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Sample records for engineering intracellular active

  1. Engineering intracellular active transport systems as in vivo biomolecular tools.

    SciTech Connect

    Bachand, George David; Carroll-Portillo, Amanda

    2006-11-01

    Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptional regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further

  2. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  3. Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility.

    PubMed

    Ng, Chee Ping; Goodman, Thomas T; Park, In-Kyu; Pun, Suzie H

    2009-02-01

    Intracellular transport after endosomal escape presents one of the major barriers for efficient non-viral gene delivery because plasmid DNA and synthetic nanoparticulate carriers suffer from significantly restricted diffusion in the cytoplasm. We postulate that forces generated by actin polymerization, a mechanism used by several bacterial pathogens such as Listeria monocytogenes, can be harnessed to propel nanoparticles within the cytoplasm and thereby overcome diffusional limitations associated with gene transport in the cell cytoplasm. In this work, we synthesized and characterized plasmid DNA-containing nanoparticles modified with ActA protein, the single protein in L. monocytogenes responsible for activating actin polymerization and initiating actin comet-tail propulsion. The motility of the ActA-modified nanoparticles was assessed in Xenopus laevis cytoplasmic extract supplemented with fluorescently labeled actin. Nanoparticle motility was monitored using multi-color, time-lapse fluorescence microscopy for the formation of actin comet tails attached to the fluorescently labeled vehicle. We observed particle motility with velocities approximately 0.06 microm/s with anionic-charged plasmid carriers formed from either poly(lactic-co-glycolic acid) (PLGA) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, but interestingly not with cationic particles assembled by encapsulation of plasmid with either polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) lipids. Control particles coated with albumin instead of ActA also showed no motility. Taken together, we have demonstrated the feasibility of translating the comet-tail propulsion mechanism to synthetic drug carriers as a potential approach to overcome intracellular transport barriers, and also have identified appropriate gene delivery systems that can be employed for this mechanism.

  4. Engineering of obligate intracellular bacteria: progress, challenges and paradigms.

    PubMed

    McClure, Erin E; Chávez, Adela S Oliva; Shaw, Dana K; Carlyon, Jason A; Ganta, Roman R; Noh, Susan M; Wood, David O; Bavoil, Patrik M; Brayton, Kelly A; Martinez, Juan J; McBride, Jere W; Valdivia, Raphael H; Munderloh, Ulrike G; Pedra, Joao H F

    2017-09-01

    It is estimated that approximately one billion people are at risk of infection with obligate intracellular bacteria, but little is known about the underlying mechanisms that govern their life cycles. The difficulty in studying Chlamydia spp., Coxiella spp., Rickettsia spp., Anaplasma spp., Ehrlichia spp. and Orientia spp. is, in part, due to their genetic intractability. Recently, genetic tools have been developed; however, optimizing the genomic manipulation of obligate intracellular bacteria remains challenging. In this Review, we describe the progress in, as well as the constraints that hinder, the systematic development of a genetic toolbox for obligate intracellular bacteria. We highlight how the use of genetically manipulated pathogens has facilitated a better understanding of microbial pathogenesis and immunity, and how the engineering of obligate intracellular bacteria could enable the discovery of novel signalling circuits in host-pathogen interactions.

  5. Activities of Antimicrobial Agents against Intracellular Pneumococci

    PubMed Central

    Mandell, Gerald L.; Coleman, Elizabeth J.

    2000-01-01

    Pneumococci can enter and survive inside human lung alveolar carcinoma cells. We examined the activity of azithromycin, gentamicin, levofloxacin, moxifloxacin, penicillin G, rifampin, telithromycin, and trovafloxacin against pneumococci inside and outside cells. We found that moxifloxacin, trovafloxacin, and telithromycin were the most active, but only telithromycin killed all intracellular organisms. PMID:10952618

  6. Intracellular angiotensin II activates rat myometrium

    PubMed Central

    Deliu, Elena; Tica, Andrei A.; Motoc, Dana; Brailoiu, G. Cristina

    2011-01-01

    Angiotensin II is a modulator of myometrial activity; both AT1 and AT2 receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca2+ concentration ([Ca2+]i) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT1 receptor antagonist losartan but not by the AT2 receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca2+]i. Blockade of AT1 receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca2+]i produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca2+-free saline, indicating a major involvement of Ca2+ release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP3) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP3 pathway. Angiotensin II-induced increase in [Ca2+]i was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT1-like receptors on lysosomes and activates PLC-IP3-dependent Ca2+ release from endoplasmic reticulum; the response is further augmented by a Ca2+-induced Ca2+ release mechanism via ryanodine receptors activation. PMID:21613610

  7. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  8. Intracellular mechanisms of lymphoid cell activation.

    PubMed

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells.

  9. Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

    PubMed Central

    García, Isabel; Pascual, Alvaro; Ballesta, Sofía; Joyanes, Providencia; Perea, Evelio J.

    2000-01-01

    The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus. PMID:11036051

  10. Engineering intracellular biomineralization and biosensing by a magnetic protein

    PubMed Central

    Matsumoto, Yuri; Chen, Ritchie; Anikeeva, Polina; Jasanoff, Alan

    2015-01-01

    Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 107 variants induces threefold greater cellular iron loading than mammalian heavy chain Ft, over fivefold higher contrast in magnetic resonance imaging, and robust retention on magnetic separation columns. Mechanistic studies of mutant Ft proteins indicate that improved magnetism arises in part from increased iron oxide nucleation efficiency. Molecular-level iron loading in engineered Ft enables detection of individual particles inside cells and facilitates creation of Ft-based intracellular magnetic devices. We demonstrate construction of a magnetic sensor actuated by gene expression in yeast. PMID:26522873

  11. Engineering intracellular biomineralization and biosensing by a magnetic protein.

    PubMed

    Matsumoto, Yuri; Chen, Ritchie; Anikeeva, Polina; Jasanoff, Alan

    2015-11-02

    Remote measurement and manipulation of biological systems can be achieved using magnetic techniques, but a missing link is the availability of highly magnetic handles on cellular or molecular function. Here we address this need by using high-throughput genetic screening in yeast to select variants of the iron storage ferritin (Ft) that display enhanced iron accumulation under physiological conditions. Expression of Ft mutants selected from a library of 10(7) variants induces threefold greater cellular iron loading than mammalian heavy chain Ft, over fivefold higher contrast in magnetic resonance imaging, and robust retention on magnetic separation columns. Mechanistic studies of mutant Ft proteins indicate that improved magnetism arises in part from increased iron oxide nucleation efficiency. Molecular-level iron loading in engineered Ft enables detection of individual particles inside cells and facilitates creation of Ft-based intracellular magnetic devices. We demonstrate construction of a magnetic sensor actuated by gene expression in yeast.

  12. Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications.

    PubMed

    Gaur, Shuchi; Bhargava-Shah, Aarohi; Hori, Sharon; Afjei, Rayhaneh; Sekar, Thillai V; Gambhir, Sanjiv S; Massoud, Tarik F; Paulmurugan, Ramasamy

    2017-09-15

    Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention both by modifying the N-terminal secretory signal peptide and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminus of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors

  13. Impact of Photosensitizers Activation on Intracellular Trafficking and Viscosity

    PubMed Central

    Aubertin, Kelly; Bonneau, Stéphanie; Silva, Amanda K. A.; Bacri, Jean-Claude; Gallet, François; Wilhelm, Claire

    2013-01-01

    The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact. PMID:24386423

  14. Impact of photosensitizers activation on intracellular trafficking and viscosity.

    PubMed

    Aubertin, Kelly; Bonneau, Stéphanie; Silva, Amanda K A; Bacri, Jean-Claude; Gallet, François; Wilhelm, Claire

    2013-01-01

    The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact.

  15. Uptake and intracellular activity of fluconazole in human polymorphonuclear leukocytes.

    PubMed Central

    Pascual, A; García, I; Conejo, C; Perea, E J

    1993-01-01

    The penetration of fluconazole into human polymorphonuclear leukocytes (PMNs) and tissue culture epithelial cells (McCoy) was evaluated. At different extracellular concentrations (0.5 to 10 mg/liter), fluconazole reached cell-associated concentrations greater than the extracellular ones in either human PMNs (intracellular concentration to extracellular concentration ratio, > or = 2.2) or McCoy cells (intracellular concentration to extracellular concentration ratio, > or = 1.3). The uptake of fluconazole by PMNs was rapid and reversible but was not energy dependent. The intracellular penetration of fluconazole was not affected by environmental pH or temperature. Ingestion of opsonized zymosan and opsonized Candida albicans did not significantly increase the amount of PMN-associated fluconazole. At therapeutic extracellular concentrations, the intracellular activity of fluconazole against C. albicans in PMNs was significantly lower than that of amphotericin B. It was concluded that fluconazole reaches high intracellular concentrations within PMNs but shows moderate activity against intracellular C. albicans in vitro. PMID:8452347

  16. Intracellular activity of azithromycin against bacterial enteric pathogens.

    PubMed Central

    Rakita, R M; Jacques-Palaz, K; Murray, B E

    1994-01-01

    Azithromycin, a new azalide antibiotic, is active in vitro against a variety of enteric bacterial pathogens. Since it is concentrated inside human neutrophils and other cells, it might be particularly useful in the treatment of infections caused by enteropathogens that invade host tissues. The intracellular activity of azithromycin against several enteric pathogens that had been phagocytosed by neutrophils was determined. Azithromycin was effective in reducing the intracellular viabilities of almost all strains tested, including representative strains of Salmonella, Shigella, and enteroinvasive, enteropathogenic, enterotoxigenic, and enterohemorrhagic Escherichia coli. Erythromycin was also effective in this model system, although azithromycin was generally more effective than erythromycin against strains of invasive enteric pathogens. Cefotaxime reduced intracellular bacterial viability to a lesser extent than either azithromycin or erythromycin. The presence of neutrophils did not significantly affect the activity of azithromycin in this system. Azithromycin may be a useful agent for the treatment of bacterial diarrhea, and clinical trials should be considered. PMID:7810998

  17. Utilizing Natural and Engineered Peroxiredoxins As Intracellular Peroxide Reporters.

    PubMed

    Van Laer, Koen; Dick, Tobias P

    2016-01-01

    It is increasingly apparent that nature evolved peroxiredoxins not only as H2O2 scavengers but also as highly sensitive H2O2 sensors and signal transducers. Here we ask whether the H2O2 sensing role of Prx can be exploited to develop probes that allow to monitor intracellular H2O2 levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular H2O2 concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of H2O2 levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redox-sensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for H2O2 probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular H2O2 homeostasis and Prx signaling.

  18. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery

    PubMed Central

    Wang, Ming; Altinoglu, Sarah; Takeda, Yuji S.; Xu, Qiaobing

    2015-01-01

    Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes. PMID:26529317

  19. Subcompartmentalized Nanoreactors as Artificial Organelle with Intracellular Activity.

    PubMed

    Thingholm, Bo; Schattling, Philipp; Zhang, Yan; Städler, Brigitte

    2016-04-06

    Cell mimicry is an approach which aims at substituting missing or lost activity. In this context, the goal of artificial organelles is to provide intracellularly active nanoreactors to affect the cellular performance. So far, only a handful of reports discuss concepts addressing this challenge based on single-component reactors. Here, the assembly of nanoreactors equipped with glucose oxidase (GOx)-loaded liposomal subunits coated with a poly(dopamine) polymer layer and RGD targeting units is reported. When comparing different surface modifications, the uptake of the nanoreactors by endothelial cells and macrophages with applied shear stress is confirmed without inherent cytotoxicity. Furthermore, the encapsulation and preserved activity of GOx within the nanoreactors is shown. The intracellular activity is demonstrated by exposing macrophages with internalized nanoreactors to glucose and assessment of the cell viability after 6 and 24 h. The macrophage viability is found to be reduced due to the intracellularly produced hydrogen peroxide by GOx. This report on the first intracellular active subcompartmentalized nanoreactors is a considerable step in therapeutic cell mimicry. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  1. Activity of 10 antimicrobial agents against intracellular Rhodococcus equi.

    PubMed

    Giguère, Steeve; Berghaus, Londa J; Lee, Elise A

    2015-08-05

    Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocyte-derived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Engineers and Active Responsibility.

    PubMed

    Pesch, Udo

    2015-08-01

    Knowing that technologies are inherently value-laden and systemically interwoven with society, the question is how individual engineers can take up the challenge of accepting the responsibility for their work? This paper will argue that engineers have no institutional structure at the level of society that allows them to recognize, reflect upon, and actively integrate the value-laden character of their designs. Instead, engineers have to tap on the different institutional realms of market, science, and state, making their work a 'hybrid' activity combining elements from the different institutional realms. To deal with this institutional hybridity, engineers develop routines and heuristics in their professional network, which do not allow societal values to be expressed in a satisfactory manner. To allow forms of 'active' responsibility, there have to be so-called 'accountability forums' that guide moral reflections of individual actors. The paper will subsequently look at the methodologies of value-sensitive design (VSD) and constructive technology assessment (CTA) and explore whether and how these methodologies allow engineers to integrate societal values into the design technological artifacts and systems. As VSD and CTA are methodologies that look at the process of technological design, whereas the focus of this paper is on the designer, they can only be used indirectly, namely as frameworks which help to identify the contours of a framework for active responsibility of engineers.

  3. Nature's engines: active matter

    NASA Astrophysics Data System (ADS)

    Yeomans, Julia M.

    2017-03-01

    Active materials, bacteria, molecular motors, and self-propelled colloids, continuously transform chemical energy from the environment to mechanical work. Dense active matter, from layers of cells to flocks of birds, self-assembles into intricate patterns. Nature's engines are complex and efficient, and we would like to exploit her ideas to fabricate nano-machines.

  4. Intracellular water motion decreases in apoptotic macrophages after caspase activation.

    PubMed

    Hortelano, S; García-Martín, M L; Cerdán, S; Castrillo, A; Alvarez, A M; Boscá, L

    2001-10-01

    Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.

  5. Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

    PubMed Central

    Blair, Nathaniel T.; Kaczmarek, J. Stefan

    2009-01-01

    TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation. PMID:19398778

  6. Anhydrobiotic engineering of bacterial and mammalian cells: is intracellular trehalose sufficient?

    PubMed

    Tunnacliffe, A; García de Castro, A; Manzanera, M

    2001-09-01

    Anhydrobiotic engineering aims to confer a high degree of desiccation tolerance on otherwise sensitive living organisms and cells by adopting the strategies of anhydrobiosis. Nonreducing disaccharides such as trehalose and sucrose are thought to play a pivotal role in resistance to desiccation stress in many microorganisms, invertebrates, and plants, and in vitro trehalose is known to confer stability on dried biomolecules and biomembranes. We have therefore tested the hypothesis that intracellular trehalose (or a similar molecule) may be not only necessary for anhydrobiosis but also sufficient. High concentrations of trehalose were produced in bacteria by osmotic preconditioning, and in mammalian cells by genetic engineering, but in neither system was desiccation tolerance similar to that seen in anhydrobiotic organisms, suggesting that trehalose alone is not sufficient for anhydrobiosis. In Escherichia coli such desiccation tolerance was achievable, but only when bacteria were dried in the presence of both extracellular trehalose and intracellular trehalose. In mouse L cells, improved osmotolerance was observed with up to 100 mM intracellular trehalose, but desiccation was invariably lethal even with extracellular trehalose present. We conclude that anhydrobiotic engineering of at least some microorganisms is achievable with present technology, but that further advances are needed for similar desiccation tolerance of mammalian cells. Copyright 2001 Elsevier Science (USA).

  7. Intracellular chemiluminescence activates targeted photodynamic destruction of leukaemic cells

    PubMed Central

    Laptev, R; Nisnevitch, M; Siboni, G; Malik, Z; Firer, M A

    2006-01-01

    Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps target-cell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf–Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf–Hp at intracellular chemiluminescence. This strategy was then used to obviate the use of external radiation for Ps activation by incubating the cells with luminol either before or together with Tf–Hp. This novel chemical means of PDT activation induced cytotoxicity in 95% of cells. These combined approaches provide an opportunity to develop broader and more effective applications of PDT. PMID:16819545

  8. Structural rearrangement of the intracellular domains during AMPA receptor activation

    PubMed Central

    Zachariassen, Linda G.; Katchan, Ljudmila; Jensen, Anna G.; Pickering, Darryl S.; Plested, Andrew J. R.

    2016-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  9. Engineering a growth sensor to select intracellular antibodies in the cytosol of mammalian cells.

    PubMed

    Nguyen, Thuy Duong; Takasuka, Hitoshi; Kaku, Yoshihiro; Inoue, Satoshi; Nagamune, Teruyuki; Kawahara, Masahiro

    2017-03-16

    Intracellular antibodies (intrabodies) are expected to function as therapeutics as well as tools for elucidating in vivo function of proteins. In this study, we propose a novel intrabody selection method in the cytosol of mammalian cells by utilizing a growth signal, induced by the interaction of the target antigen and an scFv-c-kit growth sensor. Here, we challenge this method to select specific intrabodies against rabies virus nucleoprotein (RV-N) for the first time. As a result, we successfully select antigen-specific intrabodies from a naïve synthetic library using phage panning followed by our growth sensor-based intracellular selection method, demonstrating the feasibility of the method. Additionally, we succeed in improving the response of the growth sensor by re-engineering the linker region of its construction. Collectively, the described selection method utilizing a growth sensor may become a highly efficient platform for selection of functional intrabodies in the future.

  10. Enzyme-activated intracellular drug delivery with tubule clay nanoformulation

    NASA Astrophysics Data System (ADS)

    Dzamukova, Maria R.; Naumenko, Ekaterina A.; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2015-05-01

    Fabrication of stimuli-triggered drug delivery vehicle s is an important milestone in treating cancer. Here we demonstrate the selective anticancer drug delivery into human cells with biocompatible 50-nm diameter halloysite nanotube carriers. Physically-adsorbed dextrin end stoppers secure the intercellular release of brilliant green. Drug-loaded nanotubes penetrate through the cellular membranes and their uptake efficiency depends on the cells growth rate. Intercellular glycosyl hydrolases-mediated decomposition of the dextrin tube-end stoppers triggers the release of the lumen-loaded brilliant green, which allowed for preferable elimination of human lung carcinoma cells (A549) as compared with hepatoma cells (Hep3b). The enzyme-activated intracellular delivery of brilliant green using dextrin-coated halloysite nanotubes is a promising platform for anticancer treatment.

  11. Diverse intracellular pathogens activate type III interferon expression from peroxisomes.

    PubMed

    Odendall, Charlotte; Dixit, Evelyn; Stavru, Fabrizia; Bierne, Helene; Franz, Kate M; Durbin, Ann Fiegen; Boulant, Steeve; Gehrke, Lee; Cossart, Pascale; Kagan, Jonathan C

    2014-08-01

    Type I interferon responses are considered the primary means by which viral infections are controlled in mammals. Despite this view, several pathogens activate antiviral responses in the absence of type I interferons. The mechanisms controlling type I interferon-independent responses are undefined. We found that RIG-I like receptors (RLRs) induce type III interferon expression in a variety of human cell types, and identified factors that differentially regulate expression of type I and type III interferons. We identified peroxisomes as a primary site of initiation of type III interferon expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust type III interferon responses in human cells. These findings highlight the importance of different intracellular organelles in specific innate immune responses.

  12. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  13. Chemical Engineering Division Activities

    ERIC Educational Resources Information Center

    Chemical Engineering Education, 1978

    1978-01-01

    The 1978 ASEE Chemical Engineering Division Lecturer was Theodore Vermeulen of the University of California at Berkeley. Other chemical engineers who received awards or special recognition at a recent ASEE annual conference are mentioned. (BB)

  14. The Mechanical Environment Modulates Intracellular Calcium Oscillation Activities of Myofibroblasts

    PubMed Central

    Godbout, Charles; Follonier Castella, Lysianne; Smith, Eric A.; Talele, Nilesh; Chow, Melissa L.; Garonna, Adriano; Hinz, Boris

    2013-01-01

    Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair. PMID:23691248

  15. Platelet activating factor raises intracellular calcium ion concentration in macrophages

    PubMed Central

    1986-01-01

    Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in

  16. Engineering Salmonella as intracellular factory for effective killing of tumour cells.

    PubMed

    Camacho, Eva María; Mesa-Pereira, Beatriz; Medina, Carlos; Flores, Amando; Santero, Eduardo

    2016-07-28

    Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria. Here we have developed an inducible autolysis system based in the lysis operon of the lambda phage that, in response to anhydrotetracycline, lysates Salmonella thus releasing its content. The system was combined with a salicylate cascade system that allows efficient production of therapeutic molecules in response to aspirin and with a sifA mutation that liberates bacteria from the vacuoles to a cytosolic location. The combination of these three elements makes this strain a putative powerful instrument in cancer treatment. We have used this engineered strain for the intracellular production and delivery of Cp53 peptide. The engineered strain is able to sequentially produce and release the cytotoxic peptide while proliferating inside tumour cells, thus inducing host cell death. Our results show that temporal separation of protein production from protein release is essential to efficiently kill tumour cells. The combined system is a further step in the engineering of more efficient bacteria for cancer therapy.

  17. Engineering Salmonella as intracellular factory for effective killing of tumour cells

    PubMed Central

    Camacho, Eva María; Mesa-Pereira, Beatriz; Medina, Carlos; Flores, Amando; Santero, Eduardo

    2016-01-01

    Salmonella have many desirable properties as antitumour-agent due to its ability to proliferate inside tumours and induce tumour regression. Additionally, this bacterium can be genetically engineered to deliver therapeutic proteins intratumourally. The main limitation of this approach is the efficient release of therapeutic molecules from intratumoural bacteria. Here we have developed an inducible autolysis system based in the lysis operon of the lambda phage that, in response to anhydrotetracycline, lysates Salmonella thus releasing its content. The system was combined with a salicylate cascade system that allows efficient production of therapeutic molecules in response to aspirin and with a sifA mutation that liberates bacteria from the vacuoles to a cytosolic location. The combination of these three elements makes this strain a putative powerful instrument in cancer treatment. We have used this engineered strain for the intracellular production and delivery of Cp53 peptide. The engineered strain is able to sequentially produce and release the cytotoxic peptide while proliferating inside tumour cells, thus inducing host cell death. Our results show that temporal separation of protein production from protein release is essential to efficiently kill tumour cells. The combined system is a further step in the engineering of more efficient bacteria for cancer therapy. PMID:27464652

  18. Activation of the Homeostatic Intracellular Repair Response during Cardiac Surgery

    PubMed Central

    Jahania, Salik M.; Sengstock, David; Vaitkevicius, Peter; Andres, Allen; Ito, Bruce R.; Gottlieb, Roberta A.; Mentzer, Robert M.

    2013-01-01

    Introduction The homeostatic intracellular repair response (HIR2) is an endogenous beneficial pathway that eliminates damaged mitochondria and dysfunctional proteins in response to stress. The underlying mechanism is adaptive autophagy. The purpose of this study was to determine whether the HIR2 response is activated in the heart in patients undergoing cardiac surgery and to assess whether it is associated with the duration of ischemic arrest and predicted surgical outcome. Methods Autophagy was assessed in 19 patients undergoing coronary artery bypass or valve surgery requiring cardiopulmonary bypass (CPB). Biopsies of the right atrial appendage obtained before initiation of CPB and after weaning from CPB were analyzed for autophagy by immunoblotting for LC3, Beclin-1, Atg5-12, and p62. Changes in p62, a marker of autophagic flux, were correlated with duration of ischemia and with the mortality/morbidity risk scores obtained from the Society of Thoracic Surgeons Adult Cardiac Surgery Database (v2.73). Results Heart surgery was associated with a robust increase in autophagic flux indicated by depletion of LC3-I, LC3-II, Beclin-1, and Atg5-12; the magnitude of change for each of these factors correlated significantly with changes in the flux marker p62. Moreover, changes in p62 correlated directly with cross clamp time and inversely with the mortality/morbidity risk scores. Conclusion These findings are consistent with preclinical studies indicating that HIR2 is cardioprotective, and reveal that it is activated in patients in response to myocardial ischemic stress. Strategies designed to amplify HIR2 during conditions of cardiac stress may have therapeutic utility and represent an entirely new approach to myocardial protection in patients undergoing heart surgery. PMID:23415552

  19. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

    PubMed

    Wang, Meng; Li, Sijin; Zhao, Huimin

    2016-01-01

    The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

  20. Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins

    PubMed Central

    Butler, David C.; McLear, Julie A.; Messer, Anne

    2013-01-01

    The engineered antibody approach to Huntington's disease (HD) therapeutics is based on the premise that significantly lowering the levels of the primary misfolded mutant protein will reduce abnormal protein interactions and direct toxic effects of the misfolded huntingtin (HTT). This will in turn reduce the pathologic stress on cells, and normalize intrinsic proteostasis. Intracellular antibodies (intrabodies) are single-chain (scFv) and single-domain (dAb; nanobody) variable fragments that can retain the affinity and specificity of full-length antibodies, but can be selected and engineered as genes. Functionally, they represent a protein-based approach to the problem of aberrant mutant protein folding, post-translational modifications, protein-protein interactions, and aggregation. Several intrabodies that bind on either side of the expanded polyglutamine tract of mutant HTT have been reported to improve the mutant phenotype in cell and organotypic cultures, fruit flies, and mice. Further refinements to the difficult challenges of intraneuronal delivery, cytoplasmic folding, and long-term efficacy are in progress. This review covers published studies and emerging approaches on the choice of targets, selection and engineering methods, gene and protein delivery options, and testing of candidates in cell and animal models. The resultant antibody fragments can be used as direct therapeutics and as target validation/drug discovery tools for HD, while the technology is also applicable to a wide range of neurodegenerative and other diseases that are triggered by toxic proteins. PMID:22120646

  1. Evaluation of Boldine Activity against Intracellular Amastigotes of Leishmania amazonensis.

    PubMed

    Salama, Isabel Cristina; Arrais-Lima, Cristina; Arrais-Silva, Wagner Welber

    2017-06-01

    Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate in vitro activity of boldine against Leishmania amazonensis murine cell infection. Boldine ((S)-2,9-dihydroxy-1,10-dimethoxy-aporphine) is an aporphine alkaloid found abundantly in the leaves/bark of boldo (Peumus boldus Molina), a widely distributed tree native to Chile. The in vitro system consisted of murine macrophage infection with amastigotes of L. amazonensis treated with different concentrations from 50 to 600 μg/ml of boldine for 24 hr. Intracellular parasite destruction was assessed by morphological examination and boldine cytotoxicity to macrophages was tested by the MTT viability assay. When cells were treated with 100 μg/ml of boldine the reduction of parasite infection was 81% compared with untreated cultures cells. Interestingly, boldine-treatment caused a concentration-dependent decrease of macrophage infection that culminated with 96% of reduction when cells were submitted to 600 μg/ml of boldine. Cell cultures exposed to 100 μg/ml of boldine and 300 μg/ml of Glucantime(®) during 24 hr showed a significant reduction of 50% in parasitized cells compared with cell cultures exposed just to Glucantime(®). The study showed that treatment with boldine produces a better effect than treatment with the reference antimonial drug, glucantime, in L. amazonensis infected macrophage. Our results suggest that boldine is a potentially useful agent for the treatment of leishmaniasis.

  2. Evaluation of Boldine Activity against Intracellular Amastigotes of Leishmania amazonensis

    PubMed Central

    Salama, Isabel Cristina; Arrais-Lima, Cristina; Arrais-Silva, Wagner Welber

    2017-01-01

    Leishmaniasis is a neglected and endemic disease that affects poorest population mainly in developing countries. A lack of adequate and definitive chemotherapeutic agents to fight against this infection has led to the investigation of numerous compounds. The aim of this study was to investigate in vitro activity of boldine against Leishmania amazonensis murine cell infection. Boldine ((S)-2,9-dihydroxy-1,10-dimethoxy-aporphine) is an aporphine alkaloid found abundantly in the leaves/bark of boldo (Peumus boldus Molina), a widely distributed tree native to Chile. The in vitro system consisted of murine macrophage infection with amastigotes of L. amazonensis treated with different concentrations from 50 to 600 μg/ml of boldine for 24 hr. Intracellular parasite destruction was assessed by morphological examination and boldine cytotoxicity to macrophages was tested by the MTT viability assay. When cells were treated with 100 μg/ml of boldine the reduction of parasite infection was 81% compared with untreated cultures cells. Interestingly, boldine-treatment caused a concentration-dependent decrease of macrophage infection that culminated with 96% of reduction when cells were submitted to 600 μg/ml of boldine. Cell cultures exposed to 100 μg/ml of boldine and 300 μg/ml of Glucantime® during 24 hr showed a significant reduction of 50% in parasitized cells compared with cell cultures exposed just to Glucantime®. The study showed that treatment with boldine produces a better effect than treatment with the reference antimonial drug, glucantime, in L. amazonensis infected macrophage. Our results suggest that boldine is a potentially useful agent for the treatment of leishmaniasis. PMID:28719960

  3. Zn2+ activates large conductance Ca2+-activated K+ channel via an intracellular domain.

    PubMed

    Hou, Shangwei; Vigeland, Leif E; Zhang, Guangping; Xu, Rong; Li, Min; Heinemann, Stefan H; Hoshi, Toshinori

    2010-02-26

    Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn(2+) functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn(2+) signaling are not well known. We show here that intracellular Zn(2+) potently and reversibly activates large-conductance voltage- and Ca(2+)-activated Slo1 K(+) (BK) channels. The full effect of Zn(2+) requires His(365) in the RCK1 (regulator of conductance for K(+)) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp(367) and Glu(399), also reduced activation of the channel by Zn(2+), suggesting a possible structural arrangement for Zn(2+) binding by the aforementioned residues. Extracellular Zn(2+) activated Slo1 BK channels when coexpressed with Zn(2+)-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn(2+) signaling and may participate in sculpting cellular response to an increase in intracellular Zn(2+) concentration.

  4. Intracellular sodium ion activity and sodium transport in rabbit urinary bladder.

    PubMed Central

    Eaton, D C

    1981-01-01

    1. Intracellular potentials and the intracellular activities of Na+ and K+ were examined using conventional and ion-selective micro-electrodes. 2. In animals on a normal diet, the intracellular Na+ activity was 8.6 +/- 2.9 mM (mean +/- S.D.) with a mean short-circuit current of 2.8 +/- 0.9 microA/cm2. 3. In animals on a low-Na+ diet, the intracellular Na+ activity was 18.5 +/- 9.9 mM with a short-circuit current of 4.5 +/- 1.3 microA/cm2 (mean +/- S.D.). 4. There was a correlation between short-circuit current and intracellular Na+ activity which could be fitted by a saturating hyperbolic relationship. 5. Treatment of the issue with ouabain and amiloride produced an increase and a decrease, respectively, in the intracellular Na+ activity. 6. Treatment with aldosterone produced a large increase in short-circuit current with a substantial increase in intracellular Na+ activity. 7. Intracellular Na+ activity does not seem to affect apical membrane permeability directly. PMID:7320880

  5. Intracellular activities of RP 59500 (quinupristin-dalfopristin) and sparfloxacin against Enterococcus faecium.

    PubMed Central

    Herrera-Insúa, I; Jacques-Palaz, K; Murray, B E; Rakita, R M

    1996-01-01

    RP 59500, a combination of the streptogramins quinupristin and dalfopristin, and sparfloxacin are new antibiotics with good in vitro activities against Enterococcus faecium, which is an increasingly important nosocomial pathogen with resistance to multiple antimicrobials. Since fluoroquinolones and related macrolides have displayed high intracellular concentrations inside host cells, we evaluated the intracellular activities of these agents inside neutrophils against three strains each of vancomycin-susceptible E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF). At concentrations equal to four times the MIC, RP 59500 and sparfloxacin decreased the number of intracellular VSEF organisms, while both antibiotics were at best bacteriostatic against intracellular VREF strains. At concentrations equal to one-fourth of the MIC, both antibiotics were bacteriostatic against intracellular VSEF strains but were ineffective in inhibiting the growth of VREF strains. Despite their anticipated markedly higher intracellular human neutrophil (PMN) concentrations, RP 59500 and sparfloxacin activities in medium alone were equal to or greater than those inside PMNs against almost all strains. We conclude that the intracellular PMN concentrations of these antibiotics may not be directly related to their intracellular activities in our assay. The reason for the differences in their activities against VSEF versus VREF remains undefined. PMID:8849245

  6. Pharmacology of the intracellular pathways activated by amyloid beta protein.

    PubMed

    Balleza-Tapia, Hugo; Peña, Fernando

    2009-06-01

    Alzheimer's disease (AD) is a late-life cognitive disorder associated, among other things, to the presence of extracellular aggregates of fibrillar amyloid beta protein (Abeta). However, there is growing evidence that early stages of AD may be due to neuronal network dysfunction produced by the actions of soluble forms of Abeta. Therefore, the development of new therapeutic strategies to treat AD, at least during its first stages, may be focused on preventing or reversing, the deleterious effects that soluble Abeta exerts on neuronal circuit function. In order to do so, it is necessary to elucidate the pathophysiological processes involved in Abeta-induced neuronal network dysfunction and the molecular processes underlying such dysfunction. Over the last decades, there has been extensive research about the molecular mechanisms involved in the effects of Abeta as well as possible neuroprotective strategies against such effects. Here we are going to review some of the intracellular pathways triggered by Abeta, which involve membrane receptors such as nicotinic-R, NMDA-R, integrins, TNF-R1, RAGE, FPRL and p75NTR and their intracellular mediators such as GSK3, PKC, PI3K, Akt, FAK, MAPK family, Src family and cdk5. Several of these pathways may constitute therapeutic targets for the treatment of the Abeta-induced neuronal network dysfunction which is, at least in part, the basis for cognitive dysfunction in AD.

