; Zhang, Yan; Jing, Renwei; Feng, Lijun

    2013-08-01

    Akirin is a recently described nuclear protein that is thought to be required for the NF-κB signaling pathway in insects and vertebrates. Here, functional investigations of akirin are described in the basal chordate amphioxus Branchiostoma belcheri tsingtauense in an attempt to link this gene between insect and vertebrate lineages. Phylogenetic analysis indicated that amphioxus akirin represented a true ortholog of the two characterized vertebrate akirin paralogs. Amphioxus akirin, coding 219 amino acids with two nuclear localization signal (NLS) sequences and one 14-3-3 binding motif, was widely expressed in various tissues and up-regulated in response to Escherichia coli (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium) challenges. Furthermore, amphioxus akirin was strictly localized to the nucleus of HEK293T cells in a confocal analysis. Our work identified and characterized for the first time an amphioxus akirin homolog and will promote a better understanding of the evolution and transcriptional network of the akirin gene family.

  15. Synthesis and antiplasmodial activity of betulinic acid and ursolic acid analogues.

    PubMed

    Innocente, Adrine M; Silva, Gloria N S; Cruz, Laura Nogueira; Moraes, Miriam S; Nakabashi, Myna; Sonnet, Pascal; Gosmann, Grace; Garcia, Célia R S; Gnoatto, Simone C B

    2012-10-12

    More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC₅₀ = 220 and 175 nM, respectively) while 1a and b demonstrated good activity (IC₅₀ = 4 and 5 μM, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC₅₀ of 4 μM and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

  16. A CgIFNLP receptor from Crassostrea gigas and its activation of the related genes in human JAK/STAT signaling pathway.

    PubMed

    Zhang, Ran; Liu, Rui; Xin, Lusheng; Chen, Hao; Li, Chenghua; Wang, Lingling; Song, Linsheng

    2016-12-01

    Interferon is a highly pleiotropic cytokine, once binding to its receptors, can activate JAK kinases and STAT transcription factors to initiate the transcription of genes downstream from interferon-stimulated response elements. In the present study, a cytokine receptor-like 3 molecule was selected and cloned from oyster Crassostrea gigas, which contained a fibronectin type III domain (designed CgIFNR-3). The expression pattern of CgIFNR-3 mRNA was detected in all the tested tissues including mantle, gills, hepatopancreas, muscle, and hemocytes, with the highest expression level in hemocytes. After poly (I: C) stimulation, the expression level of CgIFNR-3 in hemocytes was observed to significantly increase at 3 h (13.06-fold, p < 0.01). CgIFNR-3 was indicated to interact with CgIFNLP by in vitro GST pull-down assay, and to activate the expression of transcription factors including ISRE, STAT3 and GAS, in human Janus kinase signal transducer and activator of transcription (JAK/STAT) pathway after co-transfection in HEK-293T cells in the reporter luciferase activity assay. These results suggested that CgIFNR-3 could bind to CgIFNLP as an interferon receptor and participate in the activation of JAK/STAT pathway in human, which will benefit for intensive studies of interferon signaling pathway in mollusc. PMID:27373517

  17. Comparative photochemistry of animal type 1 and type 4 cryptochromes.

    PubMed

    Ozturk, Nuri; Selby, Christopher P; Song, Sang-Hun; Ye, Rui; Tan, Chuang; Kao, Ya-Ting; Zhong, Dongping; Sancar, Aziz

    2009-09-15

    Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

  18. The first characterization of multidrug and toxin extrusion (MATE/SLC47) proteins in zebrafish (Danio rerio)

    PubMed Central

    Lončar, Jovica; Popović, Marta; Krznar, Petra; Zaja, Roko; Smital, Tvrtko

    2016-01-01

    Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endogenous compounds and xenobiotics across the plasma membrane. They are conserved from bacteria to mammals, with different numbers of genes within groups. Here, we present the first data on identification and functional characterization of Mate proteins in zebrafish (Danio rerio). Phylogenetic analysis revealed six Mates in teleost fish, annotated as Mate3–8, which form a distinct cluster separated from the tetrapod MATEs/Mates. Synteny analysis showed that zebrafish mate genes are orthologous to human MATEs. Gene expression analysis revealed that all the mate transcripts were constitutively and differentially expressed during embryonic development, followed by pronounced and tissue-specific expression in adults. Functional analyses were performed using transport activity assays with model substrates after heterologous overexpression of five zebrafish Mates in HEK293T cells. The results showed that zebrafish Mates interact with both physiological and xenobiotic substances but also substantially differ with respect to the interacting compounds and interaction strength in comparison to mammalian MATEs/Mates. Taken together, our data clearly indicate a potentially important role for zebrafish Mate transporters in zebrafish embryos and adults and provide a basis for detailed functional characterizations of single zebrafish Mate transporters. PMID:27357367

  19. Cationic lipids containing protonated cyclen and different hydrophobic groups linked by uracil-PNA monomer: synthesis and application for gene delivery.

    PubMed

    Liu, Jun-Liang; Ma, Qing-Ping; Huang, Qing-Dong; Yang, Wei-Han; Zhang, Ji; Wang, Jing-Yi; Zhu, Wen; Yu, Xiao-Qi

    2011-09-01

    In this report, as candidates for non-viral gene vectors, cationic lipids L1, L2 and L3 based on protonated cyclen and different hydrophobic groups (cholesterol, dodecanol or diosgenin) linked by PNA monomer were designed and synthesized. Their liposomes were easily prepared by mixing the synthesized lipids with dioleoylphosphatidyl ethanolamine (DOPE) under appropriate mole ratios. Agarose gel retardation and fluorescent titration by ethidium bromide (EB) showed the strong DNA-binding ability with the K(sv) values of 1.21 × 10(7), 3.76 × 10(6) and 2.90 × 10(6) M(-1) for the liposomes formed from L1-L3, respectively. These liposomes could retard pDNA at an N/P ratio of 3 and form lipoplexes with sizes around 200-300 nm and zeta-potential values of +20-50 mV at N/P ratio from 4 to 10. Besides, the cytotoxicity of the three lipoplexes assayed by MTT is quite different. The results from in vitro transfection in HEK 293T and A549 cell lines showed that the transfection efficiency of L3/DOPE/DNA lipoplex at an N/P ratio of 6 and lipid/DOPE mole ratio of 1:2 is slightly higher than that of Lipofectamine 2000™, indicating that the title PNA monomer-based cationic lipids have great potential to be efficient non-viral gene vector.

  20. Ca2+-dependent facilitation of Cav1.3 Ca2+ channels by densin and Ca2+/calmodulin-dependent protein kinase II

    PubMed Central

    Jenkins, Meagan A.; Christel, Carl J.; Jiao, Yuxia; Abiria, Sunday; Kim, Kristin Y.; Usachev, Yuriy M.; Obermair, Gerald J.; Colbran, Roger J.; Lee, Amy

    2010-01-01

    Cav1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca2+ signaling in neurons. CaMKII directly potentiates the activity of Cav1.2 and Cav1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein, densin, is required for Ca2+-dependent facilitation of Cav1.3 channels. While neither CaMKII nor densin independently affect Cav1.3 properties in transfected HEK293T cells, the two together augment Cav1.3 Ca2+ currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca2+, CaMKII activation and its association with densin, as well as densin binding to the Cav1.3 α1 subunit C-terminal domain. Cav1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca2+-dependent facilitation that may intensify postsynaptic Ca2+ signals during high-frequency stimulation. PMID:20392935

  1. Bioinspired synthesis of polydopamine/Ag nanocomposite particles with antibacterial activities.

    PubMed

    Wu, Chengjiao; Zhang, Guoxing; Xia, Tian; Li, Zhenni; Zhao, Kai; Deng, Ziwei; Guo, Dingzong; Peng, Bo

    2015-10-01

    Mussel-inspired chemistry (polydopamine) offers great opportunities to develop inexpensive and efficient process for many types of materials with complex shapes and functions in a mild and friendly environment. This paper describes a facile, yet green approach to synthesize polydopamine/silver (PDA/Ag) nanocomposite particles with a combination use of polydopamine chemistry and electroless metallization of Ag. In this approach, monodisperse spherical polydopamine particles are first synthesized by the oxidation and self-polymerization of dopamine (monomer) in an alkaline water-ethanol solution at room temperature, which are served as the active templates for secondary reactions due to the abundant catechol and amine groups on the surface. Subsequently, the silver precursor-[Ag(NH3)2](+) ions introduced are easily absorbed onto the surface of the PDA particles, and are immediately in situ reduced to metallic Ag nanoparticles with the help of these active catechol and amine groups. During the preparation, no additional reductants, toxic reagents and intricate instruments are needed. These as-synthesized PDA/Ag nanocomposite particles are ideal candidates for antibacterial application because they do not show significant cytotoxicity against HEK293T human embryonic kidney cells in the in vitro cytotoxicity assay, whereas demonstrate enhanced antibacterial abilities against Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) in the antibacterial assays. Owing to their excellent cytocompatibilities and antibacterial activities, these PDA/Ag nanocomposite particles can be considered as the promising antibacterial materials for future biomedical applications.