  7. Mechanisms Associated with Activation of Intracellular Metabotropic Glutamate Receptor, mGluR5.

    PubMed

    Jong, Yuh-Jiin I; O'Malley, Karen L

    2017-01-01

    The group 1 metabotropic glutamate receptor, mGluR5, is found on the cell surface as well as on intracellular membranes where it can mediate both overlapping and unique signaling effects. Previously we have shown that glutamate activates intracellular mGluR5 by entry through sodium-dependent transporters and/or cystine glutamate exchangers. Calibrated antibody labelling suggests that the glutamate concentration within neurons is quite high (~10 mM) raising the question as to whether intracellular mGluR5 is maximally activated at all times or whether a different ligand might be responsible for receptor activation. To address this issue, we used cellular, optical and molecular techniques to show that intracellular glutamate is largely sequestered in mitochondria; that the glutamate concentration necessary to activate intracellular mGluR5 is about ten-fold higher than what is necessary to activate cell surface mGluR5; and uncaging caged glutamate within neurons can directly activate the receptor. Thus these studies further the concept that glutamate itself serves as the ligand for intracellular mGluR5.

  8. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

    PubMed

    Liszewski, M Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G; Fara, Antonella F; Subias, Marta; Pickering, Matthew C; Drouet, Christian; Meri, Seppo; Arstila, T Petteri; Pekkarinen, Pirkka T; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P; Kemper, Claudia

    2013-12-12

    Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

  9. tert-Butylhydroquinone mobilizes intracellular-bound zinc to stabilize Nrf2 through inhibiting phosphatase activity.

    PubMed

    Chen, Yunfang; Wang, Sheng; Fu, Xin; Zhou, Wenqu; Hong, Wei; Zou, Dongting; Li, Xichong; Liu, Jinbao; Ran, Pixin; Li, Bing

    2015-08-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular "free" zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation. Copyright © 2015 the American Physiological Society.

  10. Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Tseng, Peter

    Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to

  11. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    PubMed

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

  12. Alterations in intracellular cations and cell membrane ATPase activity in patients with malignant hypertension.

    PubMed

    Touyz, R M; Milne, F J

    1995-08-01

    To determine whether cellular cation concentrations and cell membrane ATPase activity are altered in patients with malignant hypertension. Sixteen black patients with malignant hypertension were studied and compared with age- and sex-matched essential hypertensive and normotensive subjects. Calcium, magnesium, sodium and potassium concentrations and cell membrane Na,K-ATPase, Ca-ATPase and Mg-ATPase activities were determined in platelets and erythrocytes. Intracellular concentrations of total magnesium and calcium were measured by atomic absorption spectrophotometry, and those of sodium and potassium by flame photometry. Cell membrane ATPase activity was measured by a colorimetric method. The intracellular calcium level was significantly elevated and intracellular magnesium and potassium levels and cell membrane ATPase activity significantly decreased in the hypertensive group. These changes were more marked in patients with malignant hypertension than in patients with benign essential hypertension. In the malignant hypertensive group, mean arterial pressure was negatively correlated with intracellular magnesium and positively correlated with intracellular calcium and sodium levels. Cellular cation changes in malignant hypertension may be related to altered transmembrane transport mechanisms and activation of the renin-angiotensin system. These alterations may be more pronounced in the malignant than in the benign phase of hypertension.

  13. Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

    PubMed

    Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

    2015-12-01

    Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival.

  14. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-09

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  15. The influence of cell growth and enzyme activity changes on intracellular metabolite dynamics in AGE1.HN.AAT cells.

    PubMed

    Rath, Alexander G; Rehberg, Markus; Janke, Robert; Genzel, Yvonne; Scholz, Sebastian; Noll, Thomas; Rose, Thomas; Sandig, Volker; Reichl, Udo

    2014-05-20

    Optimization of bioprocesses with mammalian cells mainly concentrates on cell engineering, cell screening and medium optimization to achieve enhanced cell growth and productivity. For improving cell lines by cell engineering techniques, in-depth understandings of the regulation of metabolism and product formation as well as the resulting demand for the different medium components are needed. In this work, the relationship of cell specific growth and uptake rates and of changes in maximum in vitro enzyme activities with intracellular metabolite pools of glycolysis, pentose phosphate pathway, citric acid cycle and energy metabolism were determined for batch cultivations with AGE1.HN.AAT cells. Results obtained by modeling cell growth and consumption of main substrates showed that the dynamics of intracellular metabolite pools is primarily linked to the dynamics of specific glucose and glutamine uptake rates. By analyzing maximum in vitro enzyme activities we found low activities of pyruvate dehydrogenase and pyruvate carboxylase which suggest a reduced metabolite transfer into the citric acid cycle resulting in lactate release (Warburg effect). Moreover, an increase in the volumetric lactate production rate during the transition from exponential to stationary growth together with a transient accumulation of fructose 1,6-bisphosphate, fructose 1-phosphate and ribose 5-phosphate point toward an upregulation of PK via FBP. Glutaminase activity was about 44-fold lower than activity of glutamine synthetase. This seemed to be sufficient for the supply of intermediates for biosynthesis but might lead to unnecessary dissipation of ATP. Taken together, our results elucidate regulation of metabolic networks of immortalized mammalian cells by changes of metabolite pools over the time course of batch cultivations. Eventually, it enables the use of cell engineering strategies to improve the availability of building blocks for biomass synthesis by increasing glucose as well as

  16. Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

    PubMed Central

    Zhang, Yumin; Wang, Hong; Li, Jianrong; Jimenez, Daniel A.; Levitan, Edwin S.; Aizenman, Elias; Rosenberg, Paul A.

    2010-01-01

    Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study, we observed that TPEN [N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine], a zinc chelator, protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor, diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition, we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX), p38 mitogen-activated protein kinase (MAPK), and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX, generation of reactive oxygen species (ROS), and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX, ROS accumulation, p38 activation, and caspase-3 activation. Therefore, therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation, stroke, and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. PMID:15564577

  17. Intracellular gold nanoparticles increase neuronal excitability and aggravate seizure activity in the mouse brain.

    PubMed

    Jung, Seungmoon; Bang, Minji; Kim, Byung Sun; Lee, Sungmun; Kotov, Nicholas A; Kim, Bongsoo; Jeon, Daejong

    2014-01-01

    Due to their inert property, gold nanoparticles (AuNPs) have drawn considerable attention; their biological application has recently expanded to include nanomedicine and neuroscience. However, the effect of AuNPs on the bioelectrical properties of a single neuron remains unknown. Here we present the effect of AuNPs on a single neuron under physiological and pathological conditions in vitro. AuNPs were intracellularly applied to hippocampal CA1 neurons from the mouse brain. The electrophysiological property of CA1 neurons treated with 5- or 40-nm AuNPs was assessed using the whole-cell patch-clamp technique. Intracellular application of AuNPs increased both the number of action potentials (APs) and input resistance. The threshold and duration of APs and the after hyperpolarization (AHP) were decreased by the intracellular AuNPs. In addition, intracellular AuNPs elicited paroxysmal depolarizing shift-like firing patterns during sustained repetitive firings (SRF) induced by prolonged depolarization (10 sec). Furthermore, low Mg2+-induced epileptiform activity was aggravated by the intracellular AuNPs. In this study, we demonstrated that intracellular AuNPs alter the intrinsic properties of neurons toward increasing their excitability, and may have deleterious effects on neurons under pathological conditions, such as seizure. These results provide some considerable direction on application of AuNPs into central nervous system (CNS).

  18. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens.

  19. ARE MACROPHAGES ACTIVATED AND INDUCE PULMONARY INJURY BY INTRACELLULARLY BIOAVAILABLE IRON?

    EPA Science Inventory

    ARE MACROPHAGES ACTIVATED AND INDUCE PULMONARY INJURY BY INTRACELLULARLY BIOAVAILABLE IRON? UP Kodavanti1, MCJ Schladweiler1, S Becker2, DL Costa1, P Mayer3, A Ziesenis3, WG Kreyling3, 1ETD, 2HSDivision, NHEERL, USEPA, Research Triangle Park, NC, USA, and 3GSF, Inhalation Biology...

  20. Intracellular activity of tedizolid phosphate and ACH-702 versus Mycobacterium tuberculosis infected macrophages

    PubMed Central

    2014-01-01

    Background Due to the emergency of multidrug-resistant strains of Mycobacterium tuberculosis, is necessary the evaluation of new compounds. Findings Tedizolid, a novel oxazolidinone, and ACH-702, a new isothiazoloquinolone, were tested against M. tuberculosis infected THP-1 macrophages. These two compounds significantly decreased the number of intracellular mycobacteria at 0.25X, 1X, 4X and 16X the MIC value. The drugs were tested either in nanoparticules or in free solution. Conclusion Tedizolid and ACH-702 have a good intracellular killing activity comparable to that of rifampin or moxifloxacin. PMID:24708819

  1. NMDA receptor-mediated epileptiform persistent activity requires calcium release from intracellular stores in prefrontal neurons.

    PubMed

    Gao, Wen-Jun; Goldman-Rakic, Patricia S

    2006-02-01

    Various normal and pathological forms of synchronized population activity are generated by recurrent excitation among pyramidal neurons in the neocortex. However, the intracellular signaling mechanisms underlying this activity remain poorly understood. In this study, we have examined the cellular properties of synchronized epileptiform activity in the prefrontal cortex with particular emphasis on a potential role of intracellular calcium stores. We find that the zero-magnesium-induced synchronized activity is blocked by inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPases, phospholipase C (PLC), the inositol 1,4,5-trisphosphate (IP3) receptor, and the ryanodine receptor. This same activity is, however, not affected by application of metabotropic glutamatergic receptor (mGluR) agonists, nor by introduction of an mGluR antagonist. These results suggest that persistent synchronized activity in vitro is dependent upon calcium release from internal calcium stores through the activation of PLC-IP3 receptor pathway. Our findings also raise the possibility that intracellular calcium release may be involved in the generation of pathologic synchronized activity in epilepsy in vivo and in physiological forms of synchronized cortical activity.

  2. Bactericidal and intracellular activity of β-lactams against Mycobacterium abscessus.

    PubMed

    Lefebvre, Anne-Laure; Dubée, Vincent; Cortes, Mélanie; Dorchêne, Delphine; Arthur, Michel; Mainardi, Jean-Luc

    2016-06-01

    Cefoxitin and imipenem are the sole recommended β-lactams for the treatment of Mycobacterium abscessus pulmonary infections. Here, we investigated whether one of these drugs displays superiority in terms of killing and intracellular activity. We have also evaluated whether the use of a β-lactamase inhibitor could improve their activity. The impact of the β-lactamase BlaMab on the activity of β-lactams was assessed by comparing M. abscessus CIP104536 and its β-lactamase-deficient ΔblaMab derivative, as well as by using the β-lactamase inhibitor avibactam. The activity of cefoxitin, imipenem, amoxicillin and ceftaroline, alone and in various combinations including amikacin, was compared based on determination of time-kill curves and of intracellular proliferation in human macrophages. Imipenem was superior to cefoxitin in both the time-kill and macrophage assays. Production of BlaMab limited the activity of imipenem. The combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. Deletion of blaMab extended the spectrum of β-lactams active against M. abscessus to include amoxicillin and ceftaroline. In the absence of BlaMab, amoxicillin was as active as imipenem. These drugs were more active than ceftaroline and cefoxitin was the least active. Avibactam increased the intracellular activity of ceftaroline, but inhibition of BlaMab was only partial, as previously reported for amoxicillin. Evaluation of the killing and intracellular activities of β-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drugs potentially useful to treat pulmonary infections. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Status of the intracellular gate in the activated-not-open state of shaker K+ channels.

    PubMed

    del Camino, Donato; Kanevsky, Max; Yellen, Gary

    2005-11-01

    Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421-439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389-414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the

  4. Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

    PubMed Central

    del Camino, Donato; Kanevsky, Max; Yellen, Gary

    2005-01-01

    Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process

  5. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin

    PubMed Central

    Sanematsu, Keisuke; Kitagawa, Masayuki; Yoshida, Ryusuke; Nirasawa, Satoru; Shigemura, Noriatsu; Ninomiya, Yuzo

    2016-01-01

    Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL. PMID:26960429

  6. Modeling spontaneous activity in the developing spinal cord using activity-dependent variations of intracellular chloride.

    PubMed

    Marchetti, Cristina; Tabak, Joel; Chub, Nikolai; O'Donovan, Michael J; Rinzel, John

    2005-04-06

    We investigated how spontaneous activity is generated in developing, hyperexcitable networks. We focused our study on the embryonic chick spinal cord, a preparation that exhibits rhythmic discharge on multiple timescales: slow episodes (lasting minutes) and faster intraepisode cycling (approximately 1 Hz frequency). For this purpose, we developed a mean field model of a recurrent network with slow chloride dynamics and a fast depression variable. We showed that the model, in addition to providing a biophysical mechanism for the slow dynamics, was able to account for the experimentally observed activity. The model made predictions on how interval and duration of episodes are affected when changing chloride-mediated synaptic transmission or chloride flux across cell membrane. These predictions guided experiments, and the model results were compared with experimental data obtained with electrophysiological recordings. We found agreement when transmission was affected through changes in synaptic conductance and good qualitative agreement when chloride flux was varied through changes in external chloride concentration or in the rate of the Na+-K+-2Cl- cotransporter. Furthermore, the model made predictions about the time course of intracellular chloride concentration and chloride reversal potential and how these are affected by changes in synaptic conductance. Based on the comparison between modeling and experimental results, we propose that chloride dynamics could be an important mechanism in rhythm generation in the developing chick spinal cord.

  7. Activation of tumoricidal properties in human blood monocytes by muramyl dipeptide requires specific intracellular interaction

    SciTech Connect

    Fogler, W.E.; Fidler, I.J.

    1986-03-15

    The purpose of this study was to identify the mechanism by which muramyl dipeptide (MDP) activates antitumor cytotoxic properties in normal and interferon-..gamma.. (IFN-..gamma..)-primed human peripheral blood monocytes. The structurally and functionally active MDP analog, nor-muramyl dipeptide (nor-MDP), and (/sup 3/H)nor-MDP were used as reference glycopeptides. Direct activation of normal, noncytotoxic monocytes by nor-MDP was enhanced its encapsulation within multilamellar vesicles (MLV). Studies with (/sup 3/H)nor-MDP revealed that the activation of monocytes by nor-MDP was not attributable to its interaction with a specific cell surface receptor, nor did it result merely from the internalization by monocytes of glycopeptide. Subthreshold concentrations of nor-MDP could activate tumor cytotoxic properties in IFN-..gamma..-primed monocytes. The intracellular interaction of (/sup 3/H)nor-MDP with IFN-..gamma..-primed monocytes was specific in that intracellular levels of radiolabeled material could be displaced and recovered as intact molecules by unlabeled nor-MDP, but not by a biologically inactive MDP stereoisomer. Collectively, these results suggest that the activation of tumoricidal properties in human blood monocytes by MDP occurs subsequent to intracellular interaction with specific MDP receptors.

  8. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  9. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  10. The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition.

    PubMed

    Bruning, Marc; Barsukov, Igor; Franke, Barbara; Barbieri, Sonia; Volk, Martin; Leopoldseder, Sonja; Ucurum, Zöhre; Mayans, Olga

    2012-05-01

    Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.

  11. Separation of synaptic and spike activity in intracellular recordings for selective analysis.

    PubMed

    Hedwig, B; Knepper, M

    1992-04-01

    A software spike filter has been developed which allows the separation of synaptic activity and action potentials in intracellular recordings. The algorithm uses the different velocities of the membrane potential during synaptic and spike activity and a time window to identify action potentials. When spikes are recognized, they are removed and the membrane potential is substituted by interpolated values. The spike filter makes possible a separate quantitative evaluation of postsynaptic potentials and spike activity. Thus a comprehensive characterization of neuron activity can be obtained. The spike filter is part of a modular software package designed for the evaluation of neurobiological data.

  12. Visual Snapshots of Intracellular Kinase Activity At The Onset of Mitosis

    PubMed Central

    Dai, Zhaohua; Dulyaninova, Natalya G.; Kumar, Sanjai; Bresnick, Anne R.; Lawrence, David S.

    2007-01-01

    Summary Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell division cycle. Robust protein kinase activity transpires just prior to, but not immediately following, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC β isoform with subsequent NEB. PMID:18022564

  13. Two helices in the third intracellular loop determine anoctamin 1 (TMEM16A) activation by calcium.

    PubMed

    Lee, Jesun; Jung, Jooyoung; Tak, Min Ho; Wee, Jungwon; Lee, Byeongjoon; Jang, Yongwoo; Chun, Hyeyeon; Yang, Dong-Jin; Yang, Young Duk; Park, Sang Ho; Han, Byung Woo; Hyun, Soonsil; Yu, Jaehoon; Cho, Hawon; Hartzell, H Criss; Oh, Uhtaek

    2015-08-01

    Anoctamin 1 (ANO1)/TMEM16A is a Cl(-) channel activated by intracellular Ca(2+) mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca(2+). Here, we demonstrate that two helices, "reference" and "Ca(2+) sensor" helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca(2+)-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca(2+)-dependent activation because neutralization of these charges change the Ca(2+) sensitivity. We now predict that the Ca(2+) sensor helix attaches to the reference helix in the resting state, and as intracellular Ca(2+) rises, Ca(2+) acts on the sensor helix, which repels it from the reference helix. This Ca(2+)-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.

  14. PIP2 Activates TRPV5 and Releases Its Inhibition by Intracellular Mg2+

    PubMed Central

    Lee, Jason; Cha, Seung-Kuy; Sun, Tie-Jun; Huang, Chou-Long

    2005-01-01

    The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C–activating hormones via alteration of the sensitivity to intracellular Mg2+. PMID:16230466

  15. Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium.

    PubMed

    Vallabhapurapu, Subrahmanya D; Blanco, Víctor M; Sulaiman, Mahaboob K; Vallabhapurapu, Swarajya Lakshmi; Chu, Zhengtao; Franco, Robert S; Qi, Xiaoyang

    2015-10-27

    Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS.

  16. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

  17. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  18. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine.

    PubMed

    Peifer, Susanne; Barduhn, Tobias; Zimmet, Sarah; Volmer, Dietrich A; Heinzle, Elmar; Schneider, Konstantin

    2012-10-24

    Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol g(CDW)⁻¹. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol g(CDW)⁻¹). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol g(CDW)⁻¹) derived from IMP degradation. The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization.

  19. Intracellular reactive oxygen species in monocytes generated by photosensitive chromophores activated with blue light.

    PubMed

    Bouillaguet, Serge; Owen, Brandi; Wataha, John C; Campo, Marino A; Lange, Norbert; Schrenzel, Jacques

    2008-08-01

    Disinfection of the tooth pulp-canal system is imperative to successful endodontic therapy. Yet, studies suggest that 30-50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death mediated by generated reactive oxygen species (ROS) has been reported, but red-light also may thermally damage resident tissues. In the current study, we tested the hypothesis that several blue light chromophores (380-500 nm) generate intracellular reactive oxygen species but are not cytotoxic to mammalian cells. THP1 monocytes were exposed to 10 microM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (27J/cm(2), 60s). After activation, intracellular ROS were measured using a dihydrofluorescein diacetate technique, and cytotoxicity was determined by measuring mitochondrial activity with the MTT method. All photosensitizers produced intracellular ROS levels that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Photosensitizers except riboflavin exhibited cytotoxicity above 10 microM, and all except pheophorbide-a-PLL were more cytotoxic after blue light irradiation. The current study demonstrated the possible utility of blue light chromophores as producers of ROS that would be useful for endodontic disinfection.

  20. Clioquinol and pyrithione activate TRPA1 by increasing intracellular Zn2+.

    PubMed

    Andersson, David A; Gentry, Clive; Moss, Sian; Bevan, Stuart

    2009-05-19

    The antifungal and amoebicidal drug clioquinol (CQ) was withdrawn from the market when it was linked to an epidemic of subacute myelo-optico-neuropathy (SMON). Clioquinol exerts its anti-parasitic actions by acting as a Cu/Zn chelator and ionophore. Here we show that local injections of CQ produce mechanical hyperalgesia and cold hypersensitivity through a mechanism involving TRPA1 in mice. We also show that CQ activates TRPA1 in a Zn(2+)-dependent manner. Using a different Zn(2+)-ionophore, zinc pyrithione (ZnPy), we demonstrate that low, nanomolar concentrations of intracellular Zn(2+) ([Zn(2+)](i)) stimulate TRPA1. Direct application of Zn(2+) to the intracellular face of excised, inside-out patches activates TRPA1 with an EC(50) value of 7.5 +/- 1 nM. TRPA1 is expressed in a subpopulation of nociceptive dorsal root ganglion (DRG) neurons, where it acts as a sensory receptor for environmental irritants and oxidants. Using cultured DRG neurons from wild-type and TRPA1-deficient mice, we demonstrate that TRPA1 is the principal excitatory receptor for increased [Zn(2+)](i) in DRG neurons. In conclusion, we have discovered that TRPA1 acts a sensor of intracellular Zn(2+), and that Zn(2+) ionophores, such as CQ and ZnPy, activate TRPA1 by increasing [Zn(2+)](i). We also demonstrate that CQ-evoked mechanical hyperalgesia and cold allodynia require TRPA1 in vivo.

  1. Activation of Oral Trigeminal Neurons by Fatty Acids is Dependent upon Intracellular Calcium

    PubMed Central

    Yu, Tian; Shah, Bhavik P.; Hansen, Dane R.; Park-York, MieJung; Gilbertson, Timothy A.

    2012-01-01

    The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that conveys the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to stimulate rat trigeminal neurons has remained unexplored. Here, we found that several free fatty acids are capable of activating trigeminal neurons with different kinetics. Further, a polyunsaturated fatty acid, linoleic acid (LA), activates trigeminal neurons by increasing intracellular calcium concentration and generating depolarizing receptor potentials. Ion substitution and pharmacological approaches reveal that intracellular calcium store depletion is crucial for LA-induced signaling in a subset of trigeminal neurons. Using pseudorabies virus (PrV) as a live cell tracer, we identified a subset of lingual nerve-innervated trigeminal neurons that respond to different subsets of fatty acids. Quantitative real-time PCR of several transient receptor potential (TRP) channel markers in individual neurons validated that PrV labeled a subset but not the entire population of lingual-innervated trigeminal neurons. We further confirmed that the LA-induced intracellular calcium rise is exclusively coming from the release of calcium stores from the endoplasmic reticulum in this subset of lingual nerve-innervated trigeminal neurons. PMID:22644615

  2. Catalytic activity of human carbonic anhydrase isoform IX is displayed both extra- and intracellularly.

    PubMed

    Klier, Michael; Jamali, Somayeh; Ames, Samantha; Schneider, Hans-Peter; Becker, Holger M; Deitmer, Joachim W

    2016-01-01

    Most carbonic anhydrases catalyse the reversible conversion of carbon dioxide to protons and bicarbonate, either as soluble cytosolic enzymes, in or at intracellular organelles, or at the extracellular face of the cell membrane as membrane-anchored proteins. Carbonic anhydrase isoform IX (CA IX), a membrane-bound enzyme with catalytic activity at the extracellular membrane surface, has come to prominence in recent years because of its association with hypoxic tissue, particularly tumours, often indicating poor prognosis. We have evaluated the catalytic activity of CA IX heterologously expressed in Xenopus laevis oocytes by measuring the amplitude and rate of cytosolic pH changes as well as pH changes at the outer membrane surface (pHs ) during addition and removal of 5% CO2 /25 mm HCO3-, and by mass spectrometry. Our results indicate both extracellular and intracellular catalytic activity of CA IX. Reduced rates of CO2 -dependent intracellular pH changes after knockdown of CA IX confirmed these findings in two breast cancer cell lines: MCF-7 and MDA-MB-231. Our results demonstrate a new function of CA IX that may be important in the search for therapeutic cancer drugs targeting CA IX.

  3. PPARdelta activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway.

    PubMed

    Lee, Kuy-Sook; Park, Jin-Hee; Lee, Seahyoung; Lim, Hyun-Joung; Park, Hyun-Young

    2009-04-01

    Peroxisome proliferator-activated receptors delta (PPARdelta) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARdelta in cardiac cells except that PPARdelta ligand treatment protects cardiac hypertrophy by inhibiting NF-kappaB activation. Thus, in the present study, we examined the effect of selective PPARdelta ligand L-165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L-165041 (10 microM) inhibited AngII-induced [(3)H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L-165041 pretreatment significantly inhibited AngII-induced intracellular Ca(2+) increase and subsequent phosphorylation of FAK. Further experiment using Ca(2+) ionophore A23187 confirmed that Ca(2+) induced FAK phosphorylation, and this was also blocked by L-165041 pretreatment. In addition, overexpression of PPARdelta using adenovirus significantly inhibited AngII-induced intracellular Ca(2+) increase and FAK expression, while PPARdelta siRNA treatment abolished the effect of L-165041. These data indicate that PPARdelta ligand L-165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca(2+)/FAK/ERK signaling pathway in a PPARdelta dependent mechanism.

  4. Intracellular ROS Protection Efficiency and Free Radical-Scavenging Activity of Curcumin

    PubMed Central

    Barzegar, Abolfazl; Moosavi-Movahedi, Ali A.

    2011-01-01

    Curcumin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of curcumin in polar solvents by a comparative study using ESR, reduction of ferric iron in aqueous medium and intracellular ROS/toxicity assays. ESR data indicated that the steric hindrance among adjacent big size groups within a galvinoxyl molecule limited the curcumin to scavenge galvinoxyl radicals effectively, while curcumin showed a powerful capacity for scavenging intracellular smaller oxidative molecules such as H2O2, HO•, ROO•. Cell viability and ROS assays demonstrated that curcumin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and lethal effects of cumene hydroperoxide. Curcumin also showed good electron-transfer capability, with greater activity than trolox in aqueous solution. Curcumin can readily transfer electron or easily donate H-atom from two phenolic sites to scavenge free radicals. The excellent electron transfer capability of curcumin is because of its unique structure and different functional groups, including a β-diketone and several π electrons that have the capacity to conjugate between two phenyl rings. Therfore, since curcumin is inherently a lipophilic compound, because of its superb intracellular ROS scavenging activity, it can be used as an effective antioxidant for ROS protection within the polar cytoplasm. PMID:22016801

  5. In vitro activity of artemisone and artemisinin derivatives against extracellular and intracellular Helicobacter pylori.

    PubMed

    Sisto, Francesca; Scaltrito, Maria Maddalena; Masia, Carla; Bonomi, Arianna; Coccè, Valentina; Marano, Giuseppe; Haynes, Richard K; Miani, Alessandro; Farronato, Giampietro; Taramelli, Donatella

    2016-07-01

    The in vitro activity of the new artemisinin derivative artemisone as well as other molecules of the same class against Helicobacter pylori and their effects when combined with standard antibiotics were evaluated. Since H. pylori can be internalised into gastric epithelial cells, the effects of artemisinin, dihydroartemisinin and artemisone against intracellular H. pylori were also investigated. Bacteriostatic [minimum inhibitory concentration (MIC)] and bactericidal [minimum bactericidal concentration (MBC)] activities were assessed against 24 clinical strains of H. pylori with different antibiotics susceptibilities. Artemisone showed MIC50 and MIC90 values of 0.25 mg/L and 0.5 mg/L, respectively, and an MBC50 value of 0.5 mg/L. Artemisone was synergistic with amoxicillin in 60% of strains, with clarithromycin in 40% and with metronidazole in 20%. There was no interaction between artemisone and omeprazole or bismuth citrate. Against intracellular H. pylori, only dihydroartemisinin at 2× MIC caused a 1 log10 CFU decrease after 18 h and 24 h of incubation. This is the first demonstration in vitro of the activity of artemisinin derivatives against intracellular H. pylori and indicates that artemisone has the potential to be efficacious for the treatment of H. pylori infection, especially in combination with antibiotics.

  6. Active intracellular transport in metastatic cells studied by spatial light interference microscopy.

    PubMed

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-01-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

  7. Identifying the structure-activity relationship of leelamine necessary for inhibiting intracellular cholesterol transport

    PubMed Central

    Gowda, Raghavendra; Inamdar, Gajanan S.; Kuzu, Omer; Dinavahi, Saketh S.; Krzeminski, Jacek; Battu, Madhu Babu; Voleti, Sreedhara R.; Amin, Shantu; Robertson, Gavin P.

    2017-01-01

    Leelamine is an anticancer chemotherapeutic agent inhibiting intracellular cholesterol transport. Cell death mediated by leelamine occurs due to the lysosomotropic property of the compound, its accumulation in the lysosome, and inhibition of cholesterol transport leading to lack of availability for key processes required for functioning of cancer cells. The present study dissects the structure-activity-relationship of leelamine using synthesized derivatives of leelamine and abietic acid, a structurally similar compound, to identify the moiety responsible for anti-cancer activity. Similar to leelamine, all active derivatives had an amino group or a similar moiety that confers a lysosomotropic property to the compound enabling its accumulation in the lysosome. Active derivatives inhibited intracellular cholesterol transport and hindered xenografted melanoma tumor development without obvious systemic toxicity. In silico studies suggested that active derivatives accumulating in lysosomes bound to NPC1, a protein responsible for cholesterol export from the lysosome, to inhibit its activity that then caused accumulation, and lack of cholesterol availability for other key cellular activities. Thus, active derivatives of leelamine or abietic acid maintained lysosomotropic properties, bound to NPC1, and disrupted cellular cholesterol transport as well as availability to retard tumor development. PMID:28423677

  8. Localization of the Intracellular Activity Domain of Pasteurella multocida Toxin to the N Terminus

    PubMed Central

    Wilson, Brenda A.; Ponferrada, Virgilio G.; Vallance, Jefferson E.; Ho, Mengfei

    1999-01-01

    We have shown that Pasteurella multocida toxin (PMT) directly causes transient activation of Gqα protein that is coupled to phosphatidylinositol-specific phospholipase Cβ1 in Xenopus oocytes (B. A. Wilson, X. Zhu, M. Ho, and L. Lu, J. Biol. Chem. 272:1268–1275, 1997). We found that antibodies directed against an N-terminal peptide of PMT inhibited the toxin-induced response in Xenopus oocytes, but antibodies against a C-terminal peptide did not. To test whether the intracellular activity domain of PMT is localized to the N terminus, we conducted a deletion mutational analysis of the PMT protein, using the Xenopus oocyte system as a means of screening for toxin activity. Using PCR and conventional cloning techniques, we cloned from a toxinogenic strain of P. multocida the entire toxA gene, encoding the 1,285-amino-acid PMT protein, and expressed the recombinant toxin as a His-tagged fusion protein in Escherichia coli. We subsequently generated a series of N-terminal and C-terminal deletion mutants and expressed the His-tagged PMT fragments in E. coli. These proteins were screened for cytotoxic activity on cultured Vero cells and for intracellular activity in the Xenopus oocyte system. Only the full-length protein without the His tag exhibited activity on Vero cells. The full-length PMT and N-terminal fragments containing the first 500 residues elicited responses in oocytes, but the C-terminal 780 amino acid fragment did not. Our results confirm that the intracellular activity domain of PMT is localized to the N-terminal 500 amino acids of the protein and that the C terminus is required for entry into cells. PMID:9864199

  9. Activation of intracellular serine proteinase in Bacillus subtilis cells during sporulation.

    PubMed Central

    Burnett, T J; Shankweiler, G W; Hageman, J H

    1986-01-01

    Cells of Bacillus subtilis 168 (trpC2) growing and sporulating in a single chemically defined medium carried out intracellular protein degradation and increased their levels of intracellular serine protease-1 in a manner very similar to what had previously been reported for cells sporulating in nutrient broth. The results were interpreted to mean that these processes are intrinsic to sporulation rather than medium dependent. To determine the cause of these increases in specific activity of proteinases, we purified the protease, prepared rabbit immunoglobulins directed against it, and monitored changes in protease antigen levels by performing rocket immunoelectrophoresis. In cells sporulating in nutrient broth, the protease antigen levels increased about 7-fold, whereas the specific activity increased about 150-fold, for an activation of about 20-fold. In cells sporulating in the single chemically defined sporulation medium, the protease antigen increased about 10-fold, whereas the specific activity increased at least 400-fold, for an activation of about 40-fold. These results were interpreted to mean that a posttranslational event activated the protease in vivo; a previously described endogenous proteinase inhibitor was confirmed to be present in the strain used. Chloramphenicol added to the cultures inhibited both the increases in antigen levels and in the specific activity of the proteinase. PMID:3079745

  10. Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55*

    PubMed Central

    Yu, Justine; Deliu, Elena; Zhang, Xue-Quian; Hoffman, Nicholas E.; Carter, Rhonda L.; Grisanti, Laurel A.; Brailoiu, G. Cristina; Madesh, Muniswamy; Cheung, Joseph Y.; Force, Thomas; Abood, Mary E.; Koch, Walter J.; Tilley, Douglas G.; Brailoiu, Eugen

    2013-01-01

    The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca2+ signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca2+ entry via L-type Ca2+ channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca2+ release. The latter signal is further amplified by Ca2+-induced Ca2+ release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca2+ release from acidic-like Ca2+ stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca2+-induced Ca2+ release via ryanodine receptors. Extracellularly applied LPI produces Ca2+-independent membrane depolarization, whereas the Ca2+ signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders. PMID:23814062

  11. Software Engineering Improvement Activities/Plan

    NASA Technical Reports Server (NTRS)

    2003-01-01

    bd Systems personnel accomplished the technical responsibilities for this reporting period, as planned. A close working relationship was maintained with personnel of the MSFC Avionics Department Software Group (ED14). Work accomplishments included development, evaluation, and enhancement of a software cost model, performing literature search and evaluation of software tools available for code analysis and requirements analysis, and participating in other relevant software engineering activities. Monthly reports were submitted. This support was provided to the Flight Software Group/ED 1 4 in accomplishing the software engineering improvement engineering activities of the Marshall Space Flight Center (MSFC) Software Engineering Improvement Plan.

  12. Software Engineering Improvement Activities/Plan

    NASA Technical Reports Server (NTRS)

    2003-01-01

    bd Systems personnel accomplished the technical responsibilities for this reporting period, as planned. A close working relationship was maintained with personnel of the MSFC Avionics Department Software Group (ED14). Work accomplishments included development, evaluation, and enhancement of a software cost model, performing literature search and evaluation of software tools available for code analysis and requirements analysis, and participating in other relevant software engineering activities. Monthly reports were submitted. This support was provided to the Flight Software Group/ED 1 4 in accomplishing the software engineering improvement engineering activities of the Marshall Space Flight Center (MSFC) Software Engineering Improvement Plan.

  13. Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

    PubMed

    Nomura, Johji; So, Alexander; Tamura, Mizuho; Busso, Nathalie

    2015-12-15

    Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation. Copyright © 2015 by The American Association of Immunologists, Inc.

  14. Intracellular alkalization causes pain sensation through activation of TRPA1 in mice

    PubMed Central

    Fujita, Fumitaka; Uchida, Kunitoshi; Moriyama, Tomoko; Shima, Asako; Shibasaki, Koji; Inada, Hitoshi; Sokabe, Takaaki; Tominaga, Makoto

    2008-01-01

    Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH–related sensory disorders whose mechanisms are largely unknown. PMID:19033673

  15. Antibody-Mediated Elimination of the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensis during Active Infection

    PubMed Central

    Winslow, Gary M.; Yager, Eric; Shilo, Konstantin; Volk, Erin; Reilly, Andrew; Chu, Frederick K.

    2000-01-01

    It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection by Ehrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms. PMID:10722619

  16. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    PubMed Central

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-01-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction. PMID:27641076

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

    PubMed

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

    2015-05-01

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

  18. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane

    NASA Astrophysics Data System (ADS)

    Li, Nan; Yang, Yong; He, Kangmin; Zhang, Fayun; Zhao, Libo; Zhou, Wei; Yuan, Jinghe; Liang, Wei; Fang, Xiaohong

    2016-09-01

    Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane. It was found that Smad3 docked to cell membrane in both unstimulated and stimulated cells, but with different diffusion rates and dissociation kinetics. The change in its membrane docking dynamics can be used to study the activation of Smad3. Our results reveal that Smad3 binds with type I TGF-β receptor (TRI) even in unstimulated cells. Its activation is regulated by TRI phosphorylation but independent of receptor endocytosis. This study offers new information on TGF-β/Smad signaling, as well as a new approach to investigate the activation of intracellular signaling proteins for a better understanding of their functions in signal transduction.

  19. Intracellular Na(+) modulates large conductance Ca(2+)-activated K (+) currents in human umbilical vein endothelial cells.

    PubMed

    Liang, Guo Hua; Kim, Moon Young; Park, Seonghee; Kim, Ji Aee; Choi, Shinkyu; Suh, Suk Hyo

    2008-10-01

    We studied the effects of Na(+) influx on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na(+) replacement by NMDG(+) or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na(+) concentration ([Na(+)](i)) and vice versa. In addition, extracellular Na(+) depletion decreased [Na(+)](i). In voltage-clamped cells, BK(Ca) currents were markedly increased by extracellular Na(+) depletion. In inside-out patches, increasing [Na(+)](i) from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK(Ca) channels and decreasing intracellular K(+) concentration ([K(+)](i)) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na(+)](i) gradually from 0 to 70 mM, by replacing K(+), markedly reduced single channel conductance and NPo. The Na(+)-Ca(2+) exchange blocker Ni(2+) or KB-R7943 decreased [Na(+)](i) and increased BK(Ca) currents simultaneously, and the Na(+) ionophore monensin completely inhibited BK(Ca) currents. BK(Ca) currents were significantly augmented by increasing extracellular K(+) concentration ([K(+)](o)) from 6 to 12 mM and significantly reduced by decreasing [K(+)](o) from 12 or 6 to 0 mM or applying the Na(+)-K(+) pump inhibitor ouabain. These results suggest that intracellular Na(+) inhibit single channel conductance of BK(Ca) channels and that intracellular K(+) increases single channel conductance and NPo.

  20. Superoxide dismutase activity of Mycobacterium avium, M. intracellulare, and M. scrofulaceum.

    PubMed Central

    Mayer, B K; Falkinham, J O

    1986-01-01

    Superoxide dismutase (EC 1.15.1.1) (SOD) activity has been detected in crude cell extracts of representative strains of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum (MAIS) group. Polyacrylamide gel electrophoresis demonstrated a single SOD activity band for each of the MAIS strains, though there were differences in mobility. All M. avium and M. intracellulare and two of five M. scrofulaceum strains demonstrated a single activity band of identical mobility (Rf = 0.83), while the SOD activity band for the three remaining M. scrofulaceum strains migrated farther (Rf = 0.85). The differences in mobility correlated with differences in sensitivity to NaN3 and H2O2. The SOD activities of the majority of the MAIS strains which displayed the slower-migrating activity band were inhibited 22 to 81% after 15 min of exposure to 5 mM H2O2, suggesting that both iron and manganese may be present in a single enzyme. The SOD activities of the three M. scrofulaceum strains which had the faster-migrating activity band were inhibited 100% after only 5 min of exposure to 5 mM H2O2 and exhibited greater sensitivity to 5 and 10 mM NaN3, characteristics of an iron-containing SOD. A concentration of 1 mM KCN did not cause inhibition of enzyme activity in any of the MAIS strains tested. Extracellular SOD activity was detected in four of six MAIS strains and was shown to be identical in mobility to the SOD activity of the crude extracts. Images PMID:3744555

  1. Cell type- and activity-dependent extracellular correlates of intracellular spiking

    PubMed Central

    Perin, Rodrigo; Buzsáki, György; Markram, Henry; Koch, Christof

    2015-01-01

    Despite decades of extracellular action potential (EAP) recordings monitoring brain activity, the biophysical origin and inherent variability of these signals remain enigmatic. We performed whole cell patch recordings of excitatory and inhibitory neurons in rat somatosensory cortex slice while positioning a silicon probe in their vicinity to concurrently record intra- and extracellular voltages for spike frequencies under 20 Hz. We characterize biophysical events and properties (intracellular spiking, extracellular resistivity, temporal jitter, etc.) related to EAP recordings at the single-neuron level in a layer-specific manner. Notably, EAP amplitude was found to decay as the inverse of distance between the soma and the recording electrode with similar (but not identical) resistivity across layers. Furthermore, we assessed a number of EAP features and their variability with spike activity: amplitude (but not temporal) features varied substantially (∼30–50% compared with mean) and nonmonotonically as a function of spike frequency and spike order. Such EAP variation only partly reflects intracellular somatic spike variability and points to the plethora of processes contributing to the EAP. Also, we show that the shape of the EAP waveform is qualitatively similar to the negative of the temporal derivative to the intracellular somatic voltage, as expected from theory. Finally, we tested to what extent EAPs can impact the lowpass-filtered part of extracellular recordings, the local field potential (LFP), typically associated with synaptic activity. We found that spiking of excitatory neurons can significantly impact the LFP at frequencies as low as 20 Hz. Our results question the common assertion that the LFP acts as proxy for synaptic activity. PMID:25995352

  2. Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

    PubMed Central

    1988-01-01

    Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+- adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity. PMID:3235975

  3. Intracellular Activation and Deactivation of Tasidotin, an Analog of Dolastatin 15: Correlation with Cytotoxicity

    PubMed Central

    Bai, Ruoli; Edler, Michael C.; Bonate, Peter L.; Copeland, Terry D.; Pettit, George R.; Ludueña, Richard F.; Hamel, Ernest

    2009-01-01

    Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [3H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [3H]P5 was not the end product of tasidotin metabolism. Large amounts of [3H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC50 of tasidotin and a marked increase in intracellular [3H]tasidotin. BCPP also caused a 4-fold increase in the IC50 of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC50 obtained for P5 in the presence of BCPP. PMID:18927208

  4. Intracellular activation and deactivation of tasidotin, an analog of dolastatin 15: correlation with cytotoxicity.

    PubMed

    Bai, Ruoli; Edler, Michael C; Bonate, Peter L; Copeland, Terry D; Pettit, George R; Ludueña, Richard F; Hamel, Ernest

    2009-01-01

    Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [(3)H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [(3)H]P5 was not the end product of tasidotin metabolism. Large amounts of [(3)H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC(50) of tasidotin and a marked increase in intracellular [(3)H]tasidotin. BCPP also caused a 4-fold increase in the IC(50) of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC(50) obtained for P5 in the presence of BCPP.

  5. The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

    PubMed

    Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

    2014-02-01

    During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

  6. Intracellular synthesis of silver nanoparticle by actinobacteria and its antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Otari, S. V.; Patil, R. M.; Ghosh, S. J.; Thorat, N. D.; Pawar, S. H.

    2015-02-01

    Intracellular synthesis of silver nanoparticles (AgNPs) using Rhodococcus spp. is demonstrated. The synthesized nanoparticles were characterized by UV-Vis spectroscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier trans-form infrared spectroscopy, and transmission electron microscopy. Transmission electron microscopy study of microorganisms' revealed synthesis of nanoparticle was occurring inside the cell, in the cytoplasm. AgNPs ranged from 5 to 50 nm. Formed nanoparticles were stable in the colloidal solution due to presence of proteins on the surface. AgNPs showed excellent bactericidal and bacteriostatic activity against pathogenic microorganisms.

  7. Activities of the Institute for Mechanical Engineering

    NASA Astrophysics Data System (ADS)

    The Institute of Mechanical Engineering (IME) is part of Canada's National Research Council. Its mission is to undertake, support, promote, and disseminate research and development in the mechanical engineering aspects of three vital sectors of the Canadian economy: transportation, resource industries, and manufacturing. The IME achieves its mission by performing research and development in its own facilities; by developing, providing, and transferring expertise and knowledge; by making its research facilities available to collaborators and clients; and by participating in international liaison and collaborative research activities. Six research programs are conducted in the IME: Advanced Manufacturing Technology; Coastal Zone Engineering; Cold Regions Engineering; Combustion and Fluids Engineering; Ground Transportation Technology; and Machinery and Engine Technology. The rationale and major research thrusts of each program are described, and specific achievements in 1991-92 are reviewed. Lists of technical reports and papers presented by IME personnel are also included.

  8. Delineation of Diverse Macrophage Activation Programs in Response to Intracellular Parasites and Cytokines

    PubMed Central

    Zhang, Shuyi; Kim, Charles C.; Batra, Sajeev; McKerrow, James H.; Loke, P'ng

    2010-01-01

    Background The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated. Methodology/Principal Findings To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines. Conclusions/Significance This study provides global gene expression data for a diverse set of biologically significant pathogens and

  9. Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation

    PubMed Central

    de Candia, Paola; Torri, Anna; Gorletta, Tatiana; Fedeli, Maya; Bulgheroni, Elisabetta; Cheroni, Cristina; Marabita, Francesco; Crosti, Mariacristina; Moro, Monica; Pariani, Elena; Romanò, Luisa; Esposito, Susanna; Mosca, Fabio; Rossetti, Grazisa; Rossi, Riccardo L.; Geginat, Jens; Casorati, Giulia; Dellabona, Paolo; Pagani, Massimiliano; Abrignani, Sergio

    2013-01-01

    Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation. PMID:24205408

  10. A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

    PubMed

    Xiao, Xunjun; Jones, Gabrielle; Sevilla, Wednesday A; Stolz, Donna B; Magee, Kelsey E; Haughney, Margaret; Mukherjee, Amitava; Wang, Yan; Lowe, Mark E

    2016-10-28

    Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

  11. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    NASA Astrophysics Data System (ADS)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  12. Fluorescence Imaging of Intracellular Telomerase Activity Using Enzyme-Free Signal Amplification.

    PubMed

    Hong, Min; Xu, Lidan; Xue, Qingwang; Li, Lu; Tang, Bo

    2016-12-20

    A novel enzyme-free signal amplification-based assay for highly sensitive in situ fluorescence imaging and detection of intracellular telomerase activity was developed by using a gold nanoflare probe-triggered mimic-hybridization chain reaction (mimic-HCR) coupled with a graphene oxide (GO) surface-anchored fluorescence signal readout pathway. The nanoflare probe consists of gold nanoparticles (AuNPs) functionalized with a dense shell of nucleic acid sequences by Au-S bond formation. The nucleic acid sequence is composed of three segments: a long thiol-labeled sequence (HS-DNA) and two short sequences (a telomerase primer sequence, "Primer-DNA", and an FAM-terminated reporter sequence, "Flare-DNA"), both of which are complementary to HS-DNA. The mimic-HCR system is formed by two FAM-modified hairpin sequences that are adsorbed on GO. Upon endocytosis of the AuNP/GO combinatorial probe, the Primer-DNA can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to inner chain substitution and not only releases the Flare-DNA to turn on the fluorescence of FAM but also initiates the subsequent signal amplification and enrichment for the mimic-HCR system anchored on GO. The proposed approach can sensitively detect telomerase activity in living cells, distinguish normal cells from cancer cells, and monitor the change in telomerase activity in response to a telomerase inhibitor.

  13. Statins have beneficial effects on platelet free radical activity and intracellular distribution of GTPases in hyperlipidaemia.

    PubMed

    Hamilton, Paul K; Hughes, Sinead M T; Plumb, Richard D; Devine, Adrian; Leahey, William; Lyons, Kristopher S; Johnston, Dennis; McVeigh, Gary E

    2010-03-01

    In addition to lowering cholesterol, statins may alter endothelial release of the vasodilator NO and harmful superoxide free radicals. Statins also reduce cholesterol intermediates including isoprenoids. These are important for post-translational modification of substances including the GTPases Rho and Rac. By altering the membrane association of these molecules, statins affect intracellular positioning and hence activity of a multitude of substances. These include eNOS(endothelial NO synthase), which produces NO (inhibited by Rho), and NADPH oxidase, which produces superoxide (dependent on Rac). Statins may improve endothelial function by enhancing production of NO while decreasing superoxide production. A total of 40 hypercholesterolaemic patients were randomized to treatment with either atorvastatin or placebo; 20 normolipidaemic patients were also studied. Platelet nitrite, NO and superoxide were examined as was the cellular distribution of the GTPases Rho and Rac at baseline and after 8 weeks of treatment.Following atorvastatin therapy, platelet NO was increased (3.2 pmol/10(8) platelets) and superoxide output was attenuated [-3.4 pmol min(-1) (10(8) platelets)(-1)] when compared with placebo. The detection of both Rho and Rac was significantly reduced in the membranes of platelets, implying reduced activity. In conclusion, the results of the present study show altered NO/superoxide production following statin therapy. A potential mechanism for this is the change in the distribution of intracellular GTPases, which was considered to be secondary to decreases in isoprenoid intermediates, suggesting that the activity of the former had been affected by atorvastatin.

  14. Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages.

    PubMed Central

    Brummer, E; Hanson, L H; Restrepo, A; Stevens, D A

    1989-01-01

    The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis. Images PMID:2744848

  15. Nano-Engineering of Active Metamaterials

    DTIC Science & Technology

    2014-10-29

    AFRL-OSR-VA-TR-2014-0295 NANO -ENGINEERING OF ACTIVE METAMATERIALS Larry Dalton UNIVERSITY OF WASHINGTON Final Report 10/29/2014 DISTRIBUTION A...Std. Z39.18 FINAL TECHNICAL REPORT (AFOSR-FA9550-09-1-0682)— Nano -Engineering of Active Metamaterials: Publications related to this AFOSR contract...photochemical stability at telecommunication wavelengths by pump -probe methods [4,11]. Development and Application of New Synthesis and Processing

  16. Rotenone decreases intracellular aldehyde dehydrogenase activity: implications for the pathogenesis of Parkinson's disease.

    PubMed

    Goldstein, David S; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J; Sharabi, Yehonatan

    2015-04-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson's disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the 'catecholaldehyde hypothesis,' buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 min), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD(+) was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. We report that rotenone, a mitochondrial complex I inhibitor that produces an animal model of Parkinson's disease, increases intracellular levels of the toxic dopamine metabolite 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), via decreased DOPAL metabolism by aldehyde dehydrogenase (ALDH) and decreased vesicular sequestration of cytoplasmic dopamine by the vesicular monoamine transporter (VMAT). The results provide a novel

  17. Intracellular RNA recognition pathway activates strong anti-viral response in human mast cells.

    PubMed

    Lappalainen, J; Rintahaka, J; Kovanen, P T; Matikainen, S; Eklund, K K

    2013-04-01

    Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti-viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double-stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti-viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF-α. In contrast, extracellular Toll-like receptor 3 (TLR)-3-activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti-viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up-regulated the expression of melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene-1 (RIG-1), and TLR-3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti-viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti-viral proteins human myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.

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

    SciTech Connect

    Ichijo, Yuta; Mochimaru, Yuta; Azuma, Morio; Satou, Kazuhiro; Negishi, Jun; Nakakura, Takashi; Oshima, Natsuki; Mogi, Chihiro; Sato, Koichi; Matsuda, Kouhei; Okajima, Fumikazu; Tomura, Hideaki

    2016-01-01

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

  19. Teaching Engineering with Autonomous Learning Activities

    ERIC Educational Resources Information Center

    Otero, Beatriz; Rodríguez, Eva; Royo, Pablo

    2015-01-01

    This paper proposes several activities that encourage self-learning in engineering courses. For each activity, the context and the pedagogical issues addressed are described emphasizing strengths and weaknesses. Specifically, this work describes and implements five activities, which are: questionnaires, conceptual maps, videos, jigsaw and…

  20. Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation

    PubMed Central

    1991-01-01

    The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin- permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID:1719125

  1. Intracellular oxidant activity, antioxidant enzyme defense system, and cell senescence in fibroblasts with trisomy 21.

    PubMed

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS.

  2. Intracellular Oxidant Activity, Antioxidant Enzyme Defense System, and Cell Senescence in Fibroblasts with Trisomy 21

    PubMed Central

    Rodríguez-Sureda, Víctor; Vilches, Ángel; Sánchez, Olga; Audí, Laura; Domínguez, Carmen

    2015-01-01

    Down's syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrial dysfunction. Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic features of DS, such as premature aging. Using cultured fibroblasts with trisomy 21 (T21F), this study aimed to ascertain whether an imbalance exists in activities, mRNA, and protein expression of the antioxidant enzymes SOD1, SOD2, glutathione-peroxidase, and catalase during the cell replication process in vitro. T21F had high SOD1 expression and activity which led to an interenzymatic imbalance in the antioxidant defense system, accentuated with replicative senescence. Intracellular ROS production and oxidized protein levels were significantly higher in T21F compared with control cells; furthermore, a significant decline in intracellular ATP content was detected in T21F. Cell senescence was found to appear prematurely in DS cells as shown by SA-β-Gal assay and p21 assessment, though not apoptosis, as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21F. These novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F, thereby linking replicative and stress-induced senescence in cultured cells to premature aging in DS. PMID:25852816

  3. Rotenone Decreases Intracellular Aldehyde Dehydrogenase Activity: Implications for the Pathogenesis of Parkinson Disease

    PubMed Central

    Goldstein, David S.; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Kopin, Irwin J.; Sharabi, Yehonatan

    2015-01-01

    Repeated systemic administration of the mitochondrial complex I inhibitor rotenone produces a rodent model of Parkinson disease (PD). Mechanisms of relatively selective rotenone-induced damage to nigrostriatal dopaminergic neurons remain incompletely understood. According to the “catecholaldehyde hypothesis,” buildup of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to PD pathogenesis. Vesicular uptake blockade increases DOPAL levels, and DOPAL is detoxified mainly by aldehyde dehydrogenase (ALDH). We tested whether rotenone interferes with vesicular uptake and intracellular ALDH activity. Endogenous and F-labeled catechols were measured in PC12 cells incubated with rotenone (0-1000 nM, 180 minutes), without or with F-dopamine (2 μM) to track vesicular uptake and catecholamine metabolism. Rotenone dose-dependently increased DOPAL, F-DOPAL, and 3,4-dihydroxyphenylethanol (DOPET) levels while decreasing dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels and the ratio of dopamine to the sum of its deaminated metabolites. In test tubes, rotenone did not affect conversion of DOPAL to DOPAC by ALDH when NAD+ was supplied, whereas the direct-acting ALDH inhibitor benomyl markedly increased DOPAL and decreased DOPAC concentrations in the reaction mixtures. We propose that rotenone builds up intracellular DOPAL by decreasing ALDH activity and attenuating vesicular sequestration of cytoplasmic catecholamines. The results provide a novel mechanism for selective rotenone-induced toxicity in dopaminergic neurons. PMID:25645689

  4. Eurycoma longifolia extract increases intracellular production activity of luteinizing hormone (LH) in pituitary

    NASA Astrophysics Data System (ADS)

    Pratomo, H.

    2017-07-01

    Administration of the boiled water (extract) of Eurycoma longifolia (E. Longifolia) 18 mg/200 g body weight (bw) actually increases basophil cells in the anterior pituitary. Meanwhile, it is observed that basophil cells in anterior pituitary are producer cells of LH and FSH. Cell activity rate producing intracellular FSH does not increase in the amount significantly after administration of the E. longifolia onto the third day. The research attempts to prove the performance of E. longifolia to producer cells of luteinizing hormone (LH) in the anterior pituitary. Applied approach by a technical method of immunohistochemistry staining uses an antibody anti-LH. Observation is established to the treatment group of the E longifolia in a dose of 18 mg/200 g a bw on the 1st day and 3rd day, compared to control group of 1 ml distilled water on the 1st day and 3rd day. Research results that administration of the extract of E longifolia onto the third day has increased the activity of producer cells of LH in the pituitary, the synthesizing intracellular LH obviously. It can be concluded that E longifolia constitutes strong trigger in producer cells of LH to synthesize LH hormone.

  5. Engineering Novel Molecular Beacon Constructs to Study Intracellular RNA Dynamics and Localization.

    PubMed

    Ma, Zhao; Wu, Xiaotian; Krueger, Christopher J; Chen, Antony K

    2017-09-21

    With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss techniques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  6. Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency

    SciTech Connect

    Lan, Ke; Murakami, Masanao; Choudhuri, Tathagata; Kuppers, Daniel A.; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a predominantly latent infection in the infected host. Importantly, during latency, only a small number of viral encoded genes are expressed. This viral gene expression pattern contributes to the establishment of long-term infection as well as the ability of the virus to evade the immune system. Previous studies have been shown that the replication and transcription activator (RTA) encoded by ORF50 activates it downstream genes and initiates viral lytic reactivation through functional interaction with RBP-J{kappa}, the major downstream effector of the Notch signaling pathway. This indicates that RTA can usurp the conserved Notch signaling pathway and mimic the activities of intracellular Notch1 to modulate gene expression. In this report, we show that the activated intracellular domain of Notch1 (ICN) is aberrantly accumulated in KSHV latently infected pleural effusion lymphoma (PEL) cells. ICN activated the RTA promoter in a dose-dependent manner, and forced expression of ICN in latently infected KSHV-positive cells initiated full blown lytic replication with the production of infectious viral progeny. However, latency-associated nuclear antigen (LANA) which is predominantly expressed during latency can specifically down-modulate ICN-mediated transactivation of RTA and so control KSHV for lytic reactivation. These results demonstrate that LANA can inhibit viral lytic replication by antagonizing ICN function and suggest that LANA is a critical component of the regulatory control mechanism for switching between viral latent and lytic replication by directly interacting with effectors of the conserved cellular Notch1 pathway.

  7. Encapsulation of Anti-Tuberculosis Drugs within Mesoporous Silica and Intracellular Antibacterial Activities

    PubMed Central

    Xia, Xin; Pethe, Kevin; Kim, Ryangyeo; Ballell, Lluis; Barros, David; Cechetto, Jonathan; Jeon, HeeKyoung; Kim, Kideok; Garcia-Bennett, Alfonso E.

    2014-01-01

    Tuberculosis is a major problem in public health. While new effective treatments to combat the disease are currently under development, they tend suffer from poor solubility often resulting in low and/or inconsistent oral bioavailability. Mesoporous materials are here investigated in an in vitro intracellular assay, for the effective delivery of compound PA-824; a poorly soluble bactericidal agent being developed against Tuberculosis (TB). Mesoporous materials enhance the solubility of PA-824; however, this is not translated into a higher antibacterial activity in TB-infected macrophages after 5 days of incubation, where similar values are obtained. The lack of improved activity may be due to insufficient release of the drug from the mesopores in the context of the cellular environment. However, these results show promising data for the use of mesoporous particles in the context of oral delivery with expected improvements in bioavailability.

  8. Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies

    PubMed Central

    Heikal, Ahmed A

    2010-01-01

    Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation–reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities. PMID:20406068

  9. Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloride-secreting epithelium.

    PubMed Central

    Welsh, M J

    1983-01-01

    Canine tracheal epithelium secretes Cl via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. To examine the mechanisms involved, intracellular chloride activity, acCl, was measured with Cl-selective intracellular microelectrodes. The results indicate that when the rate of secretion was minimal acCl was 37 mM; with stimulation of secretion the intracellular voltage depolarized, but acCl was not significantly altered, at 39 mM. These findings indicate that: (a) Cl is accumulated across the basolateral membrane under nonsecreting and secreting conditions at an activity 3.8 and 2.4 times, respectively, that predicted for an equilibrium distribution; (b) Cl exit across the apical membrane may be passive with an electrochemical driving force of 22 mV; and (c) stimulation of secretion enhanced the rate of Cl entry across the basolateral membrane, since Cl transport increased without a change in acCl. In the absence of Na in the extracellular fluid, acCl approached the value expected for an equilibrium distribution. This finding suggests that "uphill" entry of Cl into the cell against its electrochemical gradient is dependent upon, and energized by, the entry of Na down its gradient. Submucosal bumetanide, a loop diuretic, also decreased the rate of Cl secretion and decreased acCl, indicating an inhibition of Cl entry. These findings indicate that Cl entry into the cell is directed against its electrochemical gradient and is mediated by a Na-coupled, bumetanide-inhibitable, transport process at the basolateral membrane and that Cl may exit passively down a favorable electrochemical gradient across the apical membrane. PMID:6853719

  10. Engineering and Coordination of Regulatory Networks and Intracellular Complexes to Maximize Hydrogen Production by Phototrophic Microorganisms

    SciTech Connect

    James C. Liao

    2012-05-22

    This project is a collaboration with F. R. Tabita of Ohio State. Our major goal is to understand the factors and regulatory mechanisms that influence hydrogen production. The organisms to be utilized in this study, phototrophic microorganisms, in particular nonsulfur purple (NSP) bacteria, catalyze many significant processes including the assimilation of carbon dioxide into organic carbon, nitrogen fixation, sulfur oxidation, aromatic acid degradation, and hydrogen oxidation/evolution. Our part of the project was to develop a modeling technique to investigate the metabolic network in connection to hydrogen production and regulation. Organisms must balance the pathways that generate and consume reducing power in order to maintain redox homeostasis to achieve growth. Maintaining this homeostasis in the nonsulfur purple photosynthetic bacteria is a complex feat with many avenues that can lead to balance, as these organisms possess versatile metabolic capabilities including anoxygenic photosynthesis, aerobic or anaerobic respiration, and fermentation. Growth is achieved by using H{sub 2} as an electron donor and CO{sub 2} as a carbon source during photoautotrophic and chemoautotrophic growth, where CO{sub 2} is fixed via the Calvin-Benson-Bassham (CBB) cycle. Photoheterotrophic growth can also occur when alternative organic carbon compounds are utilized as both the carbon source and electron donor. Regardless of the growth mode, excess reducing equivalents generated as a result of oxidative processes, must be transferred to terminal electron acceptors, thus insuring that redox homeostasis is maintained in the cell. Possible terminal acceptors include O{sub 2}, CO{sub 2}, organic carbon, or various oxyanions. Cells possess regulatory mechanisms to balance the activity of the pathways which supply energy, such as photosynthesis, and those that consume energy, such as CO{sub 2} assimilation or N{sub 2} fixation. The major route for CO{sub 2} assimilation is the CBB

  11. Structure and intracellular antioxidant activity of pectic polysaccharide from acerola (Malpighia emarginata).

    PubMed

    Klosterhoff, Rafael Roberto; Bark, Juliana Müller; Glänzel, Nícolas Manzke; Iacomini, Marcello; Martinez, Gláucia Regina; Winnischofer, Sheila M B; Cordeiro, Lucimara M C

    2017-08-10

    Malpighia emarginata is a tropical fruit plant, found naturally in the Caribbean islands and South America that produces an edible fruit known as acerola or Barbados Cherry. Its polysaccharides were obtained by aqueous extraction, subjected to a freezing and thawing process and ultrafiltration. A homogeneous fraction (ACWS-01E) was analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analyses. The results showed an arabinan-rich pectic polysaccharide, with 6.1×10(4)g/mol and formed mainly by a high methyl esterified (DM=86%) homogalacturonan and branched arabinan. This latter is anchored in type I rhamnogalacturonan regions. The main chain of arabinan consisted of (1→5)-linked α-Araf, branched only at O-3. The potential ACWS-01E intracellular antioxidant activity against H2O2-induced oxidative stress in murine fibroblast cell line (3T3) was determined by DCFH-DA assay. The treatment with ACWS-01E significantly reduced H2O2-induced cytotoxic effect and the levels of reactive oxygen species (ROS). These findings suggested that ACWS-01E protected and improved NIH 3T3 cell viability from H2O2-induced toxicity by decreasing intracellular levels of ROS. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

    PubMed Central

    Katrukha, Eugene A.; Mikhaylova, Marina; van Brakel, Hugo X.; van Bergen en Henegouwen, Paul M.; Akhmanova, Anna; Hoogenraad, Casper C.; Kapitein, Lukas C.