  2. Context-dependent modulation of alphabetagamma and alphabetadelta GABA A receptors by penicillin: implications for phasic and tonic inhibition.

    PubMed

    Feng, Hua-Jun; Botzolakis, Emmanuel J; Macdonald, Robert L

    2009-01-01

    Penicillin, an open-channel blocker of GABA(A) receptors, was recently reported to inhibit phasic, but not tonic, currents in hippocampal neurons. To distinguish between isoform-specific and context-dependent modulation as possible explanations for this selectivity, the effects of penicillin were evaluated on recombinant GABA(A) receptors expressed in HEK293T cells. When co-applied with saturating GABA, penicillin decreased peak amplitude, induced rebound, and prolonged deactivation of currents evoked from both synaptic and extrasynaptic receptor isoforms. However, penicillin had isoform-specific effects on the extent of desensitization, reflecting its ability to differentially modulate peak (non-equilibrium) and residual (near-equilibrium) currents. This suggested that the context of activation could determine the apparent sensitivity of a given receptor isoform to penicillin. To test this hypothesis, we explored the ability of penicillin to modulate synaptic and extrasynaptic isoform currents that were activated under more physiologically relevant conditions. Interestingly, while currents evoked from synaptic isoforms under phasic conditions (transient activation by a saturating concentration of GABA) were substantially inhibited by penicillin, currents evoked from extrasynaptic isoforms under tonic conditions (prolonged application by a sub-saturating concentration of GABA) were minimally affected. We therefore concluded that the reported inability of penicillin to modulate tonic currents could not simply be attributed to insensitivity of extrasynaptic receptors, but rather, reflected an inability to modulate these receptors in their native context of activation.

  3. The first characterization of multidrug and toxin extrusion (MATE/SLC47) proteins in zebrafish (Danio rerio).

    PubMed

    Lončar, Jovica; Popović, Marta; Krznar, Petra; Zaja, Roko; Smital, Tvrtko

    2016-01-01

    Multidrug and toxin extrusion (MATE) proteins are involved in the extrusion of endogenous compounds and xenobiotics across the plasma membrane. They are conserved from bacteria to mammals, with different numbers of genes within groups. Here, we present the first data on identification and functional characterization of Mate proteins in zebrafish (Danio rerio). Phylogenetic analysis revealed six Mates in teleost fish, annotated as Mate3-8, which form a distinct cluster separated from the tetrapod MATEs/Mates. Synteny analysis showed that zebrafish mate genes are orthologous to human MATEs. Gene expression analysis revealed that all the mate transcripts were constitutively and differentially expressed during embryonic development, followed by pronounced and tissue-specific expression in adults. Functional analyses were performed using transport activity assays with model substrates after heterologous overexpression of five zebrafish Mates in HEK293T cells. The results showed that zebrafish Mates interact with both physiological and xenobiotic substances but also substantially differ with respect to the interacting compounds and interaction strength in comparison to mammalian MATEs/Mates. Taken together, our data clearly indicate a potentially important role for zebrafish Mate transporters in zebrafish embryos and adults and provide a basis for detailed functional characterizations of single zebrafish Mate transporters. PMID:27357367

  4. Identification, Synthesis and Evaluation of Substituted Benzofurazans as Inhibitors of CREB-mediated Gene Transcription

    PubMed Central

    Xie, Fuchun; Li, Bingbing X.; Broussard, Candice; Xiao, Xiangshu

    2013-01-01

    Cyclic-AMP response-element binding protein (CREB) is a stimulus-activated transcription factor. Its transcription activity requires its binding with CREB-binding protein (CBP) after CREB is phosphorylated at Ser133. The domains involved for CREB-CBP interaction are kinase-inducible domain (KID) from CREB and KID-interacting domain (KIX) from CBP. Recent studies suggest that CREB is an attractive target for novel cancer therapeutics. To identify novel chemotypes as inhibitors of KIX-KID interaction, we screened the NCI-diversity set of compounds using a split renilla luciferase assay and identified 2-[(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio]pyridine 1-oxide (compound 1) was identified as a potent inhibitor of KIX-KID interaction. However, compound 1 was not particularly selective against CREB-mediated gene transcription in living HEK 293T cells. Further structure-activity relationship studies identified 4-aniline substituted nitrobenzofurazans with improved selectivity. PMID:23953193

  5. The Human Mitochondrial DEAD-Box Protein DDX28 Resides in RNA Granules and Functions in Mitoribosome Assembly

    PubMed Central

    Tu, Ya-Ting; Barrientos, Antoni

    2015-01-01

    SUMMARY Human mitochondrial ribosomes are specialized in the synthesis of 13 proteins, which are fundamental components of the oxidative phosphorylation system. The pathway of mitoribosome biogenesis, the compartmentalization of the process and factors involved remain largely unknown. Here, we have identified the DEAD box protein DDX28 as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and the mt-LSU. RNAi-mediated DDX28 silencing in HEK293T cells does not affect mitochondrial mRNA stability, 16S rRNA processing or modification. However, it leads to reduced levels of 16S rRNA and mt-LSU proteins, impaired mt-LSU assembly, deeply attenuated mitochondrial protein synthesis and consequent failure to assemble oxidative phosphorylation complexes. Our findings identify DDX28 as essential during the early stages of mitoribosome mt-LSU biogenesis, a process that mainly takes place near the mitochondrial nucleoids, in the compartment defined by the RNA granules. PMID:25683708

  6. Paclitaxel inhibits selenoprotein S expression and attenuates endoplasmic reticulum stress.

    PubMed

    Qin, Hong-Shuang; Yu, Pei-Pei; Sun, Ying; Wang, Dan-Feng; Deng, Xiao-Fen; Bao, Yong-Li; Song, Jun; Sun, Lu-Guo; Song, Zhen-Bo; Li, Yu-Xin

    2016-06-01

    The primary effect of the endoplasmic reticulum (ER) stress response or unfolded protein response (UPR) is to reduce the load of unfolded protein and promote survival. However, prolonged and severe ER stress leads to tissue injury and serious diseases. Thus, it is important to identify drugs that can attenuate ER stress for the treatment of diseases. Natural products continue to provide lead compounds for drug discovery and front‑line pharmacotherapy for people worldwide. Previous studies have indicated that selenoprotein S (SelS) is a sensitive and ideal maker of ER stress. In the present study, a firefly luciferase reporter driven by the SelS gene promoter was used to screen for natural compounds capable of attenuating ER stress. From this, paclitaxel (PTX) was identified to efficiently inhibit the promoter activity of the SelS gene, and further results revealed that PTX significantly inhibited the tunicamycin‑induced upregulation of SelS at the mRNA and protein levels in HepG2 and HEK293T cells. In addition, PTX was able to efficiently inhibit the expression of the ER stress marker, glucose‑regulated protein 78, in ER stress, indicating that PTX may reverse ER stress. Taken together, these results suggest that PTX is able to inhibit SelS expression during ER stress and attenuate ER stress. PMID:27109260

  7. Interplay between inhibitory ferric and stimulatory curcumin regulates phosphorylation-dependent human cystic fibrosis transmembrane conductance regulator and ΔF508 activity.

    PubMed

    Wang, Guangyu

    2015-02-24

    Curcumin potentiates cystic fibrosis transmembrane conductance regulator (CFTR) activation in an ATP-independent but phosphorylation-dependent manner. The underlying molecular mechanisms are unclear. Here, HEK-293T cells cultured in an Fe(3+)-containing medium were transiently transfected with CFTR constructs, and the role of the inhibitory Fe(3+) bridge between intracellular loop 3 and the regulatory domain of CFTR in this pathway was investigated. The results showed that ethylenediaminetetraacetic acid (EDTA) stimulated phosphorylation-dependent CFTR activation and the stimulation was suppressed by the deletion of the regulatory domain or the insertion of a C832A mutation that removes the Fe(3+)-binding interface. Furthermore, curcumin potentiation of CFTR was significantly weakened not only by Fe(3+)-insensitive mutations at the interface between the regulatory domain and intracellular loop 3 but also by N-ethylmaleimide or EDTA pretreatment that removes Fe(3+). More importantly, potentiation of CFTR was completely suppressed by sufficient Fe(3+). Finally, the insertion of Fe(3+)-insensitive H950R/S768R increased the curcumin-independent activity of ΔF508 but weakened its curcumin potentiation. Thus, Fe(3+) homeostasis in epithelia may play a critical role in regulating CFTR activity, and targeting Fe(3+)-chelating potentiators may direct new therapies for cystic fibrosis.

  8. Creating Knockouts of Conserved Oligomeric Golgi Complex Subunits Using CRISPR-Mediated Gene Editing Paired with a Selection Strategy Based on Glycosylation Defects Associated with Impaired COG Complex Function.

    PubMed

    Blackburn, Jessica Bailey; Lupashin, Vladimir V

    2016-01-01

    The conserved oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. The COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex's role in glycosylation homeostasis to obtain COG complex subunit knockouts (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

  9. hKSR-2 inhibits MEKK3-activated MAP kinase and NF-kappaB pathways in inflammation.

    PubMed

    Channavajhala, Padma L; Rao, Vikram R; Spaulding, Vikki; Lin, Lih-Ling; Zhang, Y George

    2005-09-01

    Kinase suppressor of ras (KSR) and MEKK3 (MAP kinase kinase kinase) are integral members of the MAP kinase pathway. We have recently identified a new isoform of the KSR family named human kinase suppressor of ras-2 (hKSR-2), and demonstrated that hKSR-2 negatively regulates Cot, a MAP3K family member which is important in inflammation and oncogenesis [P.L. Channavajhala, L. Wu, J.W. Cuozzo, J.P. Hall, W. Liu, L.L. Lin, Y. Zhang, J. Biol. Chem. 278 (2003) 47089-47097]. In this report, we provide evidence that hKSR-2 also regulates the activity of MEKK3 (another MAP3K family member) in HEK-293T cells. We demonstrate that hKSR-2 is a negative regulator of MEKK3-mediated activation of MAP kinase (specifically ERK and JNK) and NF-kappaB pathways, and concurrently inhibits MEKK3-mediated interleukin-8 production. We find that while hKSR-2 blocks MEKK3 activation, it has little to no effect on other members of the MAP3K family, including MEKK4, TAK1, and Ras-Raf, suggesting that its effects are selective. PMID:16039990

  10. Systemic desensitization through TRPA1 channels by capsazepine and mustard oil - a novel strategy against inflammation and pain

    PubMed Central

    Kistner, Katrin; Siklosi, Norbert; Babes, Alexandru; Khalil, Mohammad; Selescu, Tudor; Zimmermann, Katharina; Wirtz, Stefan; Becker, Christoph; Neurath, Markus F.; Reeh, Peter W.; Engel, Matthias A.

    2016-01-01

    We demonstrate a novel dual strategy against inflammation and pain through body-wide desensitization of nociceptors via TRPA1. Attenuation of experimental colitis by capsazepine (CPZ) has long been attributed to its antagonistic action on TRPV1 and associated inhibition of neurogenic inflammation. In contrast, we found that CPZ exerts its anti-inflammatory effects via profound desensitization of TRPA1. Micromolar CPZ induced calcium influx in isolated dorsal root ganglion (DRG) neurons from wild-type (WT) but not TRPA1-deficient mice. CPZ-induced calcium transients in human TRPA1-expressing HEK293t cells were blocked by the selective TRPA1 antagonists HC 030031 and A967079 and involved three cysteine residues in the N-terminal domain. Intriguingly, both colonic enemas and drinking water with CPZ led to profound systemic hypoalgesia in WT and TRPV1−/− but not TRPA1−/− mice. These findings may guide the development of a novel class of disease-modifying drugs with anti-inflammatory and anti-nociceptive effects. PMID:27356469

  11. Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    PubMed Central

    Canault, Matthias; Ghalloussi, Dorsaf; Grosdidier, Charlotte; Guinier, Marie; Perret, Claire; Chelghoum, Nadjim; Germain, Marine; Raslova, Hana; Peiretti, Franck; Morange, Pierre E.; Saut, Noemie; Pillois, Xavier; Nurden, Alan T.; Cambien, François; Pierres, Anne; van den Berg, Timo K.; Kuijpers, Taco W.; Tregouet, David-Alexandre

    2014-01-01

    The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis. PMID:24958846

  12. Nuclear Localization Signal and p53 Binding Site in MAP/ERK Kinase Kinase 1 (MEKK1).

    PubMed

    Chipps, Elizabeth; Protzman, April; Muhi, M Zubayed; Ando, Shoko; Calvet, James P; Islam, M Rafiq

    2015-12-01

    Previously, we showed that Mekk1 translocates to the nucleus, interacts with tumor suppressor protein p53, and co-represses PKD1 transcription via an atypical p53 binding site on the minimal PKD1 promoter (JBC 285:38,818-38,831, 2010). In this study, we report the mechanisms of Mekk1 nuclear transport and p53 binding. Using GFP-linked constitutively active-Mekk1 (CA-Mekk1) and a deletion strategy, we identified a nuclear localization signal (HRDVK) located at amino acid (aa) residues 1,349-1,353 in the C-terminal Mekk1 catalytic domain. Deletion of this sequence in CA-Mekk1 and full-length Mekk1 significantly reduced their nuclear translocation in both HEK293T and COS-1 cells. Using co-immunoprecipitation, we identified an adjacent sequence (GANLID, aa 1,354-1,360) in Mekk1 responsible for p53 binding. Deletion of this sequence markedly reduced the interaction of Mekk1 with p53. Mekk1 does not appear to affect phosphorylation of Ser15, located in the Mdm2 interaction site, or other Ser residues in p53. However, Mekk1 mediates p53 protein stability in the presence of Mdm2 and reduces p53 ubiquitination, suggesting an interference with Mdm2-mediated degradation of p53 by the ubiquitin-proteasome pathway.

  13. CRISPR/Cas9-based genome editing in mice by single plasmid injection.

    PubMed

    Fujihara, Yoshitaka; Ikawa, Masahito

    2014-01-01

    CRISPR/Cas-mediated genome modification has opened a new era for elucidating gene function. Gene knockout mice can be generated by injecting humanized Cas9 (hCas9) mRNA and guide RNA (sgRNA) into fertilized eggs. However, delivery of RNA instead of DNA to the fertilized oocyte requires extra preparation and extra care with storage. To simplify the method of delivery, we injected the circular pX330 plasmids expressing both hCas9 and sgRNA and found that mutant mice were generated as efficiently as with RNA injection. Different from the linearized plasmid, the circular plasmid decreased the chance of integration into the host genome. We also developed the pCAG-EGxxFP reporter plasmid for evaluating the sgRNA activity by observing EGFP fluorescence in HEK293T cells. The combination of these techniques allowed us to develop a rapid, easy, and reproducible strategy for targeted mutagenesis in living mice. This chapter provides an experimental protocol for the design of sgRNAs, the construction of pX330-sgRNA and pCAG-EGxxFP-target plasmids, the validation of cleavage efficiency in vitro, and the generation of targeted gene mutant mice. These mice can be generated within a month.