    2017-01-01

    The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics. PMID:28322225

  13. Intracellular Voyeurism: Examining the Modulation of Host Cell Activities bySalmonella enterica Serovar Typhimurium.

    PubMed

    Szeto, Jason; Brumell, John H

    2005-11-01

    Salmonella spp. can infect host cells by gaining entry through phagocytosis or by inducing host cell membrane ruffling that facilitates bacterial uptake. With its wide host range, Salmonella enterica serovar Typhimurium has proven to be an important model organism for studying intracellular bacterial pathogenesis. Upon entry into host cells, serovar Typhimurium typically resides within a membrane-bound compartment termed the Salmonella-containing vacuole (SCV). From the SCV, serovar Typhimurium can inject several effector proteins that subvert many normal host cell systems, including endocytic trafficking, cytoskeletal rearrangements, lipid signaling and distribution, and innate and adaptive host defenses. The study of these intracellular events has been made possible through the use of various imaging techniques, ranging from classic methods of transmission electron microscopy to advanced livecell fluorescence confocal microscopy. In addition, DNA microarrays have now been used to provide a "snapshot" of global gene expression in serovar Typhimurium residing within the infected host cell. This review describes key aspects of Salmonella-induced subversion of host cell activities, providing examples of imaging that have been used to elucidate these events. Serovar Typhimurium engages specific host cell machinery from initial contact with the host cell to replication within the SCV. This continuous interaction with the host cell has likely contributed to the extensive arsenal that serovar Typhimurium now possesses, including two type III secretion systems, a range of ammunition in the form of TTSS effectors, and a complex genetic regulatory network that coordinates the expression of hundreds of virulence factors.

  14. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells

    PubMed Central

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-01-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  15. Active learning in transportation engineering education

    NASA Astrophysics Data System (ADS)

    Weir, Jennifer Anne

    The objectives of this research were (1) to develop experimental active-based-learning curricula for undergraduate courses in transportation engineering and (2) to assess the effectiveness of an active-learning-based traffic engineering curriculum through an educational experiment. The researcher developed a new highway design course as a pilot study to test selected active-learning techniques before employing them in the traffic engineering curriculum. Active-learning techniques, including multiple-choice questions, short problems completed by individual students or small groups, and group discussions, were used as active interludes within lectures. The researcher also collected and analyzed student performance and attitude data from control and experimental classes to evaluate the relative effectiveness of the traditional lecture (control) approach and the active-learning (experimental) approach. The results indicate that the active-learning approach adopted for the experimental class did have a positive impact on student performance as measured by exam scores. The students in the experimental class also indicated slightly more positive attitudes at the end of the course than the control class, although the difference was not significant. The author recommends that active interludes similar to those in the experimental curricula be used in other courses in civil engineering.

  16. Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists.

    PubMed

    Wielders, S J H; Bennaghmouch, A; Reutelingsperger, C P M; Bevers, E M; Lindhout, T

    2007-03-01

    Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.

  17. A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

    PubMed Central

    Guscott, Benjamin; Balklava, Zita; Safrany, Stephen T.; Wassmer, Thomas

    2016-01-01

    The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]. PIKfyve function is required for homoeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the amyloid precursor protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. In the present study, we utilize this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP intracellular domain (AICD) to the HIV TAT domain, a cell-permeable peptide allowing proteins to penetrate cells. The resultant TAT–AICD fusion protein is cell permeable and triggers an increase in PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe, we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT–AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the AICD activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease. PMID:26934981

  18. Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization.

    PubMed

    Miele, Claudia; Paturzo, Flora; Teperino, Raffaele; Sakane, Fumio; Fiory, Francesca; Oriente, Francesco; Ungaro, Paola; Valentino, Rossella; Beguinot, Francesco; Formisano, Pietro

    2007-11-02

    Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.

  19. Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

    PubMed Central

    Le Marchand, Sylvain J.; Piston, David W.

    2012-01-01

    The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca2+]i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca2+]i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on KATP channel activity but not on tetrodotoxin-sensitive Na+ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca2+]i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by KATP channel activity or reduction in α-cell [Ca2+]i. Our results demonstrate that glucose uncouples the positive relationship between [Ca2+]i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway. PMID:23077547

  20. Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange

    SciTech Connect

    Horowitz, S.B.; Tluczek, L.J. )

    1989-12-01

    We previously showed that gonadotropin increases the K{sup +} activity in Xenopus oocytes and that this is a signal for increased translation. However, K{sup +} need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring ({sup 3}H)leucine incorporation, we found that small changes in Mg{sup 2+} greatly affect translation rates. (Ca{sup 2+} had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K{sup +} and Mg{sup 2+} are the preponderant monovalent and divalent cations. Hence, increasing cellular K{sup +} activity might increase translation by causing Mg{sup 2+} activity to rise. If so, the increased translation rates produced by hormone treatment or K{sup +} injection would be prevented by EDTA, a Mg{sup 2+} chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K{sup +} activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg{sup 2+} activity and this up-regulates translation.

  1. Spatio-temporal PLC activation in parallel with intracellular Ca2+ wave propagation in mechanically stimulated single MDCK cells.

    PubMed

    Tsukamoto, Akira; Hayashida, Yasunori; Furukawa, Katsuko S; Ushida, Takashi

    2010-03-01

    Intracellular Ca2+ transients are evoked either by the opening of Ca2+ channels on the plasma membrane or by phospholipase C (PLC) activation resulting in IP3 production. Ca2+ wave propagation is known to occur in mechanically stimulated cells; however, it remains uncertain whether and how PLC activation is involved in intracellular Ca2+ wave propagation in mechanically stimulated cells. To answer these questions, it is indispensable to clarify the spatio-temporal relations between intracellular Ca2+ wave propagation and PLC activation. Thus, we visualized both cytosolic Ca2+ and PLC activation using a real-time dual-imaging system in individual Mardin-Darby Canine Kidney (MDCK) cells. This system allowed us to simultaneously observe intracellular Ca2+ wave propagation and PLC activation in a spatio-temporal manner in a single mechanically stimulated MDCK cell. The results showed that PLC was activated not only in the mechanically stimulated region but also in other subcellular regions in parallel with intracellular Ca2+ wave propagation. These results support a model in which PLC is involved in Ca2+ signaling amplification in mechanically stimulated cells.

  2. α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells.

    PubMed

    Checco, James W; Lee, Erinna F; Evangelista, Marco; Sleebs, Nerida J; Rogers, Kelly; Pettikiriarachchi, Anne; Kershaw, Nadia J; Eddinger, Geoffrey A; Belair, David G; Wilson, Julia L; Eller, Chelcie H; Raines, Ronald T; Murphy, William L; Smith, Brian J; Gellman, Samuel H; Fairlie, W Douglas

    2015-09-09

    Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

  3. Uptake and intracellular activity of trovafloxacin in human phagocytes and tissue-cultured epithelial cells.

    PubMed Central

    Pascual, A; García, I; Ballesta, S; Perea, E J

    1997-01-01

    The penetration of trovafloxacin into human polymorphonuclear leukocytes (PMNs), human peritoneal macrophages, and tissue-cultured epithelial cells (McCoy cells) was evaluated. The cellular concentration to extracellular concentration (C/E) ratios of trovafloxacin were greater than 9 for extracellular concentrations ranging from 0.5 to 25 micrograms/ml. The uptake of trovafloxacin by PMNs was rapid, reversible, nonsaturable, not energy dependent, and significantly increased at 4 degrees C. Ingestion of opsonized zymosan, but not opsonized Staphylococcus aureus, significantly increased the amount of PMN-associated trovafloxacin. This agent at concentrations of 0.5 and 1 microgram/ml induced a greater reduction in the survival of intracellular S. aureus in PMNs than ciprofloxacin and ofloxacin. It was concluded that trovafloxacin reaches concentrations within phagocytic and nonphagocytic cells several times higher than the extracellular ones, while it remains active in PMNs. PMID:9021179

  4. Plasminogen activator inhibitor 1 is an intracellular inhibitor of furin proprotein convertase.

    PubMed

    Bernot, Denis; Stalin, Jimmy; Stocker, Pierre; Bonardo, Bernadette; Scroyen, Ilse; Alessi, Marie-Christine; Peiretti, Franck

    2011-04-15

    Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.

  5. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  6. Very High Concentrations of Active Intracellular Phosphorylated Emtricitabine in Neonates (ANRS 12109 Trial, Step 2)▿

    PubMed Central

    Hirt, Déborah; Pruvost, Alain; Ekouévi, Didier K.; Urien, Saïk; Arrivé, Elise; Kone, Mamourou; Nerrienet, Eric; Nyati, Mandisa; Gray, Glenda; Kruy, Leang Sim; Blanche, Stéphane; Dabis, François; Tréluyer, Jean-Marc

    2011-01-01

    Our objective was to investigate neonatal emtricitabine (FTC) plasma and intracellular pharmacokinetics. The study was designed as a phase I/II prospective trial in two sequential steps evaluating the combination of tenofovir disoproxil fumarate (TDF) and FTC for the prevention of mother-to-child-transmission (PMTCT) of HIV. HIV-1-infected pregnant women received two tablets of TDF (300 mg) and FTC (200 mg) at onset of labor and then one tablet daily for 7 days postpartum. Based on the data obtained in the first part of the Tenofovir/Emtricitabine in Africa and Asia (TEmAA) Study, single doses of 2 mg/kg of FTC and 13 mg/kg of TDF were given to the neonates within 12 h after birth. A total of 540 FTC plasma concentrations and 44 active intracellular phosphorylated metabolite FTC-TP concentrations were taken from the 36 enrolled women and their neonates. Concentrations were measured by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and analyzed by a population approach. The proposed dose obtained by simulations based on plasma drug concentrations was confirmed. However, median FTC-TP exposures were, respectively, 5.9 and 6.8 times higher in the fetus and the neonate than in the adult. High FTC-TP concentrations were observed in the four children who had serious adverse events (SAEs), but the link between FTC-TP concentrations and SAEs in children was not formally identified. The exposure to the active form of FTC was high in neonates despite plasma drug concentrations equivalent to those in adults. Our results are similar to those obtained with zidovudine or lamivudine. PMID:21464241

  7. Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production.

    PubMed

    Ezeamuzie, Charles I; Taslim, Najla

    2004-11-01

    The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator - phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC(50): 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 microM). In the presence of phosphodiesterase inhibitor rolipram (5 microM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC(50): 55 nM vs 22.5 microM). The inactive PMA analogue, 4alpha-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 microM) and the selective inhibitor of the novel PKCdelta, rottlerin (1-3 microM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01-0.1 microM). Western blot analysis revealed the presence of six PKC isoforms (alpha, betaI, betaII, delta, iota and zeta) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCdelta isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect.

  8. Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production

    PubMed Central

    Ezeamuzie, Charles I; Taslim, Najla

    2004-01-01

    The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator – phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC50: 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 μM). In the presence of phosphodiesterase inhibitor rolipram (5 μM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC50: 55 nM vs 22.5 μM). The inactive PMA analogue, 4α-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 μM) and the selective inhibitor of the novel PKCδ, rottlerin (1–3 μM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01–0.1 μM). Western blot analysis revealed the presence of six PKC isoforms (α, βI, βII, δ, ι and ζ) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCδ isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect. PMID:15504748

  9. Extracellular NAD+ regulates intracellular calcium levels and induces activation of human granulocytes

    PubMed Central

    2005-01-01

    β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation. PMID:16225456

  10. Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei.

    PubMed

    Pérez-Pomares, F; Díaz, S; Bautista, V; Pire, C; Bravo, G; Esclapez, J; Zafrilla, B; Bonete, María-José

    2009-07-01

    Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

  11. Quercetin suppresses intracellular ROS formation, MMP activation, and cell motility in human fibrosarcoma cells.

    PubMed

    Lee, Dong Eun; Chung, Min-Yu; Lim, Tae Gyu; Huh, Won Bum; Lee, Hyong Joo; Lee, Ki Won

    2013-09-01

    Cell metastasis is a major cause of death from cancer and can arise from excessive levels of oxidative stress. The objective of this study was to investigate whether the natural flavonoid quercetin can inhibit matrix metalloproteinase (MMP)-2 and -9 activities through the attenuation of reactive oxygen species (ROS) formation, an event expected to lead to the inhibition of cell motility. To induce sustained ROS formation, cells were treated with phenazine methosulfate (PMS; 1 μM). Noncytotoxic concentrations of quercetin inhibited PMS-induced increases in cell motility in HT1080 human fibrosarcoma (HT1080) cells. While nearly 100% of cells were observed to migrate after 24 h of PMS treatment, quercetin significantly (P < 0.01) suppressed this effect. We also found that quercetin, up to 10 μg/mL, attenuated PMS-induced MMP-2 activation. We then investigated whether the decreased levels of MMP-2 activation could be attributable to lower levels of ROS formation by quercetin. We found that quercetin treatments significantly attenuated PMS-induced ROS formation (P < 0.01) and resulted in decreased cell motility associated with a reduction in MMP-2 and -9 activitiy in HT1080 cells, even in the absence of PMS treatment. Collectively, these results suggest that quercetin inhibits cell motility via the inhibition of MMP activation in HT1080 cells in the presence and absence of PMS. This is likely to be a result of the suppression of intracellular ROS formation by quercetin. © 2013 Institute of Food Technologists®

  12. Prediction of hammerhead ribozyme intracellular activity with the catalytic core fingerprint.

    PubMed

    Gabryelska, Marta Magdalena; Wyszko, Eliza; Szymański, Maciej; Popenda, Mariusz; Barciszewski, Jan

    2013-05-01

    Hammerhead ribozyme is a versatile tool for down-regulation of gene expression in vivo. Owing to its small size and high activity, it is used as a model for RNA structure-function relationship studies. In the present paper we describe a new extended hammerhead ribozyme HH-2 with a tertiary stabilizing motif constructed on the basis of the tetraloop receptor sequence. This ribozyme is very active in living cells, but shows low activity in vitro. To understand it, we analysed tertiary structure models of substrate-ribozyme complexes. We calculated six unique catalytic core geometry parameters as distances and angles between particular atoms that we call the ribozyme fingerprint. A flanking sequence and tertiary motif change the geometry of the general base, general acid, nucleophile and leaving group. We found almost complete correlation between these parameters and the decrease of target gene expression in the cells. The tertiary structure model calculations allow us to predict ribozyme intracellular activity. Our approach could be widely adapted to characterize catalytic properties of other RNAs.

  13. Scanning electrochemical microscopy coupled with intracellular standard addition method for quantification of enzyme activity in single intact cells.

    PubMed

    Gao, Ning; Wang, Xiaolei; Li, Lu; Zhang, Xiaoli; Jin, Wenrui

    2007-11-01

    This paper uses scanning electrochemical microscopy (SECM) coupled with an intracellular standard addition method to quantify enzyme activity in single intact cells. In this work, peroxidase (PO) inside human neutrophils is chosen as the model system. Cells immobilized onto a silanized coverslip are perforated with digitonin to form micropores on the cell membrane. Hydroquinone (H(2)Q) and hydrogen peroxide (H(2)O(2)) as the enzyme substrates diffuse through the micropores into the cell interior. There, H(2)Q is converted into benzoquinone (BQ) by intracellular PO. BQ diffuses with a steady flux through the micropores from the cell interior onto the cell surface. The BQ near the cell surface is detected by the Au tip of SECM held at -0.3 V. When the tip is scanned laterally along the central line over the cell, a 2-D scan curve is obtained. Then, the intracellular standard addition method using ultramicroinjection with a submicrometer-sized micropipette tip is performed. After ultramicroinjection of a standard solution, another 2-D scan curve is recorded. The intercellular enzyme activity can be calculated from both peak current on two scan curves. This method to quantify PO activity in the cell environment has several obvious advantages: high sensitivity due to signal amplification via intracellular enzyme-catalyzed reaction and no sample dilution, no electrode fouling from adsorption of intracellular biological molecules, and no interference from electro-active compounds that can be directly oxidized at the SECM tip or from oxygen in the detected solution.

  14. Role of cathepsin A and lysosomes in the intracellular activation of novel antipapillomavirus agent GS-9191.

    PubMed

    Birkus, Gabriel; Kutty, Nilima; Frey, Christian R; Shribata, Riri; Chou, Tsuifen; Wagner, Carston; McDermott, Martin; Cihlar, Tomas

    2011-05-01

    GS-9191, a bis-amidate prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), was designed as a topical agent for the treatment of papillomavirus-associated proliferative disorders, such as genital warts. In this study, we investigated the mechanism of conversion of GS-9191 to cPrPMEDAP. We observed that GS-9191 is hydrolyzed in the presence of the lysosomal carboxypeptidase cathepsin A (CatA) in vitro and is less efficiently metabolized in CatA-deficient fibroblasts than in control cells. In addition, knockdown of CatA by small interfering RNA (siRNA) reduced the intracellular accumulation of GS-9191 metabolites. However, intracellular CatA levels did not correlate with the susceptibility of tested cell lines to GS-9191, indicating that the CatA step is unlikely to be rate limiting for the activation of GS-9191. Further analysis showed that upon the hydrolysis of the carboxylester bond in one of the GS-9191 amidate moieties, the unmasked carboxyl group displaces L-phenylalanine 2-methylpropyl ester from the other amidate moiety. The cPrPMEDAP-L-phenylalanine conjugate (cPrPMEDAP-Phe) formed is not metabolized by Hint1 (histidine triad nucleotide binding protein 1) phosphoramidase but undergoes spontaneous degradation to cPrPMEDAP in acidic pH that can be significantly enhanced by the addition of SiHa cell extract. Pretreatment of SiHa cells with bafilomycin A or chloroquine resulted in an 8-fold increase in the intracellular concentration of cPrPMEDAP-Phe metabolite and the accumulation of GS-9191 metabolites in the lysosomal/endosomal fraction. Together, these observations indicate that the conversion of GS-9191 to cPrPMEDAP occurs in lysosomes via CatA-mediated ester cleavage, followed by the release of cPrPMEDAP, most likely through the combination of enzyme-driven and spontaneous pH-driven hydrolysis of a cPrPMEDAP-Phe intermediate.

  15. Antibacterial activities of gemifloxacin, levofloxacin, gatifloxacin, moxifloxacin and erythromycin against intracellular Legionella pneumophila and Legionella micdadei in human monocytes.

    PubMed

    Baltch, Aldona L; Bopp, Lawrence H; Smith, Raymond P; Michelsen, Phyllis B; Ritz, William J

    2005-07-01

    The antibacterial activity of a new fluoroquinolone, gemifloxacin, was tested against intracellular Legionella pneumophila and Legionella micdadei and was compared with the activities of levofloxacin, gatifloxacin, moxifloxacin and erythromycin. For intracellular assays, bacteria were used to infect human monocyte-derived macrophages prepared from heparinized blood of healthy volunteers. Antibiotics were added following phagocytosis. Numbers of viable bacteria were determined at 0, 24, 48, 72 and 96 h. The intracellular antibacterial activity of gemifloxacin was concentration- and time-dependent. All of the quinolones had similar activities against L. pneumophila and L. micdadei at 10 x MIC, but there were minor differences: at 24 h moxifloxacin was significantly more active than the other quinolones against L. pneumophila, while gemifloxacin was more active against L. micdadei (P < 0.01). All of the quinolones were markedly more active than erythromycin (P < 0.01). The antibacterial effect of gemifloxacin against L. pneumophila following drug removal at 24 h persisted for 72 h at 20 x MIC but not at 10 x MIC, while for L. micdadei the antibacterial effect persisted for 24 h at 10 x MIC. All of the quinolones had similar activities against intracellular L. pneumophila and L. micdadei and were markedly more effective than erythromycin.

  16. Content Analysis in Systems Engineering Acquisition Activities

    DTIC Science & Technology

    2016-04-30

    qÜáêíÉÉåíÜ=^ååì~ä= ^Åèìáëáíáçå=oÉëÉ~êÅÜ= póãéçëáìã= tÉÇåÉëÇ~ó=pÉëëáçåë= sçäìãÉ=f= = Content Analysis in Systems Engineering Acquisition Activities Karen...of Design Thinking Ronald Giachetti, Chair and Professor, NPS Clifford Whitcomb, Professor, NPS Content Analysis in Systems Engineering...Åèìáëáíáçå=oÉëÉ~êÅÜ=mêçÖê~ãW= `êÉ~íáåÖ=póåÉêÖó=Ñçê=fåÑçêãÉÇ=`Ü~åÖÉ= - 57 - Content Analysis in Systems Engineering Acquisition Activities

  17. Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

    PubMed

    Suzuki, Atsuo; Miyawaki, Yuhri; Okuyama, Eriko; Murata, Moe; Ando, Yumi; Kato, Io; Takagi, Yuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2013-01-01

    Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

  18. Potassium permeability activated by intracellular calcium ion concentration in the pancreatic beta-cell.

    PubMed Central

    Atwater, I; Dawson, C M; Ribalet, B; Rojas, E

    1979-01-01

    1. Membrane potentials and input resistance were measured in beta-cells from mouse pancreatic islets of Langerhans in a study designed to assess the role of a K permeability specifically blocked by quinine or quinidine and activated by intracellular calcium ion concentration ([Ca2+])i-activated PK). 2. Addition of 100 microM-quinine to the perifusion medium resulted in a 10--30 mV depolarization of the membrane and an increase in the input resistance of ca. 4.10(7) omega. 3. In the absence of glucose, 100 microM-quinine induced electrical activity. 4. In the presence of glucose, 100 microM-quinine abolished the burst pattern of electrical activity and very much reduced the graded response of spike frequency normally seen with different concentrations of glucose. 5. Addition of mitochondrial inhibitors, KCN, NaN3, DNP, CCCP, FCCP, to the perifusion medium containing glucose rapidly hyperpolarized the beta-cell membrane, inducing a concomitant decrease in input resistance. 6. In the presence of glucose, these mitochondrial inhibitors reversibly blocked electrical activity; upon removal of the inhibitor, recovery of electrical activity followed a biphasic pattern. 7. The effects of mitochondrial inhibitors were partially reversed by 100 microM-quinine. 8. It is proposed that the membrane potential of the beta-cell in the absence of glucose is predominantly controlled by the [Ca2+]i-activated PK. It is further suggested that this permeability to K controls the level for glucose stimulation and leads to the generation of the burst pattern. PMID:381636

  19. Compounds leached from quinoa seeds inhibit matrix metalloproteinase activity and intracellular reactive oxygen species.

    PubMed

    Graf, B L; Cheng, D M; Esposito, D; Shertel, T; Poulev, A; Plundrich, N; Itenberg, D; Dayan, N; Lila, M A; Raskin, I

    2015-04-01

    Quinoa (Chenopodium quinoa Willd.) is a seed crop rich in bioactive compounds including phytoecdysones (especially 20-hydroxyecdysone, 20HE), polyphenols, proteins and essential fatty acids. We previously reported a method to leach and concentrate quinoa bioactives into a complex phytochemical mixture termed quinoa leachate (QL). Here, we aimed to determine the effect of QL and its chemically distinct fractions on five biochemical endpoints relevant to skin care applications: (i) cell viability, (ii) matrix metalloproteinase (MMP) mRNA expression, (iii) MMP enzymatic activity, (iv) tyrosinase enzymatic activity and (v) intracellular reactive oxygen species (ROS) production. Quinoa leachate was fractionated and chemically characterized using column chromatography and liquid chromatography-mass spectrometry (LC-MS). Cell viability was determined using a MTT assay in four mammalian cell lines. MMP-1 mRNA expression was assessed in human dermal fibroblasts (HDF) via qRT-PCR. The enzymatic activity of MMP-9 and tyrosinase was measured using fluorometric and colorimetric in vitro assays, respectively. Lipopolysaccharide (LPS)-induced ROS production was determined in human dermal fibroblasts by fluorescence intensity of an oxidant-sensitive probe. Quinoa leachate was separated into three fractions: (i) carbohydrate-rich fraction (QL-C; 71.3% w/w of QL); (ii) phytoecdysone, polyphenol and protein-rich fraction (QL-P, 13.3% w/w of QL); (iii) oil-rich fraction (QL-O, 10.8% w/w of QL). QL did not reduce cell viability in any of the four cell lines tested. QL, QL-P and QL-O each significantly inhibited MMP-1 mRNA expression in HDF at a concentration of 5 μg mL(-1) . QL and QL-P also significantly inhibited MMP-9 enzymatic activity, whereas QL-P demonstrated significant tyrosinase enzymatic inhibition. Furthermore, QL, QL-P, QL-O and 20HE significantly inhibited intracellular ROS production. This study is the first to demonstrate the MMP, tyrosinase and ROS inhibiting

  20. Nonlinear Dynamic Modeling of Neuron Action Potential Threshold During Synaptically Driven Broadband Intracellular Activity

    PubMed Central

    Roach, Shane M.; Song, Dong; Berger, Theodore W.

    2012-01-01

    Activity-dependent variation of neuronal thresholds for action potential (AP) generation is one of the key determinants of spike-train temporal-pattern transformations from presynaptic to postsynaptic spike trains. In this study, we model the nonlinear dynamics of the threshold variation during synaptically driven broadband intracellular activity. First, membrane potentials of single CA1 pyramidal cells were recorded under physiologically plausible broadband stimulation conditions. Second, a method was developed to measure AP thresholds from the continuous recordings of membrane potentials. It involves measuring the turning points of APs by analyzing the third-order derivatives of the membrane potentials. Four stimulation paradigms with different temporal patterns were applied to validate this method by comparing the measured AP turning points and the actual AP thresholds estimated with varying stimulation intensities. Results show that the AP turning points provide consistent measurement of the AP thresholds, except for a constant offset. It indicates that 1) the variation of AP turning points represents the nonlinearities of threshold dynamics; and 2) an optimization of the constant offset is required to achieve accurate spike prediction. Third, a nonlinear dynamical third-order Volterra model was built to describe the relations between the threshold dynamics and the AP activities. Results show that the model can predict threshold accurately based on the preceding APs. Finally, the dynamic threshold model was integrated into a previously developed single neuron model and resulted in a 33% improvement in spike prediction. PMID:22156947

  1. Tissue Plasminogen Activator Alters Intracellular Sequestration of Zinc through Interaction with the Transporter ZIP4

    SciTech Connect

    Emmetsberger, Jaime; Mirrione, Martine M.; Zhou, Chun; Fernandez-Monreal, Monica; Siddiq, Mustafa M.; Ji, Kyungmin; Tsirka, Stella E.

    2010-09-17

    Glutamatergic neurons contain free zinc packaged into neurotransmitter-loaded synaptic vesicles. Upon neuronal activation, the vesicular contents are released into the synaptic space, whereby the zinc modulates activity of postsynaptic neurons though interactions with receptors, transporters and exchangers. However, high extracellular concentrations of zinc trigger seizures and are neurotoxic if substantial amounts of zinc reenter the cells via ion channels and accumulate in the cytoplasm. Tissue plasminogen activator (tPA), a secreted serine protease, is also proepileptic and excitotoxic. However, tPA counters zinc toxicity by promoting zinc import back into the neurons in a sequestered form that is nontoxic. Here, we identify the zinc influx transporter, ZIP4, as the pathway through which tPA mediates the zinc uptake. We show that ZIP4 is upregulated after excitotoxin stimulation of the mouse, male and female, hippocampus. ZIP4 physically interacts with tPA, correlating with an increased intracellular zinc influx and lysosomal sequestration. Changes in prosurvival signals support the idea that this sequestration results in neuroprotection. These experiments identify a mechanism via which neurons use tPA to efficiently neutralize the toxic effects of excessive concentrations of free zinc.

  2. Activity of Medicinal Plant Extracts on Multiplication of Mycobacterium tuberculosis under Reduced Oxygen Conditions Using Intracellular and Axenic Assays

    PubMed Central

    Bhatter, Purva D.; Gupta, Pooja D.; Birdi, Tannaz J.

    2016-01-01

    Aim. Test the activity of selected medicinal plant extracts on multiplication of Mycobacterium tuberculosis under reduced oxygen concentration which represents nonreplicating conditions. Material and Methods. Acetone, ethanol and aqueous extracts of the plants Acorus calamus L. (rhizome), Ocimum sanctum L. (leaf), Piper nigrum L. (seed), and Pueraria tuberosa DC. (tuber) were tested on Mycobacterium tuberculosis H37Rv intracellularly using an epithelial cell (A549) infection model. The extracts found to be active intracellularly were further studied axenically under reducing oxygen concentrations. Results and Conclusions. Intracellular multiplication was inhibited ≥60% by five of the twelve extracts. Amongst these 5 extracts, in axenic culture, P. nigrum (acetone) was active under aerobic, microaerophilic, and anaerobic conditions indicating presence of multiple components acting at different levels and P. tuberosa (aqueous) showed bactericidal activity under microaerophilic and anaerobic conditions implying the influence of anaerobiosis on its efficacy. P. nigrum (aqueous) and A. calamus (aqueous and ethanol) extracts were not active under axenic conditions but only inhibited intracellular growth of Mycobacterium tuberculosis, suggesting activation of host defense mechanisms to mediate bacterial killing rather than direct bactericidal activity. PMID:26941797

  3. Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity**

    PubMed Central

    Qian, Ziqing; Xu, Xiaohua; Amacher, Jeanine F.; Madden, Dean R.; Cormet-Boyaka, Estelle

    2015-01-01

    We report a general strategy for intracellular delivery of linear peptidyl ligands by fusing them with a cell-penetrating peptide and cyclizing the fusion peptides through a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear, biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and a CAL-PDZ domain inhibitor for potential treatment of cystic fibrosis. PMID:25785567

  4. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  5. Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules.