  14. Comparative proteomic analysis suggests that mitochondria are involved in autosomal recessive polycystic kidney disease.

    PubMed

    Li, Qing-Wei; Lu, Xiao-Yan; You, Yong; Sun, Huan; Liu, Xin-Yu; Ai, Jian-Zhong; Tan, Rui-Zhi; Chen, Tie-Lin; Chen, Mian-Zhi; Wang, Hong-Lian; Wei, Yu-Quan; Zhou, Qin

    2012-08-01

    Autosomal recessive polycystic kidney disease (ARPKD), characterized by ectatic collecting duct, is an infantile form of PKD occurring in 1 in 20 000 births. Despite having been studied for many years, little is known about the underlying mechanisms. In the current study, we employed, for the first time, a MS-based comparative proteomics approach to investigate the differently expressed proteins between kidney tissue samples of four ARPKD and five control individuals. Thirty two differently expressed proteins were identified and six of the identified protein encoding genes performed on an independent group (three ARPKD subjects, four control subjects) were verified by semi-quantitative RT-PCR, and part of them were further validated by Western blot and immunohistochemistry. Moreover, similar alteration tendency was detected after downregulation of PKHD1 by small interfering RNA in HEK293T cell. Interestingly, most of the identified proteins are associated with mitochondria. This implies that mitochondria may be implicated in ARPKD. Furthermore, the String software was utilized to investigate the biological association network, which is based on known and predicted protein interactions. In conclusion, our findings depicted a global understanding of ARPKD progression and provided a promising resource of targeting protein, and shed some light further investigation of ARPKD. PMID:22718539

  15. Functional and Structural Characterization of FAU Gene/Protein from Marine Sponge Suberites domuncula

    PubMed Central

    Perina, Dragutin; Korolija, Marina; Popović Hadžija, Marijana; Grbeša, Ivana; Belužić, Robert; Imešek, Mirna; Morrow, Christine; Marjanović, Melanija Posavec; Bakran-Petricioli, Tatjana; Mikoč, Andreja; Ćetković, Helena

    2015-01-01

    Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with “higher” metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis. PMID:26198235

  16. Reprofiled anthelmintics abate hypervirulent stationary-phase Clostridium difficile.

    PubMed

    Gooyit, Major; Janda, Kim D

    2016-01-01

    Prolonged use of broad-spectrum antibiotics disrupts the indigenous gut microbiota, which consequently enables toxigenic Clostridium difficile species to proliferate and cause infection. The burden of C. difficile infections was exacerbated with the outbreak of hypervirulent strains that produce copious amounts of enterotoxins and spores. In recent past, membrane-active agents have generated a surge of interest due to their bactericidal property with a low propensity for resistance. In this study, we capitalized on the antimicrobial property and low oral bioavailability of salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. By broth microdilution techniques, we determined the MIC values of the anthelmintics against 16 C. difficile isolates of defined PCR-ribotype. The anthelmintics broadly inhibited C. difficile growth in vitro via a membrane depolarization mechanism. Interestingly, the salicylanilides were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. The salicylanilides were poorly active against select gut commensals (Bacteroides, Bifidobacterium and Lactobacillus species), and were non-hemolytic and non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable properties for repositioning as anti-C. difficile agents. PMID:27633064

  17. The suppressor of cytokine signaling (SOCS)-7 interacts with the actin cytoskeleton through vinexin.

    PubMed

    Martens, Nele; Wery, Maxime; Wang, Ping; Braet, Filip; Gertler, Arieh; Hooghe, Robert; Vandenhaute, Jean; Hooghe-Peters, Elisabeth L

    2004-08-01

    To understand the function of the suppressor of cytokine signaling (SOCS)-7, we have looked for proteins interacting with SOCS-7 in a stringent yeast two-hybrid screen of a human leukocyte cDNA-library. We identified the cytoskeletal molecule vinexin as a partner interacting with SOCS-7. Tests with deletion mutants of SOCS-7 demonstrated that a central region of the molecule containing several proline-rich regions, N-terminal to the SH2 domain, was responsible for the binding to vinexin. It is thus likely that one of the SH3 domains of vinexin interacts with a poly-proline region of SOCS-7. The interaction with vinexin was confirmed biochemically as vinexin-alpha was co-precipitated with SOCS-7. Confocal laser-scanning microscopy in HEK293T, MCF-7, and 3T3-L1 cells showed that part of the transfected SOCS-7-green fluorescent protein (GFP) molecules merged with vinexin and with actin. Taken together, our data indicate that SOCS-7 interacts with vinexin and the actin cytoskeleton.

  18. Post-translational modification of serine/threonine kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at lysine residue 97 directly inhibits kinase activity.

    PubMed

    Calamaras, Timothy D; Lee, Charlie; Lan, Fan; Ido, Yasuo; Siwik, Deborah A; Colucci, Wilson S

    2012-12-01

    Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE. PMID:23086944

  19. Role of Go/i subgroup of G proteins in olfactory signaling of Drosophila melanogaster

    PubMed Central

    Ignatious Raja, Jennifer S; Katanayeva, Natalya; Katanaev, Vladimir L; Galizia, C Giovanni

    2014-01-01

    Intracellular signaling in insect olfactory receptor neurons remains unclear, with both metabotropic and ionotropic components being discussed. Here, we investigated the role of heterotrimeric Go and Gi proteins using a combined behavioral, in vivo and in vitro approach. Specifically, we show that inhibiting Go in sensory neurons by pertussis toxin leads to behavioral deficits. We heterologously expressed the olfactory receptor dOr22a in human embryonic kidney cells (HEK293T). Stimulation with an odor led to calcium influx, which was amplified via calcium release from intracellular stores. Subsequent experiments indicated that the signaling was mediated by the Gβγ subunits of the heterotrimeric Go/i proteins. Finally, using in vivo calcium imaging, we show that Go and Gi contribute to odor responses both for the fast (phasic) as for the slow (tonic) response component. We propose a transduction cascade model involving several parallel processes, in which the metabotropic component is activated by Go and Gi, and uses Gβγ. PMID:24443946

  20. Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity.

    PubMed

    Sakuma, Tetsushi; Ochiai, Hiroshi; Kaneko, Takehito; Mashimo, Tomoji; Tokumasu, Daisuke; Sakane, Yuto; Suzuki, Ken-ichi; Miyamoto, Tatsuo; Sakamoto, Naoaki; Matsuura, Shinya; Yamamoto, Takashi

    2013-11-29

    Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.

  1. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  2. Reprofiled anthelmintics abate hypervirulent stationary-phase Clostridium difficile.

    PubMed

    Gooyit, Major; Janda, Kim D

    2016-09-16

    Prolonged use of broad-spectrum antibiotics disrupts the indigenous gut microbiota, which consequently enables toxigenic Clostridium difficile species to proliferate and cause infection. The burden of C. difficile infections was exacerbated with the outbreak of hypervirulent strains that produce copious amounts of enterotoxins and spores. In recent past, membrane-active agents have generated a surge of interest due to their bactericidal property with a low propensity for resistance. In this study, we capitalized on the antimicrobial property and low oral bioavailability of salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. By broth microdilution techniques, we determined the MIC values of the anthelmintics against 16 C. difficile isolates of defined PCR-ribotype. The anthelmintics broadly inhibited C. difficile growth in vitro via a membrane depolarization mechanism. Interestingly, the salicylanilides were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. The salicylanilides were poorly active against select gut commensals (Bacteroides, Bifidobacterium and Lactobacillus species), and were non-hemolytic and non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable properties for repositioning as anti-C. difficile agents.

  3. The first molluscan TRIM9 is involved in the negative regulation of NF-κB activity in the Hong Kong oyster, Crassostrea hongkongensis.

    PubMed

    Liu, Ying; Li, Jun; Wang, Fuxuan; Mao, Fan; Zhang, Yuehuan; Zhang, Yang; Yu, Ziniu

    2016-09-01

    TRIM proteins are a group of highly conserved proteins participating in a variety of biological processes such as regulation of development, apoptosis, and innate immunity. However, the functions of these proteins in the mollusk are still poorly understood. In the present study, a TRIM9 homolog (named ChTRIM9) was first identified from a transcript-ome library in the Hong Kong oyster Crassostrea hongkongensis. The full-length cDNA of ChTRIM9 is 2928 bp and has a predicted Open Reading Frame ORF) encoding 721 amino acids, encoding a putative 80.2 kDa protein. SMART analysis indicated that ChTRIM9 contains the three typical TRIM domains, a RING finger, two B-boxes, and a coiled-coil domain in the N-terminal region, whereas the C-terminal region contains a SPRY domain. qRT-PCR analysis revealed a ubiquitous presence of ChTRIM9, with the highest expression in the gills. Upon bacterial challenge in vivo, the ChTRIM9 transcripts in hemocytes were significantly down-regulated, indicating its involvement in signal transduction in immune response of oysters. Furthermore, ChTRIM9 was found to be localized mainly in the cytoplasm, and its over-expression inhibited the transcriptional activity of the NF-κB gene in HEK293T cells, demonstrating its negative role in regulating NF-κB signaling. PMID:27393236

  4. Synthesis of water-based cationic polyurethane for antibacterial and gene delivery applications.

    PubMed

    Wu, Geng-Hsi; Hsu, Shan-Hui

    2016-10-01

    Cationic polymers are often used as antimicrobial materials and transfection reagents. Water-based process could reduce environmental pollution and prevent the risk of solvent residue in the final product. In this study, waterborne biodegradable cationic polyurethane (WCPU) was synthesized by reacting polycaprolactone (PCL diol), isophorone diisocyanate (IPDI), and N-methyldiethanolamine (N-MDEA) under 75°C. An aqueous dispersion of WCPU nanoparticles (NPs) could be acquired by vigorous stirring under acidic condition. The particles in the dispersion had an average size of ∼80nm and a zeta potential of ∼60mV. When cast into films, the contact angle of the film was ∼67° and the zeta potential was ∼16mV. WCPU NPs demonstrated excellent antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (100% inhibition with a contact time of 3h). Meanwhile, the antibacterial ratio of WCPU films to E. coli and S. aureus reached 100% after 24h of contact. Moreover, WCPU NPs could be used as a transfection reagent without significant toxicity for concentrations less than 1000μg/mL and showed the ability to condensate plasmid DNA. The transfection efficiency for HEK293T cells and hBMSCs was ∼60% and ∼30% at 48h, respectively, after the transfection. Therefore, the WCPU synthesized in this study has potential antibacterial and gene delivery applications. PMID:27451371

  5. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    PubMed Central

    Bolduc, Véronique; Zou, Yaqun; Ko, Dayoung; Bönnemann, Carsten G

    2014-01-01

    Congenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi) as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA) oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT) construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies. PMID:24518369

  6. Influence of cationic lipid concentration on properties of lipid–polymer hybrid nanospheres for gene delivery

    PubMed Central

    Bose, Rajendran JC; Arai, Yoshie; Ahn, Jong Chan; Park, Hansoo; Lee, Soo-Hong

    2015-01-01

    Nanoparticles have been widely used for nonviral gene delivery. Recently, cationic hybrid nanoparticles consisting of two different materials were suggested as a promising delivery vehicle. In this study, nanospheres with a poly(d,l-lactic-co-glycolic acid) (PLGA) core and cationic lipid shell were prepared, and the effect of cationic lipid concentrations on the properties of lipid polymer hybrid nanocarriers investigated. Lipid–polymer hybrid nanospheres (LPHNSs) were fabricated by the emulsion-solvent evaporation method using different concentrations of cationic lipids and characterized for size, surface charge, stability, plasmid DNA-binding capacity, cytotoxicity, and transfection efficiency. All LPHNSs had narrow size distribution with positive surface charges (ζ-potential 52–60 mV), and showed excellent plasmid DNA-binding capacity. In vitro cytotoxicity measurements with HEK293T, HeLa, HaCaT, and HepG2 cells also showed that LPHNSs exhibited less cytotoxicity than conventional transfection agents, such as Lipofectamine and polyethyleneimine–PLGA. As cationic lipid concentrations increased, the particle size of LPHNSs decreased while their ζ-potential increased. In addition, the in vitro transfection efficiency of LPHNSs increased as lipid concentration increased. PMID:26379434

  7. Identification and primary immune characteristics of an amphioxus akirin homolog.

    PubMed

    Yan, Jie; Dong, Xuan; Kong, Yu; Zhang, Yan; Jing, Renwei; Feng, Lijun

    2013-08-01

    Akirin is a recently described nuclear protein that is thought to be required for the NF-κB signaling pathway in insects and vertebrates. Here, functional investigations of akirin are described in the basal chordate amphioxus Branchiostoma belcheri tsingtauense in an attempt to link this gene between insect and vertebrate lineages. Phylogenetic analysis indicated that amphioxus akirin represented a true ortholog of the two characterized vertebrate akirin paralogs. Amphioxus akirin, coding 219 amino acids with two nuclear localization signal (NLS) sequences and one 14-3-3 binding motif, was widely expressed in various tissues and up-regulated in response to Escherichia coli (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium) challenges. Furthermore, amphioxus akirin was strictly localized to the nucleus of HEK293T cells in a confocal analysis. Our work identified and characterized for the first time an amphioxus akirin homolog and will promote a better understanding of the evolution and transcriptional network of the akirin gene family. PMID:23732845

  8. Single nucleotide polymorphisms in the 3'UTR of VPAC-1 cooperate in modulating gene expression and impact differently on the interaction with miR525-5p.

    PubMed

    Paladini, Fabiana; Porciello, Nicla; Camilli, Giorgio; Tuncer, Sinem; Cocco, Elisa; Fiorillo, Maria Teresa; Sorrentino, Rosa

    2014-01-01

    Complex immune and neurodegenerative disorders are the result of multiple interactions between common genetic variations having, individually, a weak effect on the disease susceptibility or resistance. Interestingly, some genes have been found to be associated with more than one disease although not necessarily the same SNPs are involved. In this context, single nucleotide polymorphisms in the 3'UTR region of type 1 receptor (VPAC-1) for vasoactive intestinal peptide (VIP) have been reported to be associated with some immune-mediated as well as with neurodegenerative diseases such as Alzheimer's Disease (AD). Here, we demonstrate that variations at the 3'UTR of the VPAC-1 gene act synergistically to affect the expression of the luciferase as well as of the GFP reporter genes expressed in HEK293T cells. Moreover, the miRNA 525-5p, previously shown by us to target the 3'UTR of VPAC-1, is more efficient in decreasing GFP expression when co-expressed with constructs carrying the allele C at rs896 (p<10(-3)) suggesting that this miRNA regulates VPAC-1 expression at different levels depending on rs896 polymorphism and thus adding complexity to the network of disease susceptibility.

  9. In vitro secretion deficits are common among human coagulation factor XIII subunit B missense mutants: correlations with patient phenotypes and molecular models.

    PubMed

    Biswas, Arijit; Thomas, Anne; Bevans, Carville G; Ivaskevicius, Vytautas; Oldenburg, Johannes

    2013-11-01

    Coagulation factor XIII (FXIII) proenzyme circulates in plasma as a heterotetramer composed of two each of A and B subunits. Upon activation, the B subunits dissociate from the A subunit dimer, which gains transglutaminase activity to cross-link preformed fibrin clots increasing mechanical strength and resistance to degradation. The B subunits are thought to possess a carrier/protective function before FXIII activation. Mutations in either A or B subunits are associated with pathological patient phenotypes characterized by mild to severe bleeding. In vitro expression of FXIII B subunit (FXIIIB) missense variants in HEK293T cells revealed impaired secretion for all seven variants studied. To investigate the likely molecular environments of the missense residues, we created molecular models of individual FXIIIB Sushi domains using phylogenetically similar complement factor H Sushi domain structural templates. Assessment of the local molecular environments for the models suggested surface or buried positions for each mutant residue and possible pathological mechanisms. The in vitro expression system and in silico analytical methods and models we developed can be used to further investigate the molecular basis of FXIIIB mutation pathologies. PMID:23913518

  10. Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

    PubMed Central

    Magliozzi, Roberto; Peng, Mao; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J.R.; Low, Teck Yew

    2015-01-01

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882. PMID:26217795

  11. Rational design and synthesis of novel dibenzo[b,d]furan-1,2,3-triazole conjugates as potent inhibitors of Mycobacterium tuberculosis.