    PubMed

    Sznajder, Anna; Jendrossek, Dieter

    2011-03-01

    A Rhodospirillum rubrum gene that is predicted to code for an extracellular poly(3-hydroxybutyrate) (PHB) depolymerase by the recently published polyhydroxyalkanoates (PHA) depolymerase engineering database was cloned. The gene product (PhaZ3( Rru )) was expressed in recombinant E. coli, purified and biochemically characterized. PhaZ3( Rru ) turned out, however, to share characteristics of intracellular PHB depolymerases and revealed a combination of properties that have not yet been described for other PHB depolymerases. A fusion of PhaZ3( Rru )with the enhanced cyan fluorescent protein was able to bind to PHB granules in vivo and supported the function as an intracellular PHB depolymerase. Purified PhaZ3( Rru ) was specific for short-chain-length polyhydroxyalkanoates (PHA(SCL)) and hydrolysed both untreated native PHB granules as well as trypsin-activated native PHB granules to a mixture of mono- and dimeric 3-hydroxybutyrate. Crystalline (denatured) PHB granules were not hydrolysed by PhayZ3( Rru ). Low concentrations of calcium or magnesium ions (1-5 mM) reversibly (EDTA) inhibited the enzyme. Our data suggest that PhaZ3( Rru ) is the representative of a new type of the growing number of intracellular PHB depolymerases.

  6. Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models

    PubMed Central

    Katayanagi, Mishina; Hashimoto, Fumie

    2015-01-01

    Background Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. Objective This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax, respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Methods Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Results Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. Conclusion LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis. PMID:26082583

  7. Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

    PubMed

    Li, He; Fan, Xinqi; Chen, Xing

    2016-02-01

    Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

  8. Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized

    PubMed Central

    Daskalaki, Aikaterini; Shalaby, Nevine A.; Kux, Kristina; Tsoumpekos, Giorgos; Tsibidis, George D.; Muskavitch, Marc A.T.

    2011-01-01

    DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch. PMID:22162135

  9. Stress and radiation-induced activation of multiple intracellular signaling pathways.

    PubMed

    Dent, Paul; Yacoub, Adly; Contessa, Joseph; Caron, Ruben; Amorino, George; Valerie, Kristoffer; Hagan, Michael P; Grant, Steven; Schmidt-Ullrich, Rupert

    2003-03-01

    Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others

  10. Protein tyrosine kinase activity is essential for Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes.

    PubMed Central

    Zheng, L; Nibbering, P H; Zomerdijk, T P; van Furth, R

    1994-01-01

    Our previous study revealed that the intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma receptor I (Fc gamma RI) or Fc gamma RII is a phospholipase C (PLC)-dependent process. The aim of the present study was to investigate whether protein tyrosine kinase (PTK) activity plays a role in the Fc gamma R-mediated intracellular killing of bacteria and activation of PLC in these cells. The results showed that phagocytosis of bacteria by monocytes was not affected by the PTK inhibitors genistein and tyrphostin-47. The intracellular killing of S. aureus by monocytes after cross-linking Fc gamma RII or Fc gamma RII with anti-Fc gamma R monoclonal antibody and a bridging antibody or with human immunoglobulin G (IgG) was inhibited by these compounds in a dose-dependent fashion. The production of O2- by monocytes after stimulation with IgG or IgG-opsonized S. aureus was almost completely blocked by the PTK inhibitor. These results indicate that inhibition of PTK impairs the oxygen-dependent bactericidal mechanisms of monocytes. Genistein and tyrphostin-47, which do not affect the enzymatic activity of purified PLC, prevented activation of PLC after cross-linking Fc gamma RI or Fc gamma RII, measured as an increase in the intracellular inositol 1,4,5-trisphosphate concentration. Cross-linking Fc gamma RI or Fc gamma RII induced rapid tyrosine phosphorylation of several proteins in monocytes, one of which was identified as PLC-gamma 1, and the phosphorylation could be completely blocked by PTK inhibitors, leading to the conclusion that activation of PLC after cross-linking Fc gamma R in monocytes is regulated by PTK activity. Together, these results demonstrate that PTK activity is essential for the activation of PLC which is involved in the Fc gamma R-mediated intracellular killing of S. aureus by human monocytes. Images PMID:7927687

  11. Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism.

    PubMed

    Kerridge, Caroline; Kozlova, Daria I; Nalivaeva, Natalia N; Turner, Anthony J

    2015-01-01

    While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aβ clearance and increasing the risk of development of AD.

  12. Hypoxia Affects Neprilysin Expression Through Caspase Activation and an APP Intracellular Domain-dependent Mechanism

    PubMed Central

    Kerridge, Caroline; Kozlova, Daria I.; Nalivaeva, Natalia N.; Turner, Anthony J.

    2015-01-01

    While gene mutations in the amyloid precursor protein (APP) and the presenilins lead to an accumulation of the amyloid β-peptide (Aβ) in the brain causing neurodegeneration and familial Alzheimer's disease (AD), over 95% of all AD cases are sporadic. Despite the pathologies being indistinguishable, relatively little is known about the mechanisms affecting generation of Aβ in the sporadic cases. Vascular disorders such as ischaemia and stroke are well established risk factors for the development of neurodegenerative diseases and systemic hypoxic episodes have been shown to increase Aβ production and accumulation. We have previously shown that hypoxia causes a significant decrease in the expression of the major Aβ-degrading enzyme neprilysin (NEP) which might deregulate Aβ clearance. Aβ itself is derived from the transmembrane APP along with several other biologically active metabolites including the C-terminal fragment (CTF) termed the APP intracellular domain (AICD), which regulates the expression of NEP and some other genes in neuronal cells. Here we show that in hypoxia there is a significantly increased expression of caspase-3, 8, and 9 in human neuroblastoma NB7 cells, which can degrade AICD. Using chromatin immunoprecipitation we have revealed that there was also a reduction of AICD bound to the NEP promoter region which underlies the decreased expression and activity of the enzyme under hypoxic conditions. Incubation of the cells with a caspase-3 inhibitor Z-DEVD-FMK could rescue the effect of hypoxia on NEP activity protecting the levels of AICD capable of binding the NEP promoter. These data suggest that activation of caspases might play an important role in regulation of NEP levels in the brain under pathological conditions such as hypoxia and ischaemia leading to a deficit of Aβ clearance and increasing the risk of development of AD. PMID:26617481

  13. Intracellular Erythrocyte Platelet-activating Factor Acetylhydrolase I Inactivates Aspirin in Blood*

    PubMed Central

    Zhou, Gang; Marathe, Gopal K.; Willard, Belinda; McIntyre, Thomas M.

    2011-01-01

    Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A2 with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A2 synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood. PMID:21844189

  14. Sandia Cognitive Runtime Engine with Active Memory

    SciTech Connect

    Xavier, Patrick; Chen, Michael C.; Hart, Brian; Hart, Derek; Lippitt, Carl; Wolfenbarger, Paul; Waymire, Russel

    2005-12-01

    The SCREAM (Sandia Cognitive Runtime Engine with Active memory) software implements a subset of a Cognitive Famework developed at Sandia National Laboratories. The software is implemented in the Umbra simulation and modular software framework, which is C++-based. SCREAM components include a Concept Instance Driver, Semantic Activation Network, Concept Database, Context Recognizer, Context Database, Episodic Memory, Egocentric Spatial Memory, Allocentric Spatial Memory, Comparator, and a Context to Abstract Action converter. At initialization, modules load the data files that together specify all the components of a particular cognitive model, such as concept declarations, context declarations, spreading activation weights, and context/situation-cue-patterns.

  15. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

    SciTech Connect

    Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E.; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P.; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W.

    2016-12-16

    Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

  16. Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

    PubMed

    Gunderson, Felizza F; Mallama, Celeste A; Fairbairn, Stephanie G; Cianciotto, Nicholas P

    2015-03-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

  17. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  18. Intracellular mediators of Na -K pump activity in guinea pig pancreatic acinar cells

    SciTech Connect

    Hootman, S.R.; Ochs, D.L.; Williams, J.A.

    1985-10-01

    The involvement of CaS and cyclic nucleotides in neurohormonal regulation of Na -K -ATPase (Na -K pump) activity in guinea pig pancreatic acinar cells was investigated. Changes in Na+-K+ pump activity elicited by secretagogues were assessed by (3H)ouabain binding and by ouabain-sensitive YWRb uptake. Carbachol (CCh) and cholecystokinin octapeptide (CCK-8) each stimulated both ouabain-sensitive 86Rb+ uptake and equilibrium binding of (TH)ouabain by approximately 60%. Secretin increased both indicators of Na+-K+ pump activity by approximately 40% as did forskolin, 8-bromo- and dibutyryl cAMP, theophylline, and isobutylmethylxanthine. Incubation of acinar cells in CaS -free HEPES-buffered Ringer (HR) with 0.5 mM EGTA reduced the stimulatory effects of CCh and CCK-8 by up to 90% but caused only a small reduction in the effects of secretin, forskolin, and cAMP analogues. In addition, CCh, CCK-8, secretin, and forskolin each stimulated ouabain-insensitive 86Rb+ uptake by acinar cells. The increase elicited by CCh and CCK-8 was greatly reduced in the absence of extracellular CaS , while that caused by the latter two agents was not substantially altered. The effects of secretagogues on free CaS levels in pancreatic acinar cells also were investigated with quin-2, a fluorescent CaS chelator. Basal intracellular CaS concentration ((CaS )i) was 161 nM in resting cells and increased to 713 and 803 nM within 15 s after addition of 100 microM CCh or 10 nM CCK-8, respectively.

  19. Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

    DOE PAGES

    Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; ...

    2016-12-16

    Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

  20. A new solid-state microelectrode for measuring intracellular chloride activities.

    PubMed

    Armstrong, W M; Wojtkowski, W; Bixenman, W R

    1977-02-14

    Solid-state microelectrodes from measuring intracellular Cl activity (alphaiCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 mum), coating them under vacuum with a 0.2-0.3 mum thick layer of spectrscopic grade silver, and sealing them (except for the terminal 2-5 mum of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0+/- 0.6 m V (S,E,) per decade c hange in alphaCl. Tip resistance was (77.1+/- 3.1) x 10(9) omega(n=30). Electrode response was rapid (10-20 s), was unaffected by HCO3, H2PO4, HPO42 or protein, and remained essentially unchanged over a 24-h period. AlphaiCl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, alphaiCl significantly exceeded the value corresponding to equlibrium ditribution of Cl across the cell membrane.

  1. Anti-cancer effects of cerium oxide nanoparticles and its intracellular redox activity.

    PubMed

    Pešić, Milica; Podolski-Renić, Ana; Stojković, Sonja; Matović, Branko; Zmejkoski, Danica; Kojić, Vesna; Bogdanović, Gordana; Pavićević, Aleksandra; Mojović, Miloš; Savić, Aleksandar; Milenković, Ivana; Kalauzi, Aleksandar; Radotić, Ksenija

    2015-05-05

    Data on medical applications of cerium oxide nanoparticles CeO2 (CONP) are promising, yet information regarding their action in cells is incomplete and there are conflicting reports about in vitro toxicity. Herein, we have studied cytotoxic effect of CONP in several cancer and normal cell lines and their potential to change intracellular redox status. The IC50 was achieved only in two of eight tested cell lines, melanoma 518A2 and colorectal adenocarcinoma HT-29. Self-propagating room temperature method was applied to produce CONP with an average crystalline size of 4 nm. The results confirmed presence of Ce(3+) and O(2-) vacancies. The induction of cell death by CONP and the production of reactive oxygen species (ROS) were analyzed by flow-cytometry. Free radicals related antioxidant capacity of the cells was studied by the reduction of stable free radical TEMPONE using electron spin resonance spectroscopy. CONP showed low or moderate cytotoxicity in cancer cell lines: adenocarcinoma DLD1 and multi-drug resistant DLD1-TxR, non-small cell lung carcinoma NCI-H460 and multi-drug resistant NCI-H460/R, while normal cell lines (keratinocytes HaCaT, lung fetal fibroblasts MRC-5) were insensitive. The most sensitive were 518A2 melanoma and HT-29 colorectal adenocarcinoma cell lines, with the IC50 values being between 100 and 200 μM. Decreased rate of TEMPONE reduction and increased production of certain ROS species (peroxynitrite and hydrogen peroxide anion) indicates that free radical metabolism, thus redox status was changed, and antioxidant capacity damaged in the CONP treated 518A2 and HT-29 cells. In conclusion, changes in intracellular redox status induced by CONP are partly attributed to the prooxidant activity of the nanoparticles. Further, ROS induced cell damages might eventually lead to the cell death. However, low inhibitory potential of CONP in the other human cell lines tested indicates that CONP may be safe for human usage in industry and medicine.

  2. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

    PubMed Central

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R.; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Do Heo, Won; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  3. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  4. Antagonistic and cooperative actions of Kif7 and Sufu define graded intracellular Gli activities in Hedgehog signaling.

    PubMed

    Law, Kelvin King Lo; Makino, Shigeru; Mo, Rong; Zhang, Xiaoyun; Puviindran, Vijitha; Hui, Chi-Chung

    2012-01-01

    Graded Hedgehog (Hh) signaling governs the balance of Gli transcriptional activators and repressors to specify diverse ventral cell fates in the spinal cord. It remains unclear how distinct intracellular Gli activity is generated. Here, we demonstrate that Sufu acts universally as a negative regulator of Hh signaling, whereas Kif7 inhibits Gli activity in cooperation with, and independent of, Sufu. Together, they deter naïve precursors from acquiring increasingly ventral identity. We show that Kif7 is also required to establish high intracellular Gli activity by antagonizing the Sufu-inhibition of Gli2. Strikingly, by abolishing the negative regulatory action of Sufu, diverse ventral cell fates can be specified in the absence of extracellular Hh signaling. These data suggest that Sufu is the primary regulator of graded Hh signaling and establish that the antagonistic and cooperative actions of Kif7 and Sufu are responsible for setting up distinct Gli activity in ventral cell fate specification.

  5. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  6. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed

    Gommerat, I; Gola, M

    1996-09-15

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  7. Stepwise-activable multifunctional peptide-guided prodrug micelles for cancerous cells intracellular drug release

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Li, Mengfei; Yuan, Zhefan; Wu, Dan; Chen, Jia-da; Feng, Jie

    2016-10-01

    A novel type of stepwise-activable multifunctional peptide-guided prodrug micelles (MPPM) was fabricated for cancerous cells intracellular drug release. Deca-lysine sequence (K10), a type of cell-penetrating peptide, was synthesized and terminated with azido-glycine. Then a new kind of molecule, alkyne modified doxorubicin (DOX) connecting through disulfide bond (DOX-SS-alkyne), was synthesized. After coupling via Cu-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction, reduction-sensitive peptide-guided prodrug was obtained. Due to the amphiphilic property of the prodrug, it can assemble to form micelles. To prevent the nanocarriers from unspecific cellular uptake, the prodrug micelles were subsequently modified with 2,3-dimethyl maleic anhydride to obtain MPPM with a negatively charged outer shell. In vitro studies showed that MPPM could be shielded from cells under psychological environment. However, when arriving at mild acidic tumor site, the cell-penetrating capacity of MPPM would be activated by charge reversal of the micelles via hydrolysis of acid-labile β-carboxylic amides and regeneration of K10, which enabled efficient internalization of MPPM by tumor cells as well as following glutathione- and protease-induced drug release inside the cancerous cells. Furthermore, since the guide peptide sequences can be accurately designed and synthesized, it can be easily changed for various functions, such as targeting peptide, apoptotic peptide, even aptamers, only need to be terminated with azido-glycine. This method can be used as a template for reduction-sensitive peptide-guided prodrug for cancer therapy.

  8. Cell-specific Activation of Nuclear Factor-κB by the Parasite Trypanosoma cruzi Promotes Resistance to Intracellular Infection

    PubMed Central

    Hall, Belinda S.; Tam, Winnie; Sen, Ranjan; Pereira, Miercio E. A.

    2000-01-01

    The transcription factor nuclear factor-κB (NF-κB) is central to the innate and acquired immune response to microbial pathogens, coordinating cellular responses to the presence of infection. Here we demonstrate a direct role for NF-κB activation in controlling intracellular infection in nonimmune cells. Trypanosoma cruzi is an intracellular parasite of mammalian cells with a marked preference for infection of myocytes. The molecular basis for this tissue tropism is unknown. Trypomastigotes, the infectious stage of T. cruzi, activate nuclear translocation and DNA binding of NF-κB p65 subunit and NF-κB-dependent gene expression in epithelial cells, endothelial cells, and fibroblasts. Inactivation of epithelial cell NF-κB signaling by inducible expression of the inhibitory mutant IκBaM significantly enhances parasite invasion. T. cruzi do not activate NF-κB in cells derived from skeletal, smooth, or cardiac muscle, despite the ability of these cells to respond to tumor necrosis factor-α with NF-κB activation. The in vitro infection level in these muscle-derived cells is more than double that seen in the other cell types tested. Therefore, the ability of T. cruzi to activate NF-κB correlates inversely with susceptibility to infection, suggesting that NF-κB activation is a determinant of the intracellular survival and tissue tropism of T. cruzi. PMID:10637298

  9. NHE1 is the sodium-hydrogen exchanger active in acute intracellular pH regulation in preimplantation mouse embryos.

    PubMed

    Siyanov, Violetta; Baltz, Jay M

    2013-06-01

    Sodium-hydrogen exchangers (NHE) of the Slc9 gene family are the major regulators of intracellular pH against acidosis in mammalian cells. Of five plasma membrane NHE isoforms, mouse oocytes and preimplantation embryos express mRNAs encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in oocytes through one-cell stage embryos and lower levels after the two-cell stage. NHE2 (SLC9A2) and NHE5 (SLC9A5) are not expressed. Measurements of intracellular pH during recovery from induced acidosis indicated that recovery occurred via NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). Recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over cariporide alone, indicating that NHE3 did not play a role. There was no indication of NHE4 activity. Another regulator of intracellular pH against acidosis, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. Thus, NHE1 appears to be the only significant regulator of intracellular pH in preimplantation mouse embryos. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development. Unexpectedly, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not affect embryo development to the blastocyst stage more substantially under conditions of chronic acidosis than at normal pH. Preimplantation mouse embryos thus appear to have limited capacity to resist chronic acidosis using intracellular pH regulatory mechanisms.

  10. Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

    PubMed

    Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

    2012-04-15

    Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

  11. Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols.

    PubMed

    Aires, Virginie; Adote, Sylvie; Hichami, Aziz; Moutairou, Kabirou; Boustani, Es-Saddik E; Khan, Naim A

    2004-05-01

    Opuntia ficus indica (prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+ (CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.

  12. Intracellular redox-activated anticancer drug delivery by functionalized hollow mesoporous silica nanoreservoirs with tumor specificity.

    PubMed

    Luo, Zhong; Hu, Yan; Cai, Kaiyong; Ding, Xingwei; Zhang, Quan; Li, Menghuan; Ma, Xing; Zhang, Beilu; Zeng, Yongfei; Li, Peizhou; Li, Jinghua; Liu, Junjie; Zhao, Yanli

    2014-09-01

    In this study, a type of intracellular redox-triggered hollow mesoporous silica nanoreservoirs (HMSNs) with tumor specificity was developed in order to deliver anticancer drug (i.e., doxorubicin (DOX)) to the target tumor cells with high therapeutic efficiency and reduced side effects. Firstly, adamantanamine was grafted onto the orifices of HMSNs using a redox-cleavable disulfide bond as an intermediate linker. Subsequently, a synthetic functional molecule, lactobionic acid-grafted-β-cyclodextrin (β-CD-LA), was immobilized on the surface of HMSNs through specific complexation with the adamantyl group, where β-CD served as an end-capper to keep the loaded drug within HMSNs. β-CD-LA on HMSNs could also act as a targeting agent towards tumor cells (i.e., HepG2 cells), since the lactose group in β-CD-LA is a specific ligand binding with the asialoglycoprotein receptor (ASGP-R) on HepG2 cells. In vitro studies demonstrated that DOX-loaded nanoreservoirs could be selectively endocytosed by HepG2 cells, releasing therapeutic DOX into cytoplasm and efficiently inducing the apoptosis and cell death. In vivo investigations further confirmed that DOX-loaded nanoreservoirs could permeate into the tumor sites and actively interact with tumor cells, which inhibited the tumor growth with the minimized side effect. On the whole, this drug delivery system exhibits a great potential as an efficient carrier for targeted tumor therapy in vitro and in vivo. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Gas turbine engine active clearance control

    NASA Technical Reports Server (NTRS)

    Deveau, Paul J. (Inventor); Greenberg, Paul B. (Inventor); Paolillo, Roger E. (Inventor)

    1985-01-01

    Method for controlling the clearance between rotating and stationary components of a gas turbine engine are disclosed. Techniques for achieving close correspondence between the radial position of rotor blade tips and the circumscribing outer air seals are disclosed. In one embodiment turbine case temperature modifying air is provided in flow rate, pressure and temperature varied as a function of engine operating condition. The modifying air is scheduled from a modulating and mixing valve supplied with dual source compressor air. One source supplies relatively low pressure, low temperature air and the other source supplies relatively high pressure, high temperature air. After the air has been used for the active clearance control (cooling the high pressure turbine case) it is then used for cooling the structure that supports the outer air seal and other high pressure turbine component parts.

  14. [A role of some intracellular signaling cascades in planarian regeneration activated under irradiation with low-temperature argon plasma].

    PubMed

    Ermakov, A M; Ermakova, O N; Maevskiĭ, E I

    2014-01-01

    Using inhibitory analysis the role of some intracellular signaling pathways in activation of planarian regeneration under the influence of low-temperature argon plasma (LTAP) has been investigated. Inactivation of specific inhibitors of intracellular signaling enzymes such as the receptor tyrosine kinase (EGFR), TGF β receptor, calmodulin, adenylate cyclase, phospholipase A2, phospholipase C, cyclin-dependent protein kinase, JAK2-protein kinase, JNK-protein kinase MEK-protein kinase led to inhibition of the head growth during its regeneration in planarians. Pretreatment with LTAP irradiation provided no inhibitory action of some cascades regulating proliferation. However, the inhibitors of the key regulators of regeneration: TGF β receptor, calmodulin and MEK-protein kinase completely suppressed the activating effect of plasma. Thus, by the example of regenerating planarians it is shown, that biological activity of low-temperature argon plasma LTAP is caused by modulation of a plurality of cellular signaling systems.

  15. Active Learning in Engineering Education: A (Re)Introduction

    ERIC Educational Resources Information Center

    Lima, Rui M.; Andersson, Pernille Hammar; Saalman, Elisabeth

    2017-01-01

    The informal network "Active Learning in Engineering Education" (ALE) has been promoting Active Learning since 2001. ALE creates opportunity for practitioners and researchers of engineering education to collaboratively learn how to foster learning of engineering students. The activities in ALE are centred on the vision that learners…

  16. Active Learning in Engineering Education: A (Re)Introduction

    ERIC Educational Resources Information Center

    Lima, Rui M.; Andersson, Pernille Hammar; Saalman, Elisabeth

    2017-01-01

    The informal network "Active Learning in Engineering Education" (ALE) has been promoting Active Learning since 2001. ALE creates opportunity for practitioners and researchers of engineering education to collaboratively learn how to foster learning of engineering students. The activities in ALE are centred on the vision that learners…

  17. Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays.

    PubMed

    Gupta, Pooja; Bhatter, Purva; D'souza, Desiree; Tolani, Monica; Daswani, Poonam; Tetali, Pundarikakshudu; Birdi, Tannaz

    2014-03-04

    In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy.

  18. Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays

    PubMed Central

    2014-01-01

    Background In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. Methods Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 μg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. Results 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. Conclusion A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy. PMID:24592852

  19. Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: The case of the polyhydroxyalkanoates

    PubMed Central

    Martínez, Virginia; Herencias, Cristina; Jurkevitch, Edouard; Prieto, M. Auxiliadora

    2016-01-01

    This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures. PMID:27087466

  20. Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery: The case of the polyhydroxyalkanoates.

    PubMed

    Martínez, Virginia; Herencias, Cristina; Jurkevitch, Edouard; Prieto, M Auxiliadora

    2016-04-18

    This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered were polyhydroxyalkanoates (PHAs) produced naturally by Pseudomonas putida and Cupriavidus necator, or by recombinant Escherichia coli strains. B. bacteriovorus with a mutated PHA depolymerase gene to prevent the unwanted breakdown of the bio-product allowed the recovery of up to 80% of that accumulated by the prey bacteria, even at high biomass concentrations. This innovative downstream process highlights how B. bacteriovorus can be used as a novel, biological lytic agent for the inexpensive, industrial scale recovery of intracellular products from different Gram-negative prey cultures.

  1. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca(2+)) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca(2+)) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca(2+) concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca(2+) concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca(2+) could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  2. Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

    PubMed Central

    2015-01-01

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

  3. Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

    PubMed

    Sengupta, Aritra; Kelly, Sean C; Dwivedi, Nishant; Thadhani, Naresh; Prausnitz, Mark R

    2014-03-25

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5-9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability.

  4. Cannabinoid Receptor Activation Modifies NMDA Receptor Mediated Release of Intracellular Calcium: Implications for Endocannabinoid Control of Hippocampal Neural Plasticity

    PubMed Central

    Hampson, Robert E.; Miller, Frances; Palchik, Guillermo; Deadwyler, Sam A.

    2011-01-01

    Chronic activation or inhibition of cannabinoid receptors (CB1) leads to continuous suppression of neuronal plasticity in hippocampus and other brain regions, suggesting that endocannabinoids may have a functional role in synaptic processes that produce state-dependent transient modulation of hippocampal cell activity. In support of this, it has previously been shown in vitro that cannabinoid CB1 receptors modulate second messenger systems in hippocampal neurons that can modulate intracellular ion channels, including channels which release calcium from intracellular stores. Here we demonstrate in hippocampal slices a similar endocannabinoid action on excitatory glutamatergic synapses via modulation of NMDA-receptor mediated intracellular calcium levels in confocal imaged neurons. Calcium entry through glutamatergic NMDA-mediated ion channels increases intracellular calcium concentrations via modulation of release from ryanodine-sensitive channels in endoplasmic reticulum. The studies reported here show that NMDA-elicited increases in Calcium Green fluorescence are enhanced by CB1 receptor antagonists (i.e. rimonabant), and inhibited by CB1 agonists (i.e. WIN 55,212-2). Suppression of endocannabinoid breakdown by either reuptake inhibition (AM404) or fatty-acid amide hydrolase inhibition (URB597) produced suppression of NMDA elicited calcium increases comparable to WIN 55,212-2, while enhancement of calcium release provoked by endocannabinoid receptor antagonists (Rimonabant) was shown to depend on the blockade of CB1 receptor mediated de-phosphorylation of Ryanodine receptors. Such CB1 receptor modulation of NMDA elicited increases in intracellular calcium may account for the respective disruption and enhancement by CB1 agents of trial-specific hippocampal neuron ensemble firing patterns during performance of a short-term memory task, reported previously from this laboratory. PMID:21288475

  5. Anaplasma marginale Actively Modulates Vacuolar Maturation during Intracellular Infection of Its Tick Vector, Dermacentor andersoni

    PubMed Central

    Thompson, Chelsea Wright; Schneider, David A.; Noh, Susan M.

    2016-01-01

    ABSTRACT Tick-borne transmission of bacterial pathogens in the order Rickettsiales is responsible for diverse infectious diseases, many of them severe, in humans and animals. Transmission dynamics differ among these pathogens and are reflected in the pathogen-vector interaction. Anaplasma marginale has been shown to establish and maintain infectivity within Dermacentor spp. for weeks to months while escaping the complex network of vacuolar peptidases that are responsible for digestion of the tick blood meal. How this prolonged maintenance of infectivity in a potentially hostile environment is achieved has been unknown. Using the natural vector Dermacentor andersoni, we demonstrated that A. marginale-infected tick vacuoles (AmVs) concurrently recruit markers of the early endosome (Rab5), recycling endosome (Rab4 and Rab11), and late endosome (Rab7), are maintained near neutral pH, do not fuse with lysosomes, exclude the protease cathepsin L, and engage the endoplasmic reticulum and Golgi apparatus for up to 21 days postinfection. Maintenance of this safe vacuolar niche requires active A. marginale protein synthesis; in its absence, the AmVs mature into acidic, protease-active phagolysosomes. Identification of this bacterially directed modeling of the tick midgut endosome provides a mechanistic basis for examination of the differences in transmission efficiency observed among A. marginale strains and among vector populations. IMPORTANCE Ticks transmit a variety of intracellular bacterial pathogens that cause significant diseases in humans and animals. For successful transmission, these bacterial pathogens must first gain entry into the tick midgut digestive cells, avoid digestion, and establish a replicative niche without harming the tick vector. Little is known about how this replicative niche is established and maintained. Using the ruminant pathogen A. marginale and its natural tick vector, D. andersoni, this study characterized the features of the A. marginale

  6. Intracellular and membrane-damaging activities of methyl gallate isolated from Terminalia chebula against multidrug-resistant Shigella spp.

    PubMed

    Acharyya, Saurabh; Sarkar, Prodipta; Saha, Dhira R; Patra, Amarendra; Ramamurthy, T; Bag, Prasanta K

    2015-08-01

    Shigella spp. (Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei) cause bacillary dysentery (shigellosis), which is characterized by bloody mucous diarrhoea. Although a variety of antibiotics have been effective for treatment of shigellosis, options are becoming limited due to globally emerging drug resistance. In the present study, in vitro antibacterial activity of methyl gallate (MG) isolated from Terminalia chebula was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity of MG was determined by membrane perturbation and transmission electron microscopy (TEM). Cellular drug accumulation, cell infection and assessment of intracellular activities of MG and reference antibiotics were performed using HeLa cell cultures. The bactericidal activity of MG against multidrug-resistant (MDR) Shigella spp. in comparison with other commonly used drugs including fluoroquinolone was demonstrated here. TEM findings in the present study revealed that MG caused the total disintegration of inner and outer membranes, and leakage of the cytoplasmic contents of S. dysenteriae. The level of accumulation of MG and tetracycline in HeLa cells incubated for 24  h was relatively higher than that of ciprofloxacin and nalidixic acid (ratio of intracellular concentration/extracellular concentration of antibiotic for MG and tetracycline>ciprofloxacin and nalidixic acid). The viable number of intracellular S. dysenteriae was decreased in a time-dependent manner in the presence of MG (4 × MBC) and reduced to zero within 20  h. The significant intracellular activities of MG suggested that it could potentially be used as an effective antibacterial agent for the treatment of severe infections caused by MDR Shigella spp.

  7. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  8. Intracellular delivery of nanocarriers and targeting to subcellular organelles.

    PubMed

    Jhaveri, Aditi; Torchilin, Vladimir

    2016-01-01

    Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field.

  9. Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles

    PubMed Central

    2014-01-01

    Background Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. Results Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. Conclusions Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing

  10. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  11. Active IR-applications in civil engineering

    NASA Astrophysics Data System (ADS)

    Wiggenhauser, H.

    2002-06-01

    Applications of IR-thermography in civil engineering are not limited to the identification of heat losses in building envelopes. As it is well known from other areas of non-destructive testing, active IR-thermographic methods such as cooling down or lock-in thermography improves the results in many investigations. In civil engineering these techniques have not been used widely. Mostly thermography is used in a quasi-static manner. The interpretation of moisture measurements with thermography on surfaces can be very difficult due to several overlapping effects: emissivity changes due to composition, heat transfer through wet sections of the specimen, cooling through air flow or reflected spurious radiation sources. These effects can be reduced by selectively measuring the reflection in two wavelength windows, one on an absorption band of water and another in a reference band and then combining the results in a moisture index image. Cooling down thermography can be used to identify subsurface structural deficiencies. For building materials like concrete these measurements are performed on a much longer time scale than in flash lamp experiments. A quantitative analysis of the full cooling down process over several minutes can reliably identify defects at different depths. Experiments at BAM have shown, that active thermography is capabale of identifying structural deficiencies or moist areas in building materials much more reliable than quasi-static thermography.

  12. Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO2-x NPs

    NASA Astrophysics Data System (ADS)

    Qiu, Yuan; Rojas, Elena; Murray, Richard A.; Irigoyen, Joseba; Gregurec, Danijela; Castro-Hartmann, Pablo; Fledderman, Jana; Estrela-Lopis, Irina; Donath, Edwin; Moya, Sergio E.