    PubMed

    Yempala, Thirumal; Sridevi, Jonnalagadda Padma; Yogeeswari, Perumal; Sriram, Dharmarajan; Kantevari, Srinivas

    2014-01-01

    A series of novel dibenzo[b,d]furan-1,2,3-triazole conjugates, rationally designed by reorientation of dibenzo[b,d]furan pharmacophore and alkyl/aryl groups appended on 1,2,3-triazole core, were synthesized using click chemistry. The required key intermediate, 2-ethynyl dibenzo[b,d]furan 3 was prepared from dibenzofuran-2-carboxaldehyde using Corey-Fuchs reaction. Further reaction of 3 with various alkyl/aryl azides in the presence of copper catalyst produced 1,2,3-triazole conjugates in excellent yields. Evaluation of all the new compounds for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC27294), resulted 5a (MIC: 1.56 μg/mL), 5d (MIC: 0.78 μg/mL) and 5f (MIC: 0.78 μg/mL) as promising lead analogues. Among these three compounds, 1-(4-bromobenzyl)-4-(dibenzo[b,d]furan-2-yl)-1H-1,2,3-triazole (5f) emerged as the most promising antitubercular agent with lowest cytotoxicity (selectivity index: ≫25) against the HEK-293T cell line. PMID:24292337

  12. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    PubMed

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  13. High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis.

    PubMed

    Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong

    2016-06-10

    HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. PMID:27154224

  14. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    PubMed

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  15. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

    PubMed Central

    Ukhanov, K.; Bobkov, Y.; Corey, E.A.; Ache, B.W.

    2014-01-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5 mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  16. Reprofiled anthelmintics abate hypervirulent stationary-phase Clostridium difficile

    PubMed Central

    Gooyit, Major; Janda, Kim D.

    2016-01-01

    Prolonged use of broad-spectrum antibiotics disrupts the indigenous gut microbiota, which consequently enables toxigenic Clostridium difficile species to proliferate and cause infection. The burden of C. difficile infections was exacerbated with the outbreak of hypervirulent strains that produce copious amounts of enterotoxins and spores. In recent past, membrane-active agents have generated a surge of interest due to their bactericidal property with a low propensity for resistance. In this study, we capitalized on the antimicrobial property and low oral bioavailability of salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. By broth microdilution techniques, we determined the MIC values of the anthelmintics against 16 C. difficile isolates of defined PCR-ribotype. The anthelmintics broadly inhibited C. difficile growth in vitro via a membrane depolarization mechanism. Interestingly, the salicylanilides were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. The salicylanilides were poorly active against select gut commensals (Bacteroides, Bifidobacterium and Lactobacillus species), and were non-hemolytic and non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable properties for repositioning as anti-C. difficile agents. PMID:27633064

  17. Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.

    PubMed

    Ping, Han; Liu, Xiaomei; Zhu, Dongqin; Li, Taiming; Zhang, Chun

    2015-04-01

    The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid.

  18. The first molluscan TRIM9 is involved in the negative regulation of NF-κB activity in the Hong Kong oyster, Crassostrea hongkongensis.

    PubMed

    Liu, Ying; Li, Jun; Wang, Fuxuan; Mao, Fan; Zhang, Yuehuan; Zhang, Yang; Yu, Ziniu

    2016-09-01

    TRIM proteins are a group of highly conserved proteins participating in a variety of biological processes such as regulation of development, apoptosis, and innate immunity. However, the functions of these proteins in the mollusk are still poorly understood. In the present study, a TRIM9 homolog (named ChTRIM9) was first identified from a transcript-ome library in the Hong Kong oyster Crassostrea hongkongensis. The full-length cDNA of ChTRIM9 is 2928 bp and has a predicted Open Reading Frame ORF) encoding 721 amino acids, encoding a putative 80.2 kDa protein. SMART analysis indicated that ChTRIM9 contains the three typical TRIM domains, a RING finger, two B-boxes, and a coiled-coil domain in the N-terminal region, whereas the C-terminal region contains a SPRY domain. qRT-PCR analysis revealed a ubiquitous presence of ChTRIM9, with the highest expression in the gills. Upon bacterial challenge in vivo, the ChTRIM9 transcripts in hemocytes were significantly down-regulated, indicating its involvement in signal transduction in immune response of oysters. Furthermore, ChTRIM9 was found to be localized mainly in the cytoplasm, and its over-expression inhibited the transcriptional activity of the NF-κB gene in HEK293T cells, demonstrating its negative role in regulating NF-κB signaling.

  19. Prostaglandin E receptor 4 (PTGER4) involved in host protection against immune challenge in oyster, Crassostrea hongkongensis.

    PubMed

    Qu, Fufa; Xiang, Zhiming; Wang, Fuxuan; Qi, Lin; Xu, Fengjiao; Xiao, Shu; Yu, Ziniu

    2015-02-01

    Prostaglandin E receptor 4 (PTGER4) is an essential receptor that can detect various physiological and pathological stimuli and has been implicated in a wide variety of biological processes, including the regulation of immune responses, cytokine production, and apoptosis. In this report, the first mollusk PTGER4, referred to as ChPTGER4, was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA is 1734 bp in length, including 5'- and 3'-untranslated region (UTRs) of 354 bp and 306 bp, respectively, and an open reading frame (ORF) of 1074 bp. ChPTGER4 comprises 357 amino acids and shares significant homology with its vertebrate homologs. The results of phylogenetic analysis revealed that ChPTGER4 clusters with PTGER4 from the Pacific oyster. In addition, quantitative real-time PCR analysis revealed that ChPTGER4 was constitutively expressed in all tissues examined and that its expression was significantly up-regulated in hemocytes and gills following challenge by pathogens (Vibrio alginolyticus, Staphylococcus haemolyticus and Saccharomyces cerevisiae) and pathogen-associated molecular patterns (PAMPs: lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, fluorescence microscopy analysis revealed that ChPTGER4 localized to the membrane, and its overexpression significantly enhanced NF-κB reporter gene activation in the HEK293T cell line. In summary, this study provides the first experimental evidence of a functional PTGER4 in mollusks, which suggests its involvement in the innate immune response in oyster.

  20. Activator protein-1 (AP-1) and response to pathogen infection in the Hong Kong oyster (Crassostrea hongkongensis).

    PubMed

    Xiang, Zhiming; Qu, Fufa; Li, Jun; Qi, Lin; Yang, Zhang; Kong, Xiaoyu; Yu, Ziniu

    2014-01-01

    Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells.

  1. Identification and function of an evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) from Crassostrea hongkongensis.

    PubMed

    Qu, Fufa; Xiang, Zhiming; Wang, Fuxuan; Zhang, Yang; Li, Jun; Zhang, Yuehuan; Xiao, Shu; Yu, Ziniu

    2015-11-01

    Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a multifunctional adaptor protein that plays a key role in the regulation of the oxidative phosphorylation (OXPHOS) system, bone morphogenetic protein (BMP) pathway and Toll-like receptor (TLR) signaling pathway in mammals. However, the function of ECSIT homologs in mollusks, the second most diverse group of animals, is not well understood. In this study, we identified an ECSIT homolog in the Hong Kong oyster Crassostrea hongkongensis (ChECSIT) and investigated its biological functions. The full-length cDNA of ChECSIT is 1734 bp and includes an open reading frame (ORF) of 1074 bp that encodes a polypeptide of 451 amino acids. The predicted ChECSIT protein shares similar structural characteristics with other known ECSIT family proteins. Quantitative real-time PCR analysis revealed that ChECSIT mRNA is broadly expressed in all of the examined tissues and at different stages of embryonic development; its transcript level could be significantly up-regulated by challenge with microorganisms (Vibrio alginolyticus, Staphylococcus haemolyticus and Saccharomyces cerevisiae). In addition, ChECSIT was found to be located primarily in the cytoplasm, and its overexpression stimulated the transcriptional activity of an NF-κB reporter gene in HEK293T cells. These findings suggest that ChECSIT might be involved in embryogenesis processes and immune responses in C. hongkongensis.