    2015-04-01

    Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell endosomes and lysosomes after 24 h of incubation. They also show higher co-localisation with lipid bodies when compared to unmodified CeO2-x NPs. The brush coating does not prevent CeO2-x NPs from displaying antioxidant properties.Cerium Oxide nanoparticles (CeO2-x NPs) are modified with polymer brushes of negatively charged poly (3-sulfopropylmethacrylate) (PSPM) and positively charged poly (2-(methacryloyloxy)ethyl-trimethylammonium chloride) (PMETAC) by Atom Transfer Radical Polymerisation (ATRP). CeO2-x NPs are fluorescently labelled by covalently attaching Alexa Fluor® 488/Fluorescein isothiocyanate to the NP surface prior to polymerisation. Cell uptake, intracellular distribution and the impact on the generation of intracellular Reactive Oxygen Species (ROS) with respect to CeO2-x NPs are studied by means of Raman Confocal Microscopy (CRM), Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). PSPM and PMETAC coated CeO2-x NPs show slower and less uptake compared to uncoated Brush modified NPs display a higher degree of co-localisation with cell

  13. Probing intracellular biomarkers and mediators of cell activation using nanosensors and bioorthogonal chemistry.

    PubMed

    Haun, Jered B; Devaraj, Neal K; Marinelli, Brett S; Lee, Hakho; Weissleder, Ralph

    2011-04-26

    Nanomaterials offer unique physical properties that make them ideal biosensors for scant cell populations. However, specific targeting of nanoparticles to intracellular proteins has been challenging. Here, we describe a technique to improve intracellular biomarker sensing using nanoparticles that is based on bioorthogonal chemistry. Using trans-cyclooctene-modified affinity ligands that are administered to semipermeabilized cells and revealed by cycloaddition reaction with tetrazine-conjugated nanoparticles, we demonstrate site-specific amplification of nanomaterial binding. We also show that this technique is capable of sensing protein biomarkers and phosho-protein signal mediators, both within the cytosol and nucleus, via magnetic or fluorescent modalities. We expect the described method will have broad applications in nanomaterial-based diagnostics and therapeutics.

  14. Proteomes of Host Cell Membranes Modified by Intracellular Activities of Salmonella enterica*

    PubMed Central

    Vorwerk, Stephanie; Krieger, Viktoria; Deiwick, Jörg; Hensel, Michael; Hansmeier, Nicole

    2015-01-01

    Intracellular pathogens need to establish a growth-stimulating host niche for survival and replication. A unique feature of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium is the creation of extensive membrane networks within its host. An understanding of the origin and function of these membranes is crucial for the development of new treatment strategies. However, the characterization of this compartment is very challenging, and only fragmentary knowledge of its composition and biogenesis exists. Here, we describe a new proteome-based approach to enrich and characterize Salmonella-modified membranes. Using a Salmonella mutant strain that does not form this unique membrane network as a reference, we identified a high-confidence set of host proteins associated with Salmonella-modified membranes. This comprehensive analysis allowed us to reconstruct the interactions of Salmonella with host membranes. For example, we noted that Salmonella redirects endoplasmic reticulum (ER) membrane trafficking to its intracellular niche, a finding that has not been described for Salmonella previously. Our system-wide approach therefore has the potential to rapidly close gaps in our knowledge of the infection process of intracellular pathogens and demonstrates a hitherto unrecognized complexity in the formation of Salmonella host niches. PMID:25348832

  15. Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

    PubMed

    Kim, Jung Kuk; Choi, Jung Woong; Lim, Seyoung; Kwon, Ohman; Seo, Jeong Kon; Ryu, Sung Ho; Suh, Pann-Ghill

    2011-06-01

    Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

  16. Intracellular activities related to in vitro hippocampal sharp waves are altered in CA3 pyramidal neurons of aged mice.

    PubMed

    Moradi-Chameh, H; Peng, J; Wu, C; Zhang, L

    2014-09-26

    Pyramidal neurons in the hippocampal CA3 area interconnect intensively via recurrent axonal collaterals, and such CA3-to-CA3 recurrent circuitry plays important roles in the generation of hippocampal network activities. In particular, the CA3 circuitry is able to generate spontaneous sharp waves (SPWs) when examined in vitro. These in vitro SPWs are thought to result from the network activity of GABAergic inhibitory interneurons as SPW-correlating intracellular activities are featured with strong IPSPs in pyramidal neurons and EPSPs or spikes in GABAergic interneurons. In view of accumulating evidence indicating a decrease in subgroups of hippocampal GABAergic interneurons in aged animals, we test the hypothesis that the intracellular activities related to in vitro SPWs are altered in CA3 pyramidal neurons of aged mice. Hippocampal slices were prepared from adult and aged C57 black mice (ages 3-6 and 24-28months respectively). Population and single-cell activities were examined via extracellular and whole-cell patch-clamp recordings. CA3 SPW frequencies were not significantly different between the slices of adult and aged mice but SPW-correlating intracellular activities featured weaker IPSC components in aged CA3 pyramidal neurons compared to adult neurons. It was unlikely that this latter phenomenon was due to general impairments of GABAergic synapses in the aged CA3 circuitry as evoked IPSC responses and pharmacologically isolated IPSCs were observed in aged CA3 pyramidal neurons. In addition, aged CA3 pyramidal neurons displayed more positive resting potentials and had a higher propensity of burst firing than adult neurons. We postulate that alterations of GABAergic network activity may explain the reduced IPCS contributions to in vitro SPWs in aged CA3 pyramidal neurons. Overall, our present observations are supportive of the notion that excitability of hippocampal CA3 circuitry is increased in aged mice.

  17. Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

    PubMed

    Rizaner, Nahit; Onkal, Rustem; Fraser, Scott P; Pristerá, Alessandro; Okuse, Kenji; Djamgoz, Mustafa B A

    2016-10-01

    The possible association of intracellular Ca(2+) with metastasis in human cancer cells is poorly understood. We have studied Ca(2+) signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca(2+) was measured using a membrane-permeant fluorescent Ca(2+)-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca(2+) oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca(2+) level could be induced by applying a high-K(+) solution. Various parameters of the oscillations depended on extracellular Ca(2+) and voltage-gated Na(+) channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na(+) channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca(2+) oscillations. It is concluded that the functional voltage-gated Na(+) channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca(2+) activity. Possible mechanisms and consequences of the Ca(2+) oscillations are discussed.

  18. Functionally active t1-t1 interfaces revealed by the accessibility of intracellular thiolate groups in kv4 channels.

    PubMed

    Wang, Guangyu; Shahidullah, Mohammad; Rocha, Carmen A; Strang, Candace; Pfaffinger, Paul J; Covarrubias, Manuel

    2005-07-01

    Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn(2+) coordination. Also, added Zn(2+) or a potent Zn(2+) chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is approximately 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact alpha-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1--T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn(2+) binding.

  19. Functionally Active T1-T1 Interfaces Revealed by the Accessibility of Intracellular Thiolate Groups in Kv4 Channels

    PubMed Central

    Wang, Guangyu; Shahidullah, Mohammad; Rocha, Carmen A.; Strang, Candace; Pfaffinger, Paul J.; Covarrubias, Manuel

    2005-01-01

    Gating of voltage-dependent K+ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn2+ coordination. Also, added Zn2+ or a potent Zn2+ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ∼200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact α-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1–T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn2+ binding. PMID:15955876

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

    PubMed Central

    Lina, Taslima T.; Dunphy, Paige S.; Luo, Tian

    2016-01-01

    ABSTRACT Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. PMID:27381289

  1. Prostaglandin E2 inhibits NLRP3 inflammasome activation through EP4 receptor and intracellular cAMP in human macrophages

    PubMed Central

    Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L.; Chae, Jae Jin; Shelhamer, James H.

    2015-01-01

    Prostaglandin E2 (PGE2) is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. NLRP3 inflammasome plays an important role in host defense. Uncontrolled activation of NLRP3 inflammasome, due to mutations in the NLRP3 gene causes cryopyrin-associated periodic syndromes (CAPS). Here, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through prostaglandin E receptor 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac). A specific agonist of EP4 mimicked, while its antagonist or EP4 knockdown reversed PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. PKA or Epac agonists did not mimic and their antagonists did not reverse PGE2-mediated NLRP3 inhibition. In addition, constitutive IL-1β secretion from LPS-primed PBMCs of CAPS patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or siRNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  2. pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes

    PubMed Central

    Marquis, Hélène; Hager, Elizabeth J.

    2006-01-01

    Summary Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu. During cell–cell spread, bacteria become transiently entrapped in double-membrane vacuoles. Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide. PC-PLC activation occurs in the acidified vacuolar environment. In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host. PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS–PAGE fluorography. PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3. Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected. Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis. The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells. Immunofluorescence detection of PC-PLC in infected cells was performed. When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells. These results indicate that intracellular bacteria carry pools of proPC-PLC. Upon cell–cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC. PMID:10652090

  3. n-Propyl gallate activates hypoxia-inducible factor 1 by modulating intracellular oxygen-sensing systems.

    PubMed

    Kimura, Motohide; Takabuchi, Satoshi; Tanaka, Tomoharu; Murata, Miyahiko; Nishi, Kenichiro; Oda, Seiko; Oda, Tomoyuki; Kanai, Michiyuki; Fukuda, Kazuhiko; Kizaka-Kondoh, Shinae; Adachi, Takehiko; Takabayashi, Arimichi; Semenza, Gregg L; Hirota, Kiichi

    2008-04-01

    HIF-1 (hypoxia-inducible factor 1) is a master regulator of cellular adaptive responses to hypoxia. The expression and transcriptional activity of the HIF-1alpha subunit is stringently controlled by intracellular oxygen tension through the action of prolyl and asparaginyl hydroxylases. In the present study we demonstrate that PG (n-propyl gallate) activates HIF-1 and expression of its downstream target genes under normoxic conditions in cultured cells and in mice. The stability and transcriptional activity of HIF-1alpha are increased by PG. PG treatment inhibits the interaction between HIF-1alpha and VHL (von Hippel-Lindau protein) and promotes the interaction between HIF-1alpha and p300, indicating that PG inhibits the activity of both prolyl and asparaginyl HIF-1alpha hydroxylases. We conclude that PG activates HIF-1 and enhances the resultant gene expression by directly affecting the intracellular oxygen sensing system in vitro and in vivo and that PG represents a lead compound for the development of a non-toxic activator of HIF-1.

  4. Functionalized Carbon Quantum Dots with Dopamine for Tyrosinase Activity Monitoring and Inhibitor Screening: In Vitro and Intracellular Investigation.

    PubMed

    Chai, Lujing; Zhou, Jin; Feng, Hui; Tang, Cong; Huang, Yuanyuan; Qian, Zhaosheng

    2015-10-28

    Sensitive assay of tyrosinase (TYR) activity is in urgent demand for both fundamental research and practical application, but the exploration of functional materials with good biocompatibility for its activity evaluation at the intracellular level is still challenging until now. In this work, we develop a convenient and real-time assay with high sensitivity for TYR activity/level monitoring and its inhibitor screening based on biocompatible dopamine functionalized carbon quantum dots (Dopa-CQDs). Dopamine with redox property was functionalized on the surface of carbon quantum dots to construct a Dopa-CQDs conjugate with strong bluish green fluorescence. When the dopamine moiety in Dopa-CQDs conjugate was oxidized to a dopaquinone derivative under specific catalysis of TYR, an intraparticle photoinduced electron transfer (PET) process between CQDs and dopaquinone moiety took place, and then the fluorescence of the conjugate could be quenched simultaneously. Quantitative evaluation of TYR activity was established in terms of the relationship between fluorescence quenching efficiency and TYR activity. The assay covered a broad linear range of up to 800 U/L with a low detection limit of 7.0 U/L. Arbutin, a typical inhibitor of TYR, was chosen as an example to assess its function of inhibitor screening, and positive results were observed that fluorescence quenching extent of the probe was reduced in the presence of arbutin. It is also demonstrated that Dopa-CQD conjugate possesses excellent biocompatibility, and can sensitively monitor intracellular tyrosinase level in melanoma cells and intracellular pH changes in living cells, which provides great potential in application of TYR/pH-associated disease monitoring and medical diagnostics.

  5. Inflammatory muscle pain is dependent on the activation of kinin B₁ and B₂ receptors and intracellular kinase pathways.

    PubMed

    Meotti, F C; Campos, R; da Silva, Kabs; Paszcuk, A F; Costa, R; Calixto, J B

    2012-06-01

    B(1) and B(2) kinin receptors are involved in pain transmission but they may have different roles in the muscle pain induced by intense exercise or inflammation. We investigated the contribution of each of these receptors, and the intracellular pathways involved, in the initial development and maintenance of the muscle pain associated with inflammation-induced tissue damage. Mechanical hyperalgesia was measured using the Randall-Selitto apparatus after injecting 5% formalin solution into the gastrocnemius muscle in mice treated with selective antagonists for B(1) or B(2) receptors. The expression of kinin receptors and cytokines and the activation of intracellular kinases were monitored by real-time PCR and immunohistochemistry. The i.m. injection of formalin induced an overexpression of B(1) and B(2) receptors. This overexpression was associated with the mechanical hyperalgesia induced by formalin because treatment with B(1) receptor antagonists (des-Arg(9) [Leu(8)]-BK, DALBK, and SSR240612) or B(2) receptor antagonists (HOE 140 and FR173657) prevented the hyperalgesia. Formalin increased myeloperoxidase activity, and up-regulated TNF-α, IL-1β and IL-6 in gastrocnemius. Myeloperoxidase activity and TNF-α mRNA expression were inhibited by either DALBK or HOE 140, whereas IL-6 was inhibited only by HOE 140. The hyperalgesia induced by i.m. formalin was dependent on the activation of intracellular MAPKs p38, JNK and PKC. Inflammatory muscle pain involves a cascade of events that is dependent on the activation of PKC, p38 and JNK, and the synthesis of IL-1β, TNF-α and IL-6 associated with the up-regulation of both B(1) and B(2) kinin receptors. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  6. Inflammatory muscle pain is dependent on the activation of kinin B1 and B2 receptors and intracellular kinase pathways

    PubMed Central

    Meotti, FC; Campos, R; da Silva, KABS; Paszcuk, AF; Costa, R; Calixto, JB

    2012-01-01

    BACKGROUND AND PURPOSE B1 and B2 kinin receptors are involved in pain transmission but they may have different roles in the muscle pain induced by intense exercise or inflammation. We investigated the contribution of each of these receptors, and the intracellular pathways involved, in the initial development and maintenance of the muscle pain associated with inflammation-induced tissue damage. EXPERIMENTAL APPROACH Mechanical hyperalgesia was measured using the Randall–Selitto apparatus after injecting 5% formalin solution into the gastrocnemius muscle in mice treated with selective antagonists for B1 or B2 receptors. The expression of kinin receptors and cytokines and the activation of intracellular kinases were monitored by real-time PCR and immunohistochemistry. KEY RESULTS The i.m. injection of formalin induced an overexpression of B1 and B2 receptors. This overexpression was associated with the mechanical hyperalgesia induced by formalin because treatment with B1 receptor antagonists (des-Arg9[Leu8]-BK, DALBK, and SSR240612) or B2 receptor antagonists (HOE 140 and FR173657) prevented the hyperalgesia. Formalin increased myeloperoxidase activity, and up-regulated TNF-α, IL-1β and IL-6 in gastrocnemius. Myeloperoxidase activity and TNF-α mRNA expression were inhibited by either DALBK or HOE 140, whereas IL-6 was inhibited only by HOE 140. The hyperalgesia induced by i.m. formalin was dependent on the activation of intracellular MAPKs p38, JNK and PKC. CONCLUSIONS AND IMPLICATIONS Inflammatory muscle pain involves a cascade of events that is dependent on the activation of PKC, p38 and JNK, and the synthesis of IL-1β, TNF-α and IL-6 associated with the up-regulation of both B1 and B2 kinin receptors. PMID:22220695

  7. Intracellular carbonic anhydrase activity sensitizes cancer cell pH signaling to dynamic changes in CO2 partial pressure.

    PubMed

    Hulikova, Alzbeta; Aveyard, Nicholas; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

    2014-09-12

    Carbonic anhydrase (CA) enzymes catalyze the chemical equilibration among CO2, HCO3(-) and H(+). Intracellular CA (CAi) isoforms are present in certain types of cancer, and growing evidence suggests that low levels correlate with disease severity. However, their physiological role remains unclear. Cancer cell CAi activity, measured as cytoplasmic CO2 hydration rate (kf), ranged from high in colorectal HCT116 (∼2 s(-1)), bladder RT112 and colorectal HT29, moderate in fibrosarcoma HT1080 to negligible (i.e. spontaneous kf = 0.18 s(-1)) in cervical HeLa and breast MDA-MB-468 cells. CAi activity in cells correlated with CAII immunoreactivity and enzymatic activity in membrane-free lysates, suggesting that soluble CAII is an important intracellular isoform. CAi catalysis was not obligatory for supporting acid extrusion by H(+) efflux or HCO3(-) influx, nor for maintaining intracellular pH (pHi) uniformity. However, in the absence of CAi activity, acid loading from a highly alkaline pHi was rate-limited by HCO3(-) supply from spontaneous CO2 hydration. In solid tumors, time-dependence of blood flow can result in fluctuations of CO2 partial pressure (pCO2) that disturb cytoplasmic CO2-HCO3(-)-H(+) equilibrium. In cancer cells with high CAi activity, extracellular pCO2 fluctuations evoked faster and larger pHi oscillations. Functionally, these resulted in larger pH-dependent intracellular [Ca(2+)] oscillations and stronger inhibition of the mTORC1 pathway reported by S6 kinase phosphorylation. In contrast, the pHi of cells with low CAi activity was less responsive to pCO2 fluctuations. Such low pass filtering would "buffer" cancer cell pHi from non-steady-state extracellular pCO2. Thus, CAi activity determines the coupling between pCO2 (a function of tumor perfusion) and pHi (a potent modulator of cancer cell physiology). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

    PubMed

    Delles, C; Haller, T; Dietl, P

    1995-08-01

    1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent

  9. Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

    PubMed

    González-Barriga, Anchel; Nillessen, Bram; Kranzen, Julia; van Kessel, Ingeborg D G; Croes, Huib J E; Aguilera, Begoña; de Visser, Peter C; Datson, Nicole A; Mulders, Susan A M; van Deutekom, Judith C T; Wieringa, Bé; Wansink, Derick G

    2017-04-04

    Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

  10. Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

    PubMed Central

    1985-01-01

    Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4- bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6- bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5- trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction. PMID:3874873

  11. Mycobacterium tuberculosis Differentially Activates cGAS- and Inflammasome-Dependent Intracellular Immune Responses through ESX-1.

    PubMed

    Wassermann, Ruth; Gulen, Muhammet F; Sala, Claudia; Perin, Sonia Garcia; Lou, Ye; Rybniker, Jan; Schmid-Burgk, Jonathan L; Schmidt, Tobias; Hornung, Veit; Cole, Stewart T; Ablasser, Andrea

    2015-06-10

    Cytosolic detection of microbial products is essential for the initiation of an innate immune response against intracellular pathogens such as Mycobacterium tuberculosis (Mtb). During Mtb infection of macrophages, activation of cytosolic surveillance pathways is dependent on the mycobacterial ESX-1 secretion system and leads to type I interferon (IFN) and interleukin-1β (IL-1β) production. Whereas the inflammasome regulates IL-1β secretion, the receptor(s) responsible for the activation of type I IFNs has remained elusive. We demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential for initiating an IFN response to Mtb infection. cGAS associates with Mtb DNA in the cytosol to stimulate cyclic GAMP (cGAMP) synthesis. Notably, activation of cGAS-dependent cytosolic host responses can be uncoupled from inflammasome activation by modulating the secretion of ESX-1 substrates. Our findings identify cGAS as an innate sensor of Mtb and provide insight into how ESX-1 controls the activation of specific intracellular recognition pathways.

  12. Inhibitory effects of SSRIs on IFN-γ induced microglial activation through the regulation of intracellular calcium.

    PubMed

    Horikawa, Hideki; Kato, Takahiro A; Mizoguchi, Yoshito; Monji, Akira; Seki, Yoshihiro; Ohkuri, Takatoshi; Gotoh, Leo; Yonaha, Megumi; Ueda, Tadashi; Hashioka, Sadayuki; Kanba, Shigenobu

    2010-10-01

    Microglia, which are a major glial component of the central nervous system (CNS), have recently been suggested to mediate neuroinflammation through the release of pro-inflammatory cytokines and nitric oxide (NO). Microglia are also known to play a critical role as resident immunocompetent and phagocytic cells in the CNS. Immunological dysfunction has recently been demonstrated to be associated with the pathophysiology of depression. However, to date there have only been a few studies on the relationship between microglia and depression. We therefore investigated if antidepressants can inhibit microglial activation in vitro. Our results showed that the selective serotonin reuptake inhibitors (SSRIs) paroxetine and sertraline significantly inhibited the generation of NO and tumor necrosis factor (TNF)-α from interferon (IFN)-γ-activated 6-3 microglia. We further investigated the intracellular signaling mechanism underlying NO and TNF-α release from IFN-γ-activated 6-3 microglia. Our results suggest that paroxetine and sertraline may inhibit microglial activation through inhibition of IFN-γ-induced elevation of intracellular Ca(2+). Our results suggest that the inhibitory effect of paroxetine and sertraline on microglial activation may not be a prerequisite for antidepressant function, but an additional beneficial effect.

  13. An In Vivo Selection Identifies Listeria monocytogenes Genes Required to Sense the Intracellular Environment and Activate Virulence Factor Expression

    PubMed Central

    Portnoy, Daniel A.

    2016-01-01

    Listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and ActA-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator PrfA. The environmental cues that lead to temporal and spatial control of L. monocytogenes virulence gene expression are poorly understood. In this study, we took advantage of the robust up-regulation of ActA that occurs intracellularly and expressed Cre recombinase from the actA promoter and 5’ untranslated region in a strain in which loxP sites flanked essential genes, so that activation of actA led to bacterial death. Upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for ActA expression. Strikingly, most of the genes, including gshF, spxA1, yjbH, and ohrA, are predicted to play important roles in bacterial redox regulation. The mutants identified in the genetic selection fell into three broad categories: (1) those that failed to reach the cytosolic compartment; (2) mutants that entered the cytosol, but failed to activate the master virulence regulator PrfA; and (3) mutants that entered the cytosol and activated transcription of actA, but failed to synthesize it. The identification of mutants defective in vacuolar escape suggests that up-regulation of ActA occurs in the host cytosol and not the vacuole. Moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of PrfA via increased glutathione levels and transcriptional activation of actA while the second results in translational activation of actA and requires yjbH. Although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. PMID:27414028

  14. Identification of Three Classes of Heteroaromatic Compounds with Activity against Intracellular Trypanosoma cruzi by Chemical Library Screening

    PubMed Central

    Bettiol, Esther; Samanovic, Marie; Murkin, Andrew S.; Raper, Jayne; Buckner, Frederick; Rodriguez, Ana

    2009-01-01

    The development of new drugs against Chagas disease is a priority since the currently available medicines have toxic effects, partial efficacy and are targeted against the acute phase of disease. At present, there is no drug to treat the chronic stage. In this study, we have optimized a whole cell-based assay for high throughput screening of compounds that inhibit infection of mammalian cells by Trypanosoma cruzi trypomastigotes. A 2000-compound chemical library was screened using a recombinant T. cruzi (Tulahuen strain) expressing β-galactosidase. Three hits were selected for their high activity against T. cruzi and low toxicity to host cells in vitro: PCH1, NT1 and CX1 (IC50: 54, 190 and 23 nM, respectively). Each of these three compounds presents a different mechanism of action on intracellular proliferation of T. cruzi amastigotes. CX1 shows strong trypanocidal activity, an essential characteristic for the development of drugs against the chronic stage of Chagas disease where parasites are found intracellular in a quiescent stage. NT1 has a trypanostatic effect, while PCH1 affects parasite division. The three compounds also show high activity against intracellular T. cruzi from the Y strain and against the related kinetoplastid species Leishmania major and L. amazonensis. Characterization of the anti–T. cruzi activity of molecules chemically related to the three library hits allowed the selection of two compounds with IC50 values of 2 nM (PCH6 and CX2). These values are approximately 100 times lower than those of the medicines used in patients against T. cruzi. These results provide new candidate molecules for the development of treatments against Chagas disease and leishmaniasis. PMID:19238193

  15. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  16. Magneto-optical cellular chip model for intracellular orientational-dynamic-activity detection

    NASA Astrophysics Data System (ADS)

    Miyashita, Y.; Iwasaka, M.; Kurita, S.; Owada, N.

    2012-04-01

    In the present study, a magneto-optical cellular chip model (MoCCM) was developed to detect intracellular dynamics in macromolecules by using magneto-optical effects. For the purpose of cell-measurement under strong static magnetic fields of up to 10 T, we constructed a cellular chip model, which was a thin glass plate with a well for a cell culture. A cell line of osteoblast MC3T3-E1 was incubated in the glass well, and the well, 0.3 mm in depth, was sealed by a cover glass when the MoCCM was set in a fiber optic system. An initial intensity change of the polarized light transmission, which dispersed perpendicular to the cell's attaching surface, was collected for 10 to 60 min, and then magnetic fields were applied parallel and perpendicular to the surface and light direction, respectively. The magnetic birefringence signals that originated from the magnetic orientation of intracellular molecules such as cytoskeletons apparently appeared when the magnetic fields were constant at 10 T. A statistical analysis with 15 experiments confirmed that the cellular components under 10 T magnetic fields caused a stronger alignment, which was transferred into polarizing light intensity that increased more than the case before exposure. Cellular conditions such as generation and cell density affected the magnetic birefringence signals.

  17. Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.

    PubMed

    Kuiper, Caroline; Dachs, Gabi U; Currie, Margaret J; Vissers, Margreet C M

    2014-04-01

    Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.

  18. Sonic hedgehog stimulates the proliferation of rat gastric mucosal cells through ERK activation by elevating intracellular calcium concentration

    SciTech Connect

    Osawa, Hiroyuki; Ohnishi, Hirohide . E-mail: hohnishi@jichi.ac.jp; Takano, Koji; Noguti, Takasi; Mashima, Hirosato; Hoshino, Hiroko; Kita, Hiroto; Sato, Kiichi; Matsui, Hirofumi; Sugano, Kentaro

    2006-06-02

    Sonic Hedgehog (Shh), a member of hedgehog peptides family, is expressed in gastric gland epithelium. To elucidate Shh function to gastric mucosal cells, we examined the effect of Shh on the proliferation of a rat normal gastric mucosal cell line, RGM-1. RGM-1 cells express essential components of Shh receptor system, patched-1, and smoothened. Shh enhanced DNA synthesis in RGM-1 cells and elevated intracellular calcium concentration ([Ca{sup 2+}]{sub i}). In addition, Shh as well as calcium ionophore A32187 rapidly activated ERK. However, Shh failed to activate ERK under calcium-free culture condition. Pretreatment of cells with PD98059 attenuated the DNA synthesis promoted by Shh. Moreover, when cells were pretreated with cyclopamine, Shh could not elevate [Ca{sup 2+}]{sub i}, activate ERK or promote DNA synthesis. On the other hand, although Shh induced Gli-1 nuclear accumulation in RGM-1 cells, Shh activated ERK even in cells pretreated with actinomycin D. These results indicate that Shh promotes the proliferation of RGM-1 cells through an intracellular calcium- and ERK-dependent but transcription-independent pathway via Patched/Smoothened receptor system.

  19. Intracellular Metabolism of Nucleoside/Nucleotide Analogues: a Bottleneck to Reach Active Drugs on HIV Reverse Transcriptase.

    PubMed

    Varga, Andrea; Lionne, Corinne; Roy, Béatrice

    2016-01-01

    To date, the most effective way to treat HIV is to use a highly active antiretroviral therapy (HAART) that combines three or more different drugs. The usual regimen consists of two nucleoside reverse transcriptase inhibitors and either a protease inhibitor, a non-nucleoside reverse transcriptase inhibitor, or an integrase strand transfer inhibitor. Due to the emerging resistance against the nucleoside analogues in use, there is a continuous need for the development of such therapeutic molecules with different structural features. In this review, we would like to summarize the state of knowledge of the antiretroviral nucleoside analogues intracellular metabolism. Indeed, these molecules have to be phosphorylated in the cell, a process that is often a bottleneck, to produce their pharmacologically active triphosphorylated forms. These forms can be used by the HIV reverse transcriptase. Because they lack a 3'-hydroxyl group, they block further extension of the viral DNA, and finally lead to early chain termination. Several kinases can act on the phosphorylation of these drugs; most of them have low nucleoside/nucleotide specificity. On the other hand, there are also nucleotidases in the cell, which can reverse the phosphorylation process, thus shifting the equilibrium from the active triphosphorylated state to the non-active (not-, mono- or di-phosphorylated) states of these analogues. Here, we would like to bring to the attention of the medicinal chemists that they have to take into account the limitation of the intracellular phosphorylation machinery when designing new nucleoside analogue drugs.

  20. Engineering Challenges for Active Debris Removal

    NASA Technical Reports Server (NTRS)

    Liou, Jer-Chyi

    2011-01-01

    Recent modeling studies on the instability of the debris population in the low Earth orbit (LEO) region and the collision between Iridium 33 and Cosmos 2251 have underlined the need for active debris removal. A 2009 analysis by the NASA Orbital Debris Program Office shows that, in order to maintain the LEO debris population at a constant level for the next 200 years, an active debris removal of about five objects per year is needed. The targets identified for removal are those with the highest mass and collision probability products in the environment. Many of these objects are spent upper stages with masses ranging from 1 to more than 8 metric tons, residing in several altitude regions and concentrated in about 10 inclination bands. To remove five of those objects on a yearly basis, in a cost-effective manner, represents many challenges in engineering, technology development, and operations. This paper outlines a conceptual end-to-end debris removal operation, including launch, precision tracking, rendezvous, stabilization (of the tumbling targets), capture, and deorbit of the targets; and highlights major challenges associated with the operations. Pros and cons of several proposed removal techniques are also evaluated.

  1. Effect of external sodium on intracellular chloride activity in the surface cells of frog gastric mucosa. Microelectrode studies.

    PubMed

    Curci, S; Schettino, T

    1984-06-01

    The intracellular chloride activity and its dependence on ionic substitutions in the bathing media was studied in individual surface cells of resting gastric mucosa using conventional and Cl- selective microelectrodes. When the tissue was perfused with control NaCl-Ringer the cell membrane p.d.'s, cell-lumen (psi cm) and cell-serosa (psi cs) were -40.9 +/- 0.6 mV and -66.8 +/- 0.5 mV (n = 175) respectively and the p.d. measured by the Cl- selective microelectrodes across the serosal membrane (psi csCl-) averaged -32.4 +/- 0.7 mV (n = 138). From these values an intracellular Cl- activity (acCl-) of 15.3 mmol/l can be estimated. The data indicate that chloride ion is distributed close to equilibrium at the luminal membrane while it is accumulated by an energy requiring step at the serosal membrane. Reduction (2 mmol/l) or absence of chloride from the luminal bath did not result in any detectable change of acCl-; on the other hand, after removal of Cl- from the serosal bath the intracellular Cl- activity fell to 7.1 mmol/l. When the tissue was exposed to serosal Na+-free Ringer (Na+ replaced by choline or TMA), although the acCl- remained unaffected, a marked reduction of the electrochemical gradient for Cl- at the serosal membrane was observed. These data indicate that: chloride is accumulated in the surface cells against its electrochemical potential difference at the serosal membrane; the luminal membrane has a negligible conductance to Cl-, while the serosal membrane represents a conductive pathway to chloride; the uphill entry of chloride at the serosal membrane seems to be, at least partially, Na+-dependent.

  2. Intracellular Antioxidant Activity of Grape Skin Polyphenolic Extracts in Rat Superficial Colonocytes: In situ Detection by Confocal Fluorescence Microscopy

    PubMed Central

    Giordano, M. Elena; Ingrosso, Ilaria; Schettino, Trifone; Caricato, Roberto; Giovinazzo, Giovanna; Lionetto, M. Giulia

    2016-01-01

    Colon is exposed to a number of prooxidant conditions and several colon diseases are associated with increased levels of reactive species. Polyphenols are the most abundant antioxidants in the diet, but to date no information is available about their absorption and potential intracellular antioxidant activity on colon epithelial cells. The work was addressed to study the intracellular antioxidant activity of red grape polyphenolic extracts on rat colon epithelium experimentally exposed to prooxidant conditions. The experimental model chosen was represented by freshly isolated colon explants, which closely resemble the functional, and morphological characteristics of the epithelium in vivo. The study was carried out by in situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15 min). The qualitative and quantitative polyphenolic composition of the extracts as well as their in vitro oxygen radical absorbing capacity (ORAC) was determined. The incubation of the explants with the polyphenolic extracts for 1 h produced a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. The results demonstrated the ability of red grape polyphenols to cross the plasma membrane and exert a direct intracellular antioxidant activity in surface colonocytes, inducing a protection against pro-oxidant conditions. The changes in the polyphenol composition due to ripening process was reflected in a more effective antioxidant protection. PMID:27303304

  3. Intracellular Antioxidant Activity of Grape Skin Polyphenolic Extracts in Rat Superficial Colonocytes: In situ Detection by Confocal Fluorescence Microscopy.

    PubMed

    Giordano, M Elena; Ingrosso, Ilaria; Schettino, Trifone; Caricato, Roberto; Giovinazzo, Giovanna; Lionetto, M Giulia

    2016-01-01

    Colon is exposed to a number of prooxidant conditions and several colon diseases are associated with increased levels of reactive species. Polyphenols are the most abundant antioxidants in the diet, but to date no information is available about their absorption and potential intracellular antioxidant activity on colon epithelial cells. The work was addressed to study the intracellular antioxidant activity of red grape polyphenolic extracts on rat colon epithelium experimentally exposed to prooxidant conditions. The experimental model chosen was represented by freshly isolated colon explants, which closely resemble the functional, and morphological characteristics of the epithelium in vivo. The study was carried out by in situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15 min). The qualitative and quantitative polyphenolic composition of the extracts as well as their in vitro oxygen radical absorbing capacity (ORAC) was determined. The incubation of the explants with the polyphenolic extracts for 1 h produced a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. The results demonstrated the ability of red grape polyphenols to cross the plasma membrane and exert a direct intracellular antioxidant activity in surface colonocytes, inducing a protection against pro-oxidant conditions. The changes in the polyphenol composition due to ripening process was reflected in a more effective antioxidant protection.

  4. Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BKCa activator LDD175

    PubMed Central

    Wijerathne, Tharaka Darshana; Kim, Jihyun; Yang, Dongki

    2017-01-01

    Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm. PMID:28280418

  5. Characteristics of receptor- and transducer-coupled activation of the intracellular signalling in sensory neuron revealed by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Khalisov, M. M.; Penniyaynen, V. A.; Esikova, N. A.; Ankudinov, A. V.; Krylov, B. V.

    2017-01-01

    The mechanical properties of sensory neurons upon activation of intracellular cascade processes by comenic acid binding to a membrane opioid-like receptor (receptor-coupled), as well as a very low (endogenous) concentration of ouabain (transducer-coupled), have been investigated. Using atomic force microscopy, it is established that exposure to ouabain, in contrast to the impact of comenic acid, leads to a hardening of the neuron soma. This suggests that the receptor-coupled signal transmission to the cell genome is carried out through mechanisms that are different from the transducer-coupled signal pathways.

  6. Vehicle Systems Engineering and Integration Activities

    DTIC Science & Technology

    2012-08-31

    Reliability Improvements for Selected Equipment) package. The standard RISE package includes an upgraded propulsion system ( turbocharged engine and...component of the RISE powertrain incorporated into the original M113A3 and M730A2 vehicles is the turbocharged 275 hp 6V53T engine from Detroit Diesel...Corporation. Replacing the earlier 212 hp M113A2 engine with the turbocharged 275 hp model paved the way for improvements in performance as well as

  7. Engineering enzyme-cleavable hybrid click capsules with a pH-sheddable coating for intracellular degradation.

    PubMed

    Gunawan, Sylvia T; Liang, Kang; Such, Georgina K; Johnston, Angus P R; Leung, Melissa K M; Cui, Jiwei; Caruso, Frank

    2014-10-29

    The engineering of layer-by-layer (LbL) hybrid click capsules that are responsive to biological stimuli is reported. The capsules comprise a pH-sheddable, non cross-linked outer coating that protects enzyme-cleavable inner layers. Upon cellular uptake, the outer coating is released and the capsules are enzymatically degraded. In vitro cell degradation results in rapid capsule degradation (10 min) upon cellular internalization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

    PubMed

    Li, Duo-ling; Ma, Zhi-yong; Fu, Zhi-jie; Ling, Ming-ying; Yan, Chuan-zhu; Zhang, Yun

    2014-01-01

    Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca(2+)]i) handling in Raw 264.7 macrophages. In the present study, [Ca(2+)]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+) channel blockers had no effect on the resting [Ca(2+)]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca(2+)]i transient elevation independent of extracellular Ca(2+). The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca(2+)]i transient elevation, but pinacidil and other unselective K(+) channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+)]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+)]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+)]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

  9. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    PubMed Central

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels. PMID:26682007

  10. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells.

    PubMed

    Hou, Ya-Qin; Yao, Yao; Bao, Yong-Li; Song, Zhen-Bo; Yang, Cheng; Gao, Xiu-Li; Zhang, Wen-Jing; Sun, Lu-Guo; Yu, Chun-Lei; Huang, Yan-Xin; Wang, Guan-Nan; Li, Yu-Xin

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels.

  11. A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

    PubMed

    Strege, Peter R; Bernard, Cheryl E; Mazzone, Amelia; Linden, David R; Beyder, Arthur; Gibbons, Simon J; Farrugia, Gianrico

    2015-11-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors.

  12. A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

    PubMed Central

    Strege, Peter R.; Bernard, Cheryl E.; Mazzone, Amelia; Linden, David R.; Beyder, Arthur; Gibbons, Simon J.

    2015-01-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca2+-activated Cl− channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca2+ regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-“exon 0” upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca2+ sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(−0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl− currents between isoforms with varying concentrations of intracellular Ca2+, extracellular anions, or Cl− channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(−0) in response to varying intracellular Ca2+. The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(−0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(−0). In conclusion, human Ano1 containing exon 0 imparts its Cl− current with greater sensitivity to intracellular Ca2+ and CACC inhibitors. PMID:26359375

  13. Disfacilitation and active inhibition in the neocortex during the natural sleep-wake cycle: An intracellular study

    PubMed Central

    Timofeev, Igor; Grenier, François; Steriade, Mircea

    2001-01-01

    Earlier extracellular recordings during natural sleep have shown that, during slow-wave sleep (SWS), neocortical neurons display long-lasting periods of silence, whereas they are tonically active and discharge at higher rates during waking and sleep with rapid eye movements (REMs). We analyzed the nature of long-lasting periods of neuronal silence in SWS and the changes in firing rates related to ocular movements during REM sleep and waking using intracellular recordings from electrophysiologically identified neocortical neurons in nonanesthetized and nonparalyzed cats. We found that the silent periods during SWS are associated with neuronal hyperpolarizations, which are due to a mixture of K+ currents and disfacilitation processes. Conventional fast-spiking neurons (presumably local inhibitory interneurons) increased their firing rates during REMs and eye movements in waking. During REMs, the firing rates of regular-spiking neurons from associative areas decreased and intracellular traces revealed numerous, short-lasting, low-amplitude inhibitory postsynaptic potentials (IPSPs), that were reversed after intracellular chloride infusion. In awake cats, regular-spiking neurons could either increase or decrease their firing rates during eye movements. The short-lasting IPSPs associated with eye movements were still present in waking; they preceded the spikes and affected their timing. We propose that there are two different forms of firing rate control: disfacilitation induces long-lasting periods of silence that occur spontaneously during SWS, whereas active inhibition, consisting of low-amplitude, short-lasting IPSPs, is prevalent during REMs and precisely controls the timing of action potentials in waking. PMID:11172052

  14. Practice of Organized Team-Activities on Engineering Foundations Education

    NASA Astrophysics Data System (ADS)

    Aoki, Katsuhik

    Kanazawa Institute of Technology established Engineering Foundations Education Center (EFEC) in 2000. At the almost same time, the Math. and Science Education Study Meeting (MSESM) was organized as an engine for organized team-activities of EFEC. The MSESM has investigated various problems concerning the engineering foundation education and found out various measures against them. Those measures have been applied to the curriculum of the Engineering Foundations Education and its learning support programs. Herein, the chronological data of education effects brought by the above from 2005 to 2008 are presented and the current team-activities of MSESM including Faculty Development (FD) activities of EFEC are also reported.

  15. Intracellular Parasite Invasion Strategies

    NASA Astrophysics Data System (ADS)

    Sibley, L. D.

    2004-04-01

    Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

  16. Identification of a Lambda Toxin-Negative Clostridium perfringens Strain that Processes and Activates Epsilon Prototoxin Intracellularly

    PubMed Central

    Harkness, Justine M.; Li, Jihong; McClane, Bruce A.

    2012-01-01

    Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation. PMID:22982043

  17. CIA2 deficiency results in impaired oxidative stress response and enhanced intracellular basal UPR activity in Saccharomyces cerevisiae.

    PubMed

    Zhao, Wei; Zheng, Hua-Zhen; Niu, Yu-Jie; Yuan, Yuan; Fang, Bing-Xiong; Liu, Yi-Na; Cai, Lu-Hui; Zhou, Zhong-Jun; Liu, Xin-Guang

    2015-03-01

    Yeast Cia2p is a component of the cytosolic Fe/S protein assembly (CIA) machinery. Initial studies of the CIA machinery were performed in yeast, but the precise role of Cia2p in this eukaryote is still unknown. We report that CIA2 deficiency results in impaired oxidative stress response, as evidenced by increased sensitivity to the oxidant cumene hydroperoxide (CHP), impaired activities of superoxide dismutases and aconitase and decreased replicative lifespan in the mutants. Moreover, intracellular reactive oxygen species levels were significantly increased in CIA2-deficient cells after treatment with CHP. We also show that CIA2-deficient cells display an increased resistance to tunicamycin-induced endoplasmic reticulum (ER) stress, as evidenced by the upregulated splicing of the mRNA of HAC1, which encodes a functional transcription factor that regulates the transcription of unfolded protein response (UPR) target genes, suggesting enhanced intracellular UPR activity. Furthermore, the transcription of several canonical UPR target genes is strongly induced in CIA2-deficient cells as compared with wild-type controls. Taken together, these results suggest the involvement of Cia2p in oxidative and ER stress responses in yeast. Published by Oxford University Press on behalf of FEMS 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  18. Trifluoromethyl arylamides with antileukemia effect and intracellular inhibitory activity over serine/arginine-rich protein kinases (SRPKs).

    PubMed

    Siqueira, Raoni Pais; Barros, Marcus Vinícius de Andrade; Barbosa, Éverton de Almeida Alves; Onofre, Thiago Souza; Gonçalves, Victor Hugo Sousa; Pereira, Higor Sette; Silva Júnior, Abelardo; de Oliveira, Leandro Licursi; Almeida, Márcia Rogéria; Fietto, Juliana Lopes Rangel; Teixeira, Róbson Ricardo; Bressan, Gustavo Costa

    2017-07-07

    The serine/arginine-rich protein kinases (SRPKs) have frequently been found with altered activity in a number of cancers, suggesting they could serve as potential therapeutic targets in oncology. Here we describe the synthesis of a series of twenty-two trifluoromethyl arylamides based on the known SRPKs inhibitor N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) and the evaluation of their antileukemia effects. Some derivatives presented superior cytotoxic effects against myeloid and lymphoid leukemia cell lines compared to SRPIN340. In particular, compounds 24, 30, and 36 presented IC50 values ranging between 6.0 and 35.7 μM. In addition, these three compounds were able to trigger apoptosis and autophagy, and to exhibit synergistic effects with the chemotherapeutic agent vincristine. Furthermore, compound 30 was more efficient than SRPIN340 in impairing the intracellular phosphorylation status of SR proteins as well as the expression of MAP2K1, MAP2K2, VEGF, and RON oncogenic isoforms. Therefore, novel compounds with increased intracellular effects against SRPK activity were obtained, contributing to medicinal chemistry efforts towards the development of new anticancer agents. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. Dopamine synthesis and D3 receptor activation in pancreatic β-cells regulates insulin secretion and intracellular [Ca(2+)] oscillations.

    PubMed

    Ustione, Alessandro; Piston, David W

    2012-11-01

    Pancreatic islets are critical for glucose homeostasis via the regulated secretion of insulin and other hormones. We propose a novel mechanism that regulates insulin secretion from β-cells within mouse pancreatic islets: a dopaminergic negative feedback acting on insulin secretion. We show that islets are a site of dopamine synthesis and accumulation outside the central nervous system. We show that both dopamine and its precursor l-dopa inhibit glucose-stimulated insulin secretion, and this inhibition correlates with a reduction in frequency of the intracellular [Ca(2+)] oscillations. We further show that the effects of dopamine are abolished by a specific antagonist of the dopamine receptor D3. Because the dopamine transporter and dopamine receptors are expressed in the islets, we propose that cosecretion of dopamine with insulin activates receptors on the β-cell surface. D3 receptor activation results in changes in intracellular [Ca(2+)] dynamics, which, in turn, lead to lowered insulin secretion. Because blocking dopaminergic negative feedback increases insulin secretion, expanding the knowledge of this pathway in β-cells might offer a potential new target for the treatment of type 2 diabetes.

  20. Dopamine Synthesis and D3 Receptor Activation in Pancreatic β-Cells Regulates Insulin Secretion and Intracellular [Ca2+] Oscillations

    PubMed Central

    Ustione, Alessandro

    2012-01-01

    Pancreatic islets are critical for glucose homeostasis via the regulated secretion of insulin and other hormones. We propose a novel mechanism that regulates insulin secretion from β-cells within mouse pancreatic islets: a dopaminergic negative feedback acting on insulin secretion. We show that islets are a site of dopamine synthesis and accumulation outside the central nervous system. We show that both dopamine and its precursor l-dopa inhibit glucose-stimulated insulin secretion, and this inhibition correlates with a reduction in frequency of the intracellular [Ca2+] oscillations. We further show that the effects of dopamine are abolished by a specific antagonist of the dopamine receptor D3. Because the dopamine transporter and dopamine receptors are expressed in the islets, we propose that cosecretion of dopamine with insulin activates receptors on the β-cell surface. D3 receptor activation results in changes in intracellular [Ca2+] dynamics, which, in turn, lead to lowered insulin secretion. Because blocking dopaminergic negative feedback increases insulin secretion, expanding the knowledge of this pathway in β-cells might offer a potential new target for the treatment of type 2 diabetes. PMID:22918877

  1. Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells

    PubMed Central

    1991-01-01

    Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin- Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation. PMID:1662686

  2. Engineering Design Activities and Conceptual Change in Middle School Science

    ERIC Educational Resources Information Center

    Schnittka, Christine G.

    2009-01-01

    The purpose of this research was to investigate the impact of engineering design classroom activities on conceptual change in science, and on attitudes toward and knowledge about engineering. Students were given a situated learning context and a rationale for learning science in an active, inquiry-based method, and worked in small collaborative…

  3. Engineering Design Activities and Conceptual Change in Middle School Science

    ERIC Educational Resources Information Center

    Schnittka, Christine G.

    2009-01-01

    The purpose of this research was to investigate the impact of engineering design classroom activities on conceptual change in science, and on attitudes toward and knowledge about engineering. Students were given a situated learning context and a rationale for learning science in an active, inquiry-based method, and worked in small collaborative…

  4. Human endothelial cells are activated by interferon-γ plus tumour necrosis factor-α to kill intracellular Pseudomonas aeruginosa

    PubMed Central

    De Assis, M C; Da Costa, A O; Barja-Fidalgo, T C; Plotkowski, M C

    2000-01-01

    Proinflammatory cytokines have been shown to activate endothelial cells. To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon-γ (IFN-γ) plus tumour necrosis factor-α (TNF-α) for 24 hr and exposed to P. aeruginosa suspension for 1 hr. Light microscopy showed that activated cells internalized significantly more bacteria than control cells. To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay. In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection. In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower. Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants. HUVEC anti-P. aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the l-arginine analogues aminoguanidine and NG-monomethyl-l-arginine (l-NMMA), but was significantly inhibited by catalase. Our results indicate that HUVEC can be activated by IFN-γ plus TNF-α to kill IC P. aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity. PMID:11012781

  5. Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.

    PubMed Central

    Hassan, J; Rainsford, E; Reen, D J

    1997-01-01

    A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms. Images Figure 4 PMID:9497487

  6. Green tea extract protects endothelial progenitor cells from oxidative insult through reduction of intracellular reactive oxygen species activity

    PubMed Central

    Widowati, Wahyu; Widyanto, Rahma Micho; Husin, Winsa; Ratnawati, Hana; Laksmitawati, Dian Ratih; Setiawan, Bambang; Nugrahenny, Dian; Bachtiar, Indra

    2014-01-01

    Objective(s): Many studies have reported that tea consumption decreases cardiovascular risk, but the mechanisms remain unclear. Green tea is known to have potent antioxidant and free radical scavenging activities. This study aimed to investigate whether green tea extract (GTE) can protect endothelial progenitors cells (EPCs) against oxidative stress through antioxidant mechanisms. Materials and Methods: Mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation with Ficoll. The cells were then plated on fibronectin-coated culture dishes. After 7 days of culture, EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptake and lectin binding. EPCs were further identified by assessing the expression of CD34/45, CD133, and KDR. EPCs were then treated with hydrogen peroxide (H2O2) at doses of 50, 100, 200 µM and incubated with or without GTE (25 µg/ml). The intracellular reactive oxygen species (ROS) levels were detected by flow cytometry using a 2',7'-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. Results: GTE ameliorated the cell viability of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 25.47, 22.52, and 11.96% higher than controls, respectively. GTE also decreased the intracellular ROS levels of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 84.24, 92.27, and 93.72% compared to controls, respectively. Conclusion: GTE improves cell viability by reducing the intracellular ROS accumulation in H2O2-induced EPCs. PMID:25691948

  7. Intracellular colon cancer-associated Escherichia coli promote protumoral activities of human macrophages by inducing sustained COX-2 expression.

    PubMed

    Raisch, Jennifer; Rolhion, Nathalie; Dubois, Anaëlle; Darfeuille-Michaud, Arlette; Bringer, Marie-Agnès

    2015-03-01

    Intestinal dysbiosis has been reported in patients with colorectal cancer, and there is a high prevalence of Escherichia coli belonging to B2 phylogroup and producing a genotoxin, termed colibactin. Macrophages are one of the predominant tumor-infiltrating immune cells supporting key processes in tumor progression by producing protumoral factors such as cyclooxygenase-2 (COX-2). Here, we investigated whether B2 E. coli colonizing colon tumors could influence protumoral activities of macrophages. In contrast to commensal or nonpathogenic E. coli strains that were efficiently and rapidly degraded by macrophages at 24 h after infection, colon cancer-associated E. coli were able to resist killing by human THP-1 macrophages, to replicate intracellularly, and to persist inside host cells until at least 72 h after infection. Significant increases in COX-2 expression were observed in macrophages infected with colon cancer E. coli compared with macrophages infected with commensal and nonpathogenic E. coli strains or uninfected cells at 72 h after infection. Induction of COX-2 expression required live bacteria and was not due to colibactin production, as similar COX-2 levels were observed in macrophages infected with the wild-type colon cancer-associated E. coli 11G5 strain or a clbQ mutant unable to produce colibactin. Treatment of macrophages with ofloxacin, an antibiotic with intracellular tropism, efficiently decreased the number of intracellular bacteria and suppressed bacteria-induced COX-2 expression. This study provides new insights into the understanding of how tumor- infiltrating bacteria could influence cancer progression through their interaction with immune cells. Manipulation of microbes associated with tumors could have a deep influence on the secretion of protumoral molecules by infiltrating macrophages.

  8. Synthesis, Antimalarial Activity, and Intracellular Targets of MEFAS, a New Hybrid Compound Derived from Mefloquine and Artesunate▿

    PubMed Central

    de Pilla Varotti, Fernando; Botelho, Ana Cristina C.; Andrade, Anderson Assunção; de Paula, Renata C.; Fagundes, Elaine M. S.; Valverde, Alessandra; Mayer, Lúcia M. U.; Mendonça, Jorge Souza; de Souza, Marcus V. N.; Boechat, Núbia; Krettli, Antoniana Ursine

    2008-01-01

    A new synthetic antimalarial drug, a salt derived from two antimalarial molecules, mefloquine (MQ) and artesunate (AS), here named MEFAS, has been tested for its pharmacological activity. Combinations of AS plus MQ hydrochloride are currently being used in areas with drug-resistant Plasmodium falciparum parasites; although AS clears parasitemia in shorter time periods than any other antimalarial drug, it does not cure infected patients; in addition, MQ causes side effects and is rather expensive, important problems considering that malaria affects mostly populations in poor countries. Here, we show that MEFAS is more effective than the combination of AS and MQ, tested in parallel at different mass proportions, against P. falciparum (chloroquine-resistant clone W2 and chloroquine-sensitive clone 3D7) in vitro and in mice infected with Plasmodium berghei, promoting cure of this infection. MEFAS tested against HepG2 hepatoma cells exhibited lower toxicity than the antimalarials AS and MQ alone or combined. Possible targets of MEFAS have been studied by confocal microscopy using fluorescent probes (Fluo-4 AM and BCECF-AM) in P. falciparum synchronous culture of W2-infected red blood cells. Dynamic images show that MEFAS exhibited intracellular action increasing cytoplasmic Ca2+ at 1.0 ng/ml. This effect was also observed in the presence of tapsigargin, an inhibitor of SERCA, suggesting an intracellular target distinct from the endoplasmic reticulum. Trophozoites loaded with BCECF-AM, when treated with MEFAS, were still able to mobilize protons from the digestive vacuole (DV), altering the pH gradient. However, in the presence of bafilomycin A1, an inhibitor of the H+ pump from acidic compartments of eukaryotic cells, MEFAS had no action on the DV. In conclusion, the endoplasmic reticulum and DV are intracellular targets for MEFAS in Plasmodium sp., suggesting two modes of action of this new salt. Our data support MEFAS as a candidate for treating human malaria. PMID

  9. Intracellular Loop 2 Peptides of the Human 5HT1a Receptor are Differential Activators of Gi

    PubMed Central

    Hall, Brian; Squires, Carley; Parker, Keith K.

    2012-01-01

    Peptide mimics of intracellular loop 2 (ic2) of the human 5HT1a receptor have been studied with respect to their ability to inhibit agonist binding via interference with receptor-G-protein coupling. These peptides give shallow concentration-effect relationships. Additionally, these peptides have been studied with respect to their ability to trigger the signal transduction system of this Gi-coupled receptor. Two signaling parameters have been quantified: concentration of intracellular cAMP and changes in incorporation into the G protein of a stable analog of GTP. In both cases, peptide mimics near midloop of ic2 actually show agonist activity with efficacy falling off toward both loop termini near TM 3 and TM 4. Previous results have suggested that the loop region near the TM3/ic2 interface is primarily responsible for receptor-G-protein coupling, while the current result emphasizes the mid-ic2 loop region's ability to activate the G protein following initial coupling. A limited number of peptides from the receptor's TM5/ic3 loop vicinity were also studied regarding agonist inhibition and G-protein activation. These peptides provide additional evidence that the human 5HT1a receptor, TM5/ic3 loop region, is involved in both coupling and activation actions. Overall, these results provide further information about potential pharmacological intervention and drug development with respect to the human 5HT1a receptor/G-protein system. Finally, the structural evidence generated here provides testable models pending crystallization and X-ray analysis of the receptor. PMID:22649462

  10. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

    PubMed Central

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

    2015-01-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

  11. Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens

    PubMed Central

    Passalacqua, Karla D.; Charbonneau, Marie-Eve; Donato, Nicholas J.; Showalter, Hollis D.; Sun, Duxin; Wen, Bo; He, Miao; Sun, Hanshi

    2016-01-01

    Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes. We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection. PMID:27139470

  12. Crystal structure of the plexin A3 intracellular region reveals an autoinhibited conformation through active site sequestration

    SciTech Connect

    He, Huawei; Yang, Taehong; Terman, Jonathan R.; Zhang, Xuewu

    2010-01-20

    Plexin cell surface receptors bind to semaphorin ligands and transduce signals for regulating neuronal axon guidance. The intracellular region of plexins is essential for signaling and contains a R-Ras/M-Ras GTPase activating protein (GAP) domain that is divided into two segments by a Rho GTPase-binding domain (RBD). The regulation mechanisms for plexin remain elusive, although it is known that activation requires both binding of semaphorin to the extracellular region and a Rho-family GTPase (Rac1 or Rnd1) to the RBD. Here we report the crystal structure of the plexin A3 intracellular region. The structure shows that the N- and C-terminal portions of the GAP homologous regions together form a GAP domain with an overall fold similar to other Ras GAPs. However, the plexin GAP domain adopts a closed conformation and cannot accommodate R-Ras/M-Ras in its substrate-binding site, providing a structural basis for the autoinhibited state of plexins. A comparison with the plexin B1 RBD/Rnd1 complex structure suggests that Rnd1 binding alone does not induce a conformational change in plexin, explaining the requirement of both semaphorin and a Rho GTPase for activation. The structure also identifies an N-terminal segment that is important for regulation. Both the N-terminal segment and the RBD make extensive interactions with the GAP domain, suggesting the presence of an allosteric network connecting these three domains that integrates semaphorin and Rho GTPase signals to activate the GAP. The importance of these interactions in plexin signaling is shown by both cell-based and in vivo axon guidance assays.

  13. Activation of multiple intracellular transduction signals by vasopressin in vasopressin-sensitive neurones of the rat supraoptic nucleus

    PubMed Central

    Sabatier, Nancy; Richard, Philippe; Dayanithi, Govindan

    1998-01-01

    The intracellular mechanisms activated by the binding of vasopressin to its receptor(s) and which result in the increase of [Ca2+]i were investigated in freshly dissociated supraoptic nucleus neurones. Various pharmacological agents were used to investigate the possible involvement of phospholipase C (PLC) and adenylate cyclase (AC) intracellular pathways in the transduction of the vasopressin action. Both the PLC inhibitor U-73122 and the protein kinase C (PKC) inhibitor calphostin C, reduced the [Ca2+]i rise elicited by vasopressin. The cAMP analogue, 8-Br-cAMP produced an increase in [Ca2+]i and IBMX, a phosphodiesterase inhibitor, potentiated the response to vasopressin. After pre-incubation with the AC inhibitor SQ-22536, 7 out of 18 vasopressin-sensitive neurones showed no inhibition of the vasopressin response, while the response to vasopressin was reduced by greater than 35 % in each of the other 11 neurones. The activation of protein kinase A (PKA) with Sp-cAMPS caused an increase in [Ca2+]i which was additive to the vasopressin-elicited [Ca2+]i increase. After incubation with the PKA inhibitors Rp-cAMPS or H-89, the [Ca2+]i responses triggered by Sp-cAMPS and vasopressin were, respectively, abolished and greatly reduced. A combined administration of SQ-22536 (AC inhibitor) followed by U-73122 (PLC inhibitor), or U-73122 followed by H-89 (PKA inhibitor), virtually abolished the response to vasopressin. In vasopressin-responsive neurones, the pituitary adenylate cyclase-activating polypeptide (PACAP) induced a [Ca2+]i increase similar to the response to vasopressin and in both cases the increase was inhibited to the same extent by a combination of U-73122 and Rp-cAMPS. In conclusion, we suggest that the autoregulation exerted specifically by vasopressin on vasopressin-sensitive neurones involves the activation of both PLC- and AC-linked pathways. PMID:9824711

  14. Intracellular Na+, K+ and Cl- activities in Acheta domesticus Malpighian tubules and the response to a diuretic kinin neuropeptide.

    PubMed

    Coast, Geoffrey M

    2012-08-15

    The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in malpighian tubules of Acheta domesticus by measuring intracellular Na(+), K(+) and Cl(-) activities, basolateral membrane voltage (V(b)), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K(+) and Cl(-) across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba(2+)-sensitive K(+) channels do not contribute to net transepithelial K(+) transport and fluid secretion. A basolateral Cl(-) conductance was revealed after the blockade of K(+) channels with Ba(2+), and a current carried by the passive outward movement of Cl(-) accounts for the hyperpolarization of V(b) in response to Ba(2+). Ion uptake via Na(+)/K(+)/2Cl(-) cotransport, driven by the inwardly directed Na(+) electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na(+) and K(+) transport. The Na(+) gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na(+)/K(+)-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or V(b). Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na(+), K(+) and Cl(-) in the secreted fluid. The electrochemical gradient for Cl(-) favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl(-) transport, which is attributed to the opening of an apical Cl(-) conductance, which depolarises the apical membrane voltage.

  15. Moderate increases in intracellular calcium activate neuroprotective signals in hippocampal neurons.

    PubMed

    Bickler, P E; Fahlman, C S

    2004-01-01

    Although large increases in neuronal intracellular calcium concentrations ([Ca(2+)](i)) are lethal, moderate increases in [Ca(2+)](i) of 50-200 nM may induce immediate or long-term tolerance of ischemia or other stresses. In neurons in rat hippocampal slice cultures, we determined the relationship between [Ca(2+)](i), cell death, and Ca(2+)-dependent neuroprotective signals before and after a 45 min period of oxygen and glucose deprivation (OGD). Thirty minutes before OGD, [Ca(2+)](i) was increased in CA1 neurons by 40-200 nM with 1 nM-1 microM of a Ca(2+)-selective ionophore (calcimycin or ionomycin-"Ca(2+) preconditioning"). Ca(2+) preconditioning greatly reduced cell death in CA1, CA3 and dentate during the following 7 days, even though [Ca(2+)](i) was similar (approximately 2 microM) in preconditioned and control neurons 1 h after the OGD. When pre-OGD [Ca(2+)](i) was lowered to 25 nM (10 nM ionophore in Ca(2+)-free medium) or increased to 8 microM (10 microM ionophore), more than 90% of neurons died. Increased levels of the anti-apoptotic protein protein kinase B (Akt) and the MAP kinase ERK (p42/44) were present in preconditioned slices after OGD. Reducing Ca(2+) influx, inhibiting calmodulin, and preventing Akt or MAP kinase p42/44 upregulation prevented Ca(2+) preconditioning, supporting a specific role for Ca(2+) in the neuroprotective process. Further, in continuously oxygenated cultured hippocampal/cortical neurons, preconditioning for 30 min with 10 nM ionomycin reduced cell death following a 4 microM increase in [Ca(2+)](i) elicited by 1 microM ionomycin. Thus, a zone of moderately increased [Ca(2+)](i) before a potentially lethal insult promotes cell survival, uncoupling subsequent large increases in [Ca(2+)](i) from initiating cell death processes.

  16. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  17. The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity.

    PubMed

    Fulcher, Nanette B; Holliday, Phillip M; Klem, Erich; Cann, Martin J; Wolfgang, Matthew C

    2010-05-01

    Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.

  18. Targeting of pegylated liposomal mitomycin-C prodrug to the folate receptor of cancer cells: Intracellular activation and enhanced cytotoxicity.