  2. Vps33b regulates Vwf-positive vesicular trafficking in megakaryocytes.

    PubMed

    Dai, Jing; Lu, Yeling; Wang, Conghui; Chen, Xue; Fan, Xuemei; Gu, Hao; Wu, Xiaolin; Wang, Kemin; Gartner, T Kent; Zheng, Junke; Chen, Guoqiang; Wang, Xuefeng; Liu, Junling

    2016-09-01

    Mutations of vacuolar protein sorting-associated protein 33b (VPS33B) cause arthrogryposis, renal dysfunction, and cholestasis syndrome, and a lack of platelet α-granules in the affected patients. Conditional Vps33b knockout mice were developed to investigate the function(s) of Vps33b in platelet α-granule formation. We found that early embryonic deletion of Vps33b was lethal. PF4-Cre-driven megakaryocyte-targeted Vps33b gene deletion greatly diminished Vps33b expression in platelets, but had no effect on platelet α-granule formation and protein content. Tamoxifen-induced, haematopoietic stem cell (HSC)-specific Vps33b deletion completely depleted Vps33b in platelets, caused the absence of α-granules, and increased the number of vacuoles in platelets and megakaryocytes. VPS33B association with VIPAS39, α-tubulin, and SEC22B was identified by co-immunoprecipitation, mass spectra, and immunoblotting in human embryonic kidney 293T (HEK293T) cells. Also, pull-down experiments revealed that VIPAS39 bound to intact VPS33B; in contrast, α-tubulin and SEC22B separately interacted with the sec1-like domains of VPS33B. Vps33b deficiency in megakaryocytes disturbs the redistribution of Vipas39 and Sec22b to proplatelets, and interrupted the co-localization of Sec22b with Vwf-positive vesicles. The data presented in this study suggest that Vps33b is involved in α-granule formation possibly by facilitating the Vwf-positive vesicular trafficking to α-granule-related vacuoles in megakaryocytes. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

    PubMed

    Zhang, Zhengxing; Kuo, Hsiou-Ting; Lau, Joseph; Jenni, Silvia; Zhang, Chengcheng; Zeisler, Jutta; Bénard, François; Lin, Kuo-Shyan

    2016-08-15

    Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers. PMID:27390067

  4. Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

    PubMed

    Zhang, Zhengxing; Kuo, Hsiou-Ting; Lau, Joseph; Jenni, Silvia; Zhang, Chengcheng; Zeisler, Jutta; Bénard, François; Lin, Kuo-Shyan

    2016-08-15

    Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

  5. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    PubMed Central

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  6. Transcriptional regulation of the human Liver X Receptor α gene by Hepatocyte Nuclear Factor 4α.

    PubMed

    Theofilatos, Dimitris; Anestis, Aristomenis; Hashimoto, Koshi; Kardassis, Dimitris

    2016-01-15

    Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. PMID:26692490

  7. The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in a JNK-dependent manner.

    PubMed

    Kumar, Ashok; Byun, Hoe-Sup; Bittman, Robert; Saba, Julie D

    2011-07-01

    The bioactive signaling molecule D-erythro-sphingosine-1-phosphate (S1P) is irreversibly degraded by the enzyme S1P lyase (SPL). The reaction of SPL with C18-S1P generates ethanolamine phosphate and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S1P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac1 and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S1P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling.

  8. Organometallic Titanocene–Gold Compounds as Potential Chemotherapeutics in Renal Cancer. Study of their Protein Kinase Inhibitory Properties

    PubMed Central

    2015-01-01

    Early–late transition metal TiAu2 compounds [(η-C5H5)2Ti{OC(O)CH2PPh2AuCl}2] (3) and new [(η-C5H5)2Ti{OC(O)-4-C6H4PPh2AuCl}2] (5) were evaluated as potential anticancer agents in vitro against renal and prostate cancer cell lines. The compounds were significantly more effective than monometallic titanocene dichloride and gold(I) [{HOC(O)RPPh2}AuCl] (R = −CH2– 6, −4-C6H4– 7) derivatives in renal cancer cell lines, indicating a synergistic effect of the resulting heterometallic species. The activity on renal cancer cell lines (for 5 in the nanomolar range) was considerably higher than that of cisplatin and highly active titanocene Y. Initial mechanistic studies in Caki-1 cells in vitro coupled with studies of their inhibitory properties on a panel of 35 kinases of oncological interest indicate that these compounds inhibit protein kinases of the AKT and MAPKAPK families with a higher selectivity toward MAPKAPK3 (IC503 = 91 nM, IC505 = 117 nM). The selectivity of the compounds in vitro against renal cancer cell lines when compared to a nontumorigenic human embryonic kidney cell line (HEK-293T) and the favorable preliminary toxicity profile on C57black6 mice indicate that these compounds (especially 5) are excellent candidates for further development as potential renal cancer chemotherapeutics. PMID:25435644

  9. The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region.

    PubMed

    Pulkki, Minna M; Myllymaa, Samu; Pasternack, Arja; Lun, Stanley; Ludlow, Helen; Al-Qahtani, Ahmed; Korchynskyi, Olexandr; Groome, Nigel; Juengel, Jennifer L; Kalkkinen, Nisse; Laitinen, Mika; Ritvos, Olli; Mottershead, David G

    2011-01-30

    Oocyte-derived bone morphogenetic protein-15 (BMP15) is critical for the regulation of mammalian fertility. Previously we have found that a C-terminal His(6)-tag destroys the bioactivity of growth differentiation-9 (GDF9, a homolog of BMP15). In this study we found that recombinant human BMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by C-terminal modification, specifically, fusion to a Flag tag. After purification the mature BMP15 wt is active in transcriptional reporter assays specific for Smad1/5/8 in human granulosa-luteal (hGL) and COV434 granulosa tumor cells, whereas BMP15 with a carboxy-terminal Flag tag remains inactive. Using these same cell models we found that treatment with purified mature BMP15 wt causes a rapid phosphorylation of Smad1. The purified BMP15 wt is a potent stimulator of rat granulosa cell DNA synthesis, which could be antagonized by the BMPRII ectodomain-Fc fusion molecule, whereas the BMP15C-Flag was completely inactive. Further, the BMP15 wt form is a potent stimulator of inhibin B production in hGL cells. We found that the purified BMP15 wt consists of P16 and -17, both of which are post-translationally modified forms. This is the first characterization of a purified untagged human BMP15 mature region, which is stable and highly bioactive in human and rodent granulosa cells and as such is of importance for studies on human fertility.

  10. Metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate.

    PubMed

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling, while LRRK2 is implicated in the pathogenesis of Parkinson's disease. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with (32)P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing (32)P-orthophosphate. The (32)P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography ((32)P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  11. Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

    SciTech Connect

    Duangtum, Natapol; Junking, Mutita; Sawasdee, Nunghathai; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2011-09-16

    Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.

  12. Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

    PubMed Central

    Taymans, Jean-Marc; Gao, Fangye; Baekelandt, Veerle

    2013-01-01

    Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation. PMID:24084685

  13. The Copper Transporter 1 (CTR1) is Required to Maintain the Stability of Copper Transporter 2 (CTR2)

    PubMed Central

    Tsai, Cheng-Yu; Liebig, Janika K.; Tsigelny, Igor F.; Howell, Stephen B.

    2015-01-01

    Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteosome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteosome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system. PMID:26205368

  14. The copper transporter 1 (CTR1) is required to maintain the stability of copper transporter 2 (CTR2).

    PubMed

    Tsai, Cheng-Yu; Liebig, Janika K; Tsigelny, Igor F; Howell, Stephen B

    2015-11-01

    Mammalian cells have two influx Cu transporters that form trimers in membranes. CTR1 is the high affinity transporter that resides largely in the plasma membrane, and CTR2 is the low affinity transporter that is primarily associated with vesicular structures inside the cell. The major differences between CTR1 and CTR2 are that CTR1 contains a HIS/MET-rich domain N-terminal of the METS that participate in the first two stacked rings that form the pore, and a longer C-terminal tail that includes a Cu binding HIS-CYS-HIS (HCH) motif right at the end. It has been reported that CTR1 and CTR2 are physically associated with each other in the cell. We used the CRISPR-Cas9 technology to knock out either CTR1 or CTR2 in fully malignant HEK293T and OVCAR8 human ovarian cancer cells to investigate the interaction of CTR1 and CTR2. We report here that the level of CTR2 protein is markedly decreased in CTR1 knockout clones while the CTR2 transcript level remains unchanged. CTR2 was found to be highly ubiquitinated in the CTR1 knock out cells, and inhibition of the proteasome prevented the degradation of CTR2 when CTR1 was not present while inhibition of autophagy had no effect. Re-expression of CTR1 rescued CTR2 from degradation in the CTR1 knockout cells. We conclude that CTR1 is essential to maintain the stability of CTR2 and that in the absence of CTR1 CTR2 is degraded by the proteasome. This reinforces the concept that the functions of CTR1 and CTR2 are inter-dependent within the Cu homeostasis system.

  15. Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels.

    PubMed

    Ruchala, Iwona; Cabra, Vanessa; Solis, Ernesto; Glennon, Richard A; De Felice, Louis J; Eltit, Jose M

    2014-07-01

    Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells. PMID:24854234

  16. Src family protein tyrosine kinase regulates the basolateral K channel in the distal convoluted tubule (DCT) by phosphorylation of KCNJ10 protein.

    PubMed

    Zhang, Chengbiao; Wang, Lijun; Thomas, Sherin; Wang, Kemeng; Lin, Dao-Hong; Rinehart, Jesse; Wang, Wen-Hui

    2013-09-01

    The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba(2+)-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from -65 to -43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr(8) and Tyr(9). The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr(9) did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr(9) on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

  17. Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

    PubMed Central

    Aslam, Muhammad; Paddenberg, Renate; Quanz, Karin; Chang, Chia L.; Park, Jae-Il; Gries, Barbara; Rafiq, Amir; Faulhammer, Petra; Goldenberg, Anna; Papadakis, Tamara; Noll, Thomas; Hsu, Sheau Y. T.; Weissmann, Norbert; Kummer, Wolfgang

    2009-01-01

    Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1α on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1α enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage. PMID:19684198

  18. An NF-κB-Based High-Throughput Screen Identifies Piericidins as Inhibitors of the Yersinia pseudotuberculosis Type III Secretion System

    PubMed Central

    Duncan, Miles C.; Wong, Weng Ruh; Dupzyk, Allison J.; Bray, Walter M.; Linington, Roger G.

    2014-01-01

    The type III secretion system (T3SS) is a bacterial appendage used by dozens of Gram-negative pathogens to subvert host defenses and cause disease, making it an ideal target for pathogen-specific antimicrobials. Here, we report the discovery and initial characterization of two related natural products with T3SS-inhibitory activity that were derived from a marine actinobacterium. Bacterial extracts containing piericidin A1 and the piericidin derivative Mer-A 2026B inhibited Yersinia pseudotuberculosis from triggering T3SS-dependent activation of the host transcription factor NF-κB in HEK293T cells but were not toxic to mammalian cells. As the Yersinia T3SS must be functional in order to trigger NF-κB activation, these data indicate that piericidin A1 and Mer-A 2026B block T3SS function. Consistent with this, purified piericidin A1 and Mer-A 2026B dose-dependently inhibited translocation of the Y. pseudotuberculosis T3SS effector protein YopM inside CHO cells. In contrast, neither compound perturbed bacterial growth in vitro, indicating that piericidin A1 and Mer-A 2026B do not function as general antibiotics in Yersinia. In addition, when Yersinia was incubated under T3SS-inducing culture conditions in the absence of host cells, Mer-A 2026B and piericidin A1 inhibited secretion of T3SS cargo as effectively as or better than several previously described T3SS inhibitors, such as MBX-1641 and aurodox. This suggests that Mer-A 2026B and piericidin A1 do not block type III secretion by blocking the bacterium-host cell interaction, but rather inhibit an earlier stage, such as T3SS needle assembly. In summary, the marine-derived natural products Mer-A 2026B and piericidin A1 possess previously uncharacterized activity against the bacterial T3SS. PMID:24295981

  19. SUR2 subtype (A and B)-dependent differential activation of the cloned ATP-sensitive K+ channels by pinacidil and nicorandil

    PubMed Central

    Shindo, Takashi; Yamada, Mitsuhiko; Isomoto, Shojiro; Horio, Yoshiyuki; Kurachi, Yoshihisa

    1998-01-01

    The classical ATP sensitive K+ (KATP) channels are composed of a sulphonylurea receptor (SUR) and an inward rectifying K+ channel subunit (BIR/Kir6.2). They are the targets of vasorelaxant agents called K+ channel openers, such as pinacidil and nicorandil.In order to examine the tissue selectivity of pinacidil and nicorandil, in vitro, we compared the effects of these agents on cardiac type (SUR2A/Kir6.2) and vascular smooth muscle type (SUR2B/Kir6.2) of the KATP channels heterologously expressed in HEK293T cells, a human embryonic kidney cell line, by using the patch-clamp method.In the cell-attached recordings (145 mM K+ in the pipette), pinacidil and nicorandil activated a weakly inwardly-rectifying, glibenclamide-sensitive 80 pS K+ channel in both the transfected cells.In the whole-cell configuration, pinacidil showed a similar potency in activating the SUR2B/Kir6.2 and SUR2A/Kir6.2 channels (EC50 of ∼2 and ∼10 μM, respectively). On the other hand, nicorandil activated the SUR2B/Kir6.2 channel >100 times more potently than the SUR2A/Kir6.2 (EC50 of ∼10 μM and >500 μM, respectively).Thus, nicorandil, but not pinacidil, preferentially activates the KATP channels containing SUR2B. Because SUR2A and SUR2B are diverse only in 42 amino acids at their C-terminal ends, it is strongly suggested that this short part of SUR2B may play a critical role in the action of nicorandil on the vascular type classical KATP channel. PMID:9692785

  20. Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner.

    PubMed

    Kakumani, Pavan Kumar; Rajgokul, K S; Ponia, Sanket Singh; Kaur, Inderjeet; Mahanty, Srikrishna; Medigeshi, Guruprasad R; Banerjea, Akhil C; Chopra, Arun Prasad; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2015-10-01

    RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication.

  1. Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

    PubMed

    Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

    2016-03-01

    This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to

  2. DuOx2 Promoter Regulation by Hormones, Transcriptional Factors and the Coactivator TAZ.

    PubMed

    Cardoso-Weide, L C; Cardoso-Penha, R C; Costa, M W; Ferreira, A C F; Carvalho, D P; Santisteban, P S

    2015-03-01

    The production of H2O2, which is essential to thyroid hormone synthesis, involves two NADPH oxidases: dual oxidases 1 and 2 (DuOx1 and DuOx2). A functional study with human DuOx genes and their 5'-flanking regions showed that DuOx1 and -2 promoters are different from thyroid-specific gene promoters. Furthermore, their transcriptional activities are not restricted to thyroid cells. While regulation of Tg (thyroglobulin) and TPO (thyroperoxidase) expression have been extensively studied, DuOx2 promoter regulation by hormones and transcriptional factors need to be more explored. Herein we investigated the role of TSH, insulin and insulin-like growth factor 1 (IGF-1), as well as the cAMP effect on DuOx2 promoter (ptx41) activity in transfected rat thyroid