    PubMed

    Patil, Yogita; Amitay, Yasmine; Ohana, Patricia; Shmeeda, Hilary; Gabizon, Alberto

    2016-03-10

    Mitomycin C (MMC) is a powerful anti-bacterial, anti-fungal and anti-tumor antibiotic, often active against multidrug resistant cells. Despite a broad spectrum of antitumor activity, MMC clinical use is relatively limited due to its fast clearance and dose-limiting toxicity. To exploit the potential antitumor activity of MMC and reduce its toxicity we have previously developed a formulation of pegylated liposomes with a lipophilic prodrug of MMC (PL-MLP), activated by endogenous reducing agents which are abundant in the tumor cell environment in the form of different thiols. PL-MLP has minimal in vitro cytotoxicity unless reducing agents are added to the cell culture to activate the prodrug. In the present study, we hypothesized that targeting PL-MLP via folate receptors will facilitate intracellular activation of prodrug and enhance cytotoxic activity without added reducing agents. We grafted a lipophilic folate conjugate (folate-PEG(5000)-DSPE) to formulate folate targeted liposomes (FT-PL-MLP) and examined in vitro cell uptake and cytotoxic activity in cancer cell lines with high folate receptors (HiFR). 3H-cholesterol-hexadecyl ether (3H-Chol)-radiolabeled liposomes were prepared to study liposome-cell binding in parallel to cellular uptake of prodrug MLP. 3H-Chol and MLP cell uptake levels were 4-fold and 9-fold greater in KB HiFR cells when FT-PL-MLP is compared to non-targeted PL-MLP liposomes. The cytotoxic activity of FT-PL-MLP liposomes was significantly increased up to ~5-fold compared with PL-MLP liposomes in all tested HiFR expressing cell lines. The enhanced uptake and intracytoplasmic liposome delivery was confirmed by confocal fluorescence studies with Rhodamine-labeled liposomes. In vivo, no significant differences in pharmacokinetics and biodistribution were observed when PL-MLP was compared to FT-PL-MLP by the intravenous route. However, when liposomes were directly injected into the peritoneal cavity of mice with malignant ascites of J6456 Hi

  19. Intracellular energy status regulates activity in hypocretin/orexin neurones: a link between energy and behavioural states

    PubMed Central

    Liu, Zhong-Wu; Gan, Geliang; Suyama, Shigetomo; Gao, Xiao-Bing

    2011-01-01

    Abstract The hypocretin/orexin (Hcrt)-containing neurones within the lateral hypothalamus integrate nutritional, energetic and behavioural cues to generate the final output in order to exert their functions. It is still not clear how Hcrt neurones monitor changes in energy status in animals. In brain slices from transgenic mice expressing green fluorescent protein (GFP) exclusively in Hcrt neurones, we examined the roles of intracellular levels of ATP ([ATP]i) in regulating activities in these cells with conventional and perforated whole-cell recording. By using ‘ATP clamp’ we demonstrated that membrane potential (Vm) correlated with the [ATP]i in Hcrt neurones. Perforated recording revealed a Vm of −46.1 ± 1.6 mV (n = 18), close to the level measured with an [ATP]i equal to 5–6 mm (–48.7 ± 1.4 mV, n = 16, 5 mm ATP), suggesting that a unique demand for energy is required to maintain normal functionality in Hcrt cells. A direct disruption of ATP production or reduction in ambient glucose levels resulted in an inhibition of activity in Hcrt neurones. The Vm was significantly depolarized in Hcrt neurones in sleep-deprived mice as compared with controls (P < 0.01, t test), which was eliminated by experimental manipulations causing the same level of [ATP]i and KATP channel opening in both groups, suggesting a decrease during sleep and an increase during sustained wakefulness in [ATP]i in Hcrt cells. In summary, these data demonstrate that a delicate control of activity by monitoring the availability of intracellular energy stores in Hcrt cells may serve as a novel mechanism regulating energy expenditure and behavioural state dependent upon the energy state in animals. PMID:21727218

  20. Intracellular energy status regulates activity in hypocretin/orexin neurones: a link between energy and behavioural states.

    PubMed

    Liu, Zhong-Wu; Gan, Geliang; Suyama, Shigetomo; Gao, Xiao-Bing

    2011-09-01

    The hypocretin/orexin (Hcrt)-containing neurones within the lateral hypothalamus integrate nutritional, energetic and behavioural cues to generate the final output in order to exert their functions. It is still not clear how Hcrt neurones monitor changes in energy status in animals. In brain slices from transgenic mice expressing green fluorescent protein (GFP) exclusively in Hcrt neurones, we examined the roles of intracellular levels of ATP ([ATP](i)) in regulating activities in these cells with conventional and perforated whole-cell recording. By using 'ATP clamp' we demonstrated that membrane potential (V(m)) correlated with the [ATP](i) in Hcrt neurones. Perforated recording revealed a V(m) of -46.1 ± 1.6 mV (n = 18), close to the level measured with an [ATP](i) equal to 5-6 mm (-48.7 ± 1.4 mV, n = 16, 5 mm ATP), suggesting that a unique demand for energy is required to maintain normal functionality in Hcrt cells. A direct disruption of ATP production or reduction in ambient glucose levels resulted in an inhibition of activity in Hcrt neurones. The V(m) was significantly depolarized in Hcrt neurones in sleep-deprived mice as compared with controls (P < 0.01, t test), which was eliminated by experimental manipulations causing the same level of [ATP](i) and K(ATP) channel opening in both groups, suggesting a decrease during sleep and an increase during sustained wakefulness in [ATP](i) in Hcrt cells. In summary, these data demonstrate that a delicate control of activity by monitoring the availability of intracellular energy stores in Hcrt cells may serve as a novel mechanism regulating energy expenditure and behavioural state dependent upon the energy state in animals.

  1. Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.

    PubMed

    ben Bdira, Fredj; González, Esperanza; Pluta, Paula; Laín, Ana; Sanz-Parra, Arantza; Falcon-Perez, Juan Manuel; Millet, Oscar

    2014-11-01

    Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.

  2. Altered Intracellular ATP Production by Activated CD4+ T-Cells in Very Preterm Infants

    PubMed Central

    Corvaglia, Luigi; Gabrielli, Liliana; Chiereghin, Angela; Lazzarotto, Tiziana

    2016-01-01

    Background. The neonatal immune system is not fully developed at birth; newborns have adequate lymphocytes counts but these cells lack function. Objective. To assess the activity of T-cells and the influence of the main perinatal factors in very preterm infants (birth weight < 1500 g). Design. Blood samples from 59 preterm infants (21/59 were dizygotic twins) were collected at birth and at 30 days of life to measure CD4+ T-cell activity using the ImmuKnow™ assay. Fifteen healthy adults were included as a control group. Results. CD4+ T-cell activity was lower in VLBW infants compared with adults (p < 0.001). Twins showed lower immune activity compared to singletons (p = 0.005). Infants born vaginally showed higher CD4+ T-cell activity compared to those born by C-section (p = 0.031); infants born after prolonged Premature Rupture of Membranes (pPROM) showed higher CD4+ T-cell activity at birth (p = 0.002) compared to infants born without pPROM. Low CD4+ T-cell activity at birth is associated with necrotizing enterocolitis (NEC) in the first week of life (p = 0.049). Conclusions. Preterm infants show a lack in CD4+ T-cell activity at birth. Perinatal factors such as intrauterine inflammation, mode of delivery, and zygosity can influence the adaptive immune activation capacity at birth and can contribute to exposing these infants to serious complications such as NEC. PMID:28070527

  3. Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.

    PubMed

    Byun, Yong-Soo; Yoo, Young-Sik; Kwon, Ji-Young; Joo, Jong-Soo; Lim, Sung-A; Whang, Woong-Joo; Mok, Jee-Won; Choi, Jun-Sub; Joo, Choun-Ki

    2016-02-01

    Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 μM during 48 h, but inhibited at concentrations over 2000 μM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 μM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor

  4. Application of Intracellular Alkaline Phosphatase Activity Measurement in Detection of Neutrophil Adherence In Vitro

    PubMed Central

    Bednarska, Katarzyna; Klink, Magdalena; Sulowska, Zofia

    2006-01-01

    We have proposed the use of the fluorimetric method with 4-methylumbelliferyl phosphate (4-MUP) specific substrate for the alkaline phosphatase determination in the neutrophil adhesion assay. We provide evidence that the endogenous neutrophil alkaline phosphatase (NAP) activity evaluation is reliable to quantify neutrophil adhesion at a wide range of cell numbers (104−106). The results obtained by fluorimetric NAP activity test correlate to the results of adherence evaluated using the MTT reduction assay. The fluorimetric NAP activity test may be applied for resting as well as activated neutrophils without the risk of the activators interferences into the test. The alkaline phosphatase survey with the use of 4-MUP substrate is recommended herein as a sensitive, repeatable, simple, and reliable method of the neutrophil adherence determination in vitro. PMID:17047286

  5. Intracellular ROS

    PubMed Central

    Leshem, Yehoram

    2007-01-01

    Intracellular localization of stress induced reactive oxygen species (ROS) has emerged as an important aspect towards understanding of cellular responses to environmental stimuli. Our recent study published in the PNAS (103:18008–13)1 shows that NaCl-induced ROS appear within endosomes on the way to tonoplast as part of the vacuolar vesicle trafficking. In addition to showing ROS damage to the tonoplast, this finding may shed light upon recently reported aspects of root water relations during salt stress, suggesting a new signaling role for intracellular ROS in Arabidopsis root cells, during salt stress: ROS that are compartmentalized in endosomes are delivered by the vacuolar vesicle trafficking pathway to the tonoplast, resulting in oxidative gating of TIPs water channels. The closure of the tonoplast aquaporins contributes to the observed reduction in root hydraulic conductivity during salt stress. PMID:19704741

  6. Nanoelectropulse intracellular perturbation and electropermeabilization technology: phospholipid translocation, calcium bursts, chromatin rearrangement, cardiomyocyte activation, and tumor cell sensitivity.

    PubMed

    Vernier, P Thomas; Sun, Yinghua; Wang, Jingjing; Thu, Mya Mya; Garon, Edward; Valderrabano, Miguel; Marcu, Laura; Koeffler, H Phillip; Gundersen, Martin A

    2005-01-01

    Nanosecond, megavolt-per-meter pulsed electric fields scramble the asymmetric arrangement of phospholipids in the plasma membrane, release intracellular calcium, trigger cardiomyocyte activity, and induce apoptosis in mammalian cancer cells, without the permeabilizing effects associated with longer, lower-field pulses. Dose dependencies with respect to pulse width, amplitude, and repetition rate, and total pulse count are observed for all of these phenomena. Sensitivities vary among cell types; cells of lymphoid origin growing in suspension are more susceptible to nanoelectropulse exposure than solid tumor lines. Simple electrical models of the cell are useful for first-order explanations, but more sophisticated treatments will be required for analysis and prediction at both biomolecular and tissue levels.

  7. Regulation of the Membrane Insertion and Conductance Activity of the Metamorphic Chloride Intracellular Channel Protein CLIC1 by Cholesterol

    PubMed Central

    Valenzuela, Stella M.; Alkhamici, Heba; Brown, Louise J.; Almond, Oscar C.; Goodchild, Sophia C.; Carne, Sonia; Curmi, Paul M. G.; Holt, Stephen A.; Cornell, Bruce A.

    2013-01-01

    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer. PMID:23457643

  8. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

    PubMed

    Valenzuela, Stella M; Alkhamici, Heba; Brown, Louise J; Almond, Oscar C; Goodchild, Sophia C; Carne, Sonia; Curmi, Paul M G; Holt, Stephen A; Cornell, Bruce A

    2013-01-01

    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  9. Active Engine Mount Technology for Automobiles

    NASA Technical Reports Server (NTRS)

    Rahman, Z.; Spanos, J.

    1996-01-01

    We present a narrow-band tracking control using a variant of the Least Mean Square (LMS) algorithm [1,2,3] for supressing automobile engine/drive-train vibration disturbances. The algorithm presented here has a simple structure and may be implemented in a low cost micro controller.

  10. Antioxidant, antibacterial and anti-aging activities of intracellular zinc polysaccharides from Grifola frondosa SH-05.

    PubMed

    Zhang, Chen; Gao, Zheng; Hu, Chunlong; Zhang, Jianjun; Sun, Xinyi; Rong, Chengbo; Jia, Le

    2017-02-01

    In present work, the strain of Grifola frondosa SH-05 was used as a vector of zinc biotransformation to produce the IZPS. The bioactivities including antioxidant and antibacterial activities in vitro and anti-aging properties in vivo of IZPS were investigated comparing with the IPS. The results which were in consistent with the results of histopathology assay demonstrated that the IZPS had superior antioxidant and anti-aging activities by scavenging the hydroxyl and DPPH radicals, increasing enzyme activities, decreasing the MDA contents and ameliorating the anile condition of mice. Besides, the IZPS also showed potential antibacterial activities. The IZPS with higher bioactivities was composed of were Rha, Ino and Glu with a molar ratio of 4.7:3.6:1. These conclusions indicated that the IZPS might be a potential source of natural antioxidant, antibacterial agent and anti-aging agent.

  11. Differential intracellular signalling properties of the growth hormone receptor induced by the activation of an anti-GHR antibody.

    PubMed

    Lan, Hainan; Li, Wei; Fu, Zhiling; Yang, Yanhong; Wu, Tiancheng; Liu, Yu; Zhang, Hui; Cui, Huanzhong; Li, Yumeng; Hong, Pan; Liu, Jingsheng; Zheng, Xin

    2014-06-05

    A series of studies have reported that anti-GHR antibody can function as a GHR agonist and may serve as an attractive tool for studying the mechanisms of GHR activation. However, to date, there is relatively little information about intracellular signalling triggered by anti-GHR antibody. Therefore, in this work, we have developed a panel of monoclonal antibodies to GHBP, among which one Mab, termed CG-172, was selected for further characterisation because of its signalling properties. The results from FACS assays, receptor binding and immunoprecipitation assays and western blotting demonstrated that CG-172 specifically binds to GHR expressed on target cells. Subsequently, epitope mapping studies that used receptor binding analysis showed that CG-172 specifically binds subdomain 1 of GHR ECD. We next examined the resulting signal transduction pathways triggered by this antibody in CHO-GHR638 cells and rat hepatocytes. We found that CG-172 can activate JAK2, AKT, ERK1/2 and STAT1/3 but not STAT5. The phosphorylation kinetics of STAT1/3, AKT and ERK1/2 induced by either GH or CG-172 were analysed in dose-response and time course experiments. Our observations demonstrated that an anti-GHR monoclonal antibody (CG-172) can serve as an attractive tool to study the mechanism(s) of GHR-mediated intracellular signalling pathways and may lead to the production of signal-specific molecules that are capable of inducing different biochemical responses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. A hyperspectral and toxicological analysis of protein corona impact on silver nanoparticle properties, intracellular modifications, and macrophage activation

    PubMed Central

    Shannahan, Jonathan H; Podila, Ramakrishna; Brown, Jared M

    2015-01-01

    The inevitable adsorption of biomolecules on nanomaterials results in the formation of a protein corona (PC), which modifies the nanoparticle (NP)–cell interface resulting in modified uptake, activity, clearance, and toxicity. While the physicochemical properties of the NP govern the composition of PC, the formation of PC in turn alters the characteristics of the NP by imparting a new unique “biological” identity. To assess how the PC influences AgNP properties, intracellular modifications, and cellular responses, we utilized a combination of hyperspectral and toxicological analyses. AgNPs were coated with a complex PC (multiple proteins, eg, 10% fetal bovine serum) or a simple PC (single protein, eg, bovine serum albumin [BSA]) and evaluated by hyperspectral and dynamic light scattering for modifications in AgNP properties. Mouse macrophages were exposed to AgNPs with PCs and examined for differences in uptake, cytotoxicity, and cell activation. Hyperspectral imaging revealed intracellular modifications to AgNPs that were found to spectrally match alterations in AgNPs following incubation in lysosomal fluid. Addition of the PC influenced AgNP uptake and cytotoxicity; however, hydrodynamic size and surface charge did not contribute to these responses. Assessments of all endpoints demonstrated differences between complex and BSA PC, suggesting that these responses are not purely driven by the primary protein component of the complex PC (ie, BSA). Alterations in cellular–NP uptake/interactions may be driven through cell surface receptor recognition of protein constituents that make up the PC rather than the physicochemical differences in AgNPs. PMID:26508856

  13. Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development.

    PubMed

    Phillips, K P; Baltz, J M

    1999-04-15

    We report here that at least one major pHi-regulatory mechanism, the HCO3-/Cl- exchanger, is quiescent in unfertilized mouse eggs but becomes fully activated during early development following fertilization. Zygotes (8-12 h postfertilization) exhibited a marked intracellular alkalinization upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. In addition, efflux of Cl- from eggs upon external Cl- removal was much slower than that from zygotes, indicating additional pathways for Cl- to cross the plasma membrane in zygotes. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. Following in vitro fertilization (IVF), increased HCO3-/Cl- exchanger activity was first detectable about 4 h postfertilization and reached the maximal level after about 8 h. The upregulation of HCO3-/Cl- exchanger activity after fertilization appeared to occur by activation of existing, inactive exchangers rather than by synthesis or transport of new exchangers, as the increase in activity following IVF was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. This activation may depend on the Ca2+ transients which follow fertilization, as suppression of these transients, using the Ca2+ chelator BAPTA, reduced subsequent upregulation of HCO3-/Cl- exchanger activity by about 50%. Activation of pHi-regulatory systems may be a widespread feature of the earliest period of embryonic development, not restricted to species such as marine invertebrates as previously believed.

  14. Comparative Intracellular (THP-1 Macrophage) and Extracellular Activities of β-Lactams, Azithromycin, Gentamicin, and Fluoroquinolones against Listeria monocytogenes at Clinically Relevant Concentrations

    PubMed Central

    Carryn, Stéphane; Van Bambeke, Françoise; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.

    2002-01-01

    The activities of ampicillin, meropenem, azithromycin, gentamicin, ciprofloxacin, and moxifloxacin against intracellular hemolysin-positive Listeria monocytogenes were measured in human THP-1 macrophages and were compared with the extracellular activities observed in broth. All extracellular concentrations were adjusted to explore ranges that are clinically achievable in human serum upon conventional therapy. In broth, ampicillin, meropenem, and azithromycin were only bacteriostatic, whereas gentamicin, ciprofloxacin, and moxifloxacin were strongly bactericidal in a concentration-dependent manner. In cells, ampicillin, meropenem, azithromycin, and ciprofloxacin were slightly bactericidal (0.3- to 0.8-log CFU reductions), moxifloxacin was strongly bactericidal (2.1-log CFU reduction), and gentamicin was virtually inactive. The difference in the efficacies of moxifloxacin and ciprofloxacin in cells did not result from a difference in levels of accumulation in cells (6.96 ± 1.05 versus 7.75 ± 1.03) and was only partially explainable by the difference in the MICs (0.58 ± 0.04 versus 1.40 ± 0.17 mg/liter). Further analysis showed that intracellular moxifloxacin expressed only approximately 1/7 of the activity demonstrated against extracellular bacteria and ciprofloxacin expressed only 1/15 of the activity demonstrated against extracellular bacteria. Gentamicin did not increase the intracellular activities of the other antibiotics tested. The data suggest (i) that moxifloxacin could be of potential interest for eradication of the intracellular forms of L. monocytogenes, (ii) that the cellular accumulation of an antibiotic is not the only determinant of its intracellular activity (for fluoroquinolones, it is actually a self-defeating process as far as activity is concerned), and (iii) that pharmacodynamics (activity-to-concentration relationships) need to be considered for the establishment of efficacy against intracellular bacteria, just as they are for the

  15. Engineering of chimeric catalase-Angiopep-2 for intracellular protection of brain endothelial cells against oxidative stress.

    PubMed

    Yainoy, Sakda; Houbloyfa, Patcharaporn; Eiamphungporn, Warawan; Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong

    2014-07-01

    Blood-brain barrier (BBB) disruption and brain microvascular endothelial cells (BMVECs) death caused by excessive production of hydrogen peroxide (H2O2) have been implicated in several neurological conditions. To overcome this problem, H2O2-degrading enzyme with ability to enter the BMVECs is required. In the present study, genetic fusion of gene encoding human catalase and gene encoding Angiopep-2 (AP2), a brain targeting peptide, was performed. The fusion protein was successfully expressed in Escherichia coli and purified to homogeneity. The protein retained heme content and specific enzymatic activity in the same order of magnitude as that of native enzyme. Study of the BMVECs internalization showed that 0.1μM of the fusion protein can enter the cell within 15min, while internalization of the native protein was not observed at this condition. In addition, treatment of the BMVECs with 20 units of the fusion protein for 30min showed protection against H2O2 up to 5.0mM, whereas this protective effect was not observed from treatment with the native protein. Therefore, construction of chimeric human catalase and AP2 provides an insight into the development of potential therapeutic antioxidant with ability to penetrate the BBB for protection against neurodegenerative disorders.

  16. Hexokinase and phosphofructokinase activity and intracellular distribution correlate with aggressiveness and invasiveness of human breast carcinoma.

    PubMed

    Coelho, Raquel G; Calaça, Isadora C; Celestrini, Deborah M; Correia-Carneiro, Ana Helena P; Costa, Mauricio M; Zancan, Patricia; Sola-Penna, Mauro

    2015-10-06

    Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis.

  17. Hexokinase and phosphofructokinase activity and intracellular distribution correlate with aggressiveness and invasiveness of human breast carcinoma

    PubMed Central

    Coelho, Raquel G.; Calaça, Isadora C.; Celestrini, Deborah M.; Correia-Carneiro, Ana Helena P.; Costa, Mauricio M.; Zancan, Patricia; Sola-Penna, Mauro

    2015-01-01

    Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis. PMID:26320188

  18. Isosmotic modulation of cell volume and intracellular ion activities during stimulation of single exocrine cells.

    PubMed

    Foskett, J K; Wong, M M; Sue-A-Quan, G; Robertson, M A

    1994-02-01

    Stimulation of salivary secretion is associated with a rise of [Ca2+]i in acinar cells. We examined the osmotic and ionic consequences of activation of Ca(2+)-dependent K+ and Cl- channels, by simultaneous optical determinations of cell volume and [Ca2+]i, [Cl-]i or [Na+]i during muscarinic stimulation of single salivary acinar cells, using a differential interference contrast (DIC)-fluorescence microscope. Carbachol caused a rapid rise of [Ca2+]i, as well as a substantial cell shrinkage. Despite variability in the level and kinetics of the subsequent sustained phase of the [Ca2+]i response, cell volume was correlated with [Ca2+]i in all cases. Elevated [Ca2+]i was both necessary and sufficient to cause these changes in cell volume. The proposition that changes in cell volume reflected changes in cell solute content was confirmed by simultaneously measuring [Cl-]i and cell volume. Simultaneous determinations of cell volume and [Na+]i indicated that the initial cell shrinkage was due entirely to K+ and Cl- efflux. Subsequent to the initial shrinkage, [Na+]i rose to high levels, primarily due to activation of Na+/H+ exchange. Thus, modulation of ion transport activities under isosmotic conditions results in substantial changes in cell solute content and cell volume. Subsequent to the early Ca(2+)-induced changes in these parameters, other transporters become active, but it is unclear what signals their activation. Cell swelling by osmotic dilution of the bath resulted in compensatory cell shrinkage (RVD) which was sensitive to K+ and Cl- gradients. Nevertheless, a rise of [Ca2+]i was not necessary for RVD. Osmotic shrinkage and/or cell acidification were insufficient to activate Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Intracellular delivery and activation of the genetically encoded photosensitizer Killer Red by quantum dots encapsulated in polymeric micelles.

    PubMed

    Muthiah, Muthunarayanan; Park, Seung-Hwan; Nurunnabi, Md; Lee, Jooyoung; Lee, Yong-Kyu; Park, Hansoo; Lee, Byeong-Il; Min, Jung-Joon; Park, In-Kyu

    2014-04-01

    We have prepared polymeric micelle-encapsulating quantum dots (QDots) for delivering the optically activatable protein Killer Red (KR) as a plasmid to cancer cells. QDots absorb light at a lower wavelength and emit light at a higher wavelength in the cell cytoplasm, activating the expressed KR. Once activated, KR triggers the generation of reactive oxygen species (ROS). We prepared cadmium selenide (CdSe)/zinc sulphide (ZnS) QDots and evaluated their optical properties. Subsequently, we performed morphology studies, elemental analysis, thermogravimetric analysis (TGA), and measurements of particle size and surface charge of prepared QDots encapsulated in PHEA-g-PEG-bPEI (PPP-QDot). Cellular uptake of PPP-QDot and PPP-QDot/KR nanoparticles was confirmed using confocal microscopy, and the cellular toxicity and transfection efficiency associated with uptake of PPP-QDot/KR nanoparticles were analyzed. KR expression in normal cells and cancer cells was confirmed using confocal microscopy and Western blotting. Cellular morphologies before and after intracellular activation of KR were observed using phase contrast, fluorescence, and confocal microscopy. Cell fate after exposure to blue light-emitting diode lighting was determined using apoptosis staining and a cell proliferation assay, confirming a suppression in proliferation and a reduction in metabolic activity. We determined that ROS generation contributed to cellular damage after treatment with PPP-QDot/KR nanoparticles and blue light exposure.

  20. PIAS proteins are involved in the SUMO-1 modification, intracellular translocation and transcriptional repressive activity of RET finger protein

    SciTech Connect

    Matsuura, Tetsuo; Shimono, Yohei; Kawai, Kumi; Murakami, Hideki; Urano, Takeshi; Niwa, Yasumasa; Goto, Hidemi; Takahashi, Masahide . E-mail: mtakaha@med.nagoya-u.ac.jp

    2005-08-01

    Ret finger protein (RFP) is a nuclear protein that is highly expressed in testis and in various tumor cell lines. RFP functions as a transcriptional repressor and associates with Enhancer of Polycomb 1 (EPC1), a member of the Polycomb group proteins, and Mi-2{beta}, a main component of the nucleosome remodeling and deacetylase (NuRD) complex. We show that RFP binds with PIAS (protein inhibitor of activated STAT) proteins, PIAS1, PIAS3, PIASx{alpha} and PIASy at their carboxyl-terminal region and is covalently modified by SUMO-1 (sumoylation). PIAS proteins enhance the sumoylation of RFP in a dose-dependent manner and induce the translocation of RFP into nuclear bodies reminiscent of the PML bodies. In addition, co-expression of PIAS proteins or SUMO-1 strengthened the transcriptional repressive activity of RFP. Finally, our immunohistochemical results show that RFP, SUMO-1 and PIASy localize in a characteristic nuclear structure juxtaposed with the inner nuclear membrane (XY body) of primary spermatocytes in mouse testis. These results demonstrate that the intracellular location and the transcriptional activity of RFP are modified by PIAS proteins which possess SUMO E3 ligase activities and suggest that they may play a co-operative role in spermatogenesis.

  1. Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

    PubMed Central

    Zhu, Mei Hong; Sung, Tae Sik; O'Driscoll, Kate; Koh, Sang Don

    2015-01-01

    Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca2+-activated Cl− channels. We investigated the hypothesis that the Ca2+ responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca2+ stores. ICC, obtained from the small intestine of Kit+/copGFP mice, were studied under voltage and current clamp to determine the effects of blocking Ca2+ uptake into stores and release of Ca2+ via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca2+ concentration, suggesting that pacemaker activity depends on Ca2+ dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca2+ from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC. PMID:25631870

  2. Activated Macrophages Destroy Intracellular Leishmania Major Amastigotes by an l-Arginine-Dependent Killing Mechanism

    DTIC Science & Technology

    1990-01-01

    conversion of site from one that is supportive of replication, to one that the sandfly -adapted promastigote to the amastigote form is hostile to...Inaddiion th cometiivein-room temperature for 5 min. Absorbance at 543 om was measured.activated macrophages. In addition, t e p titi e tn- No2- was qu

  3. TGF-β-Mediated Sustained ERK1/2 Activity Promotes the Inhibition of Intracellular Growth of Mycobacterium avium in Epithelioid Cells Surrogates

    PubMed Central

    L'Abbate, Carolina; Cipriano, Ivone; Pérez-Hurtado, Elizabeth Cristina; Leão, Sylvia Cardoso; Carneiro, Célia Regina Whitaker; Machado, Joel

    2011-01-01

    Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth. PMID:21731758

  4. Combustion diagnostic for active engine feedback control

    DOEpatents

    Green, Jr., Johney Boyd; Daw, Charles Stuart; Wagner, Robert Milton

    2007-10-02

    This invention detects the crank angle location where combustion switches from premixed to diffusion, referred to as the transition index, and uses that location to define integration limits that measure the portions of heat released during the combustion process that occur during the premixed and diffusion phases. Those integrated premixed and diffusion values are used to develop a metric referred to as the combustion index. The combustion index is defined as the integrated diffusion contribution divided by the integrated premixed contribution. As the EGR rate is increased enough to enter the low temperature combustion regime, PM emissions decrease because more of the combustion process is occurring over the premixed portion of the heat release rate profile and the diffusion portion has been significantly reduced. This information is used to detect when the engine is or is not operating in a low temperature combustion mode and provides that feedback to an engine control algorithm.

  5. Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C.

    PubMed Central

    Somogyi, L; Lasić, Z; Vukicević, S; Banfić, H

    1994-01-01

    Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and

  6. Summary of aerospace and nuclear engineering activities

    NASA Technical Reports Server (NTRS)

    1988-01-01

    The Texas A&M Nuclear and Aerospace engineering departments have worked on five different projects for the NASA/USRA Advanced Design Program during the 1987/88 year. The aerospace department worked on two types of lunar tunnelers that would create habitable space. The first design used a heated cone to melt the lunar regolith, and the second used a conventional drill to bore its way through the crust. Both used a dump truck to get rid of waste heat from the reactor as well as excess regolith from the tunneling operation. The nuclear engineering department worked on three separate projects. The NEPTUNE system is a manned, outer-planetary explorer designed with Jupiter exploration as the baseline mission. The lifetime requirement for both reactor and power-conversion systems was twenty years. The second project undertaken for the power supply was a Mars Sample Return Mission power supply. This was designed to produce 2 kW of electrical power for seven years. The design consisted of a General Purpose Heat Source (GPHS) utilizing a Stirling engine as the power conversion unit. A mass optimization was performed to aid in overall design. The last design was a reactor to provide power for propulsion to Mars and power on the surface. The requirements of 300 kW of electrical power output and a mass of less than 10,000 Rg were set. This allowed the reactor and power conversion unit to fit within the Space Shuttle cargo bay.

  7. Summary of aerospace and nuclear engineering activities

    NASA Technical Reports Server (NTRS)

    1988-01-01

    The Texas A&M Nuclear and Aerospace engineering departments have worked on five different projects for the NASA/USRA Advanced Design Program during the 1987/88 year. The aerospace department worked on two types of lunar tunnelers that would create habitable space. The first design used a heated cone to melt the lunar regolith, and the second used a conventional drill to bore its way through the crust. Both used a dump truck to get rid of waste heat from the reactor as well as excess regolith from the tunneling operation. The nuclear engineering department worked on three separate projects. The NEPTUNE system is a manned, outer-planetary explorer designed with Jupiter exploration as the baseline mission. The lifetime requirement for both reactor and power-conversion systems was twenty years. The second project undertaken for the power supply was a Mars Sample Return Mission power supply. This was designed to produce 2 kW of electrical power for seven years. The design consisted of a General Purpose Heat Source (GPHS) utilizing a Stirling engine as the power conversion unit. A mass optimization was performed to aid in overall design. The last design was a reactor to provide power for propulsion to Mars and power on the surface. The requirements of 300 kW of electrical power output and a mass of less than 10,000 Rg were set. This allowed the reactor and power conversion unit to fit within the Space Shuttle cargo bay.

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

    DTIC Science & Technology

    2004-03-01

    receptors are ionotropic glutamate receptors that mediate major excitatory neurotransmission. AMPA receptors mediate the majority of fast excitatory...calcium ions and ability to activate downstream calcium-dependent signal transduction processes. Phosphorylation of the AMPA receptor at Ser845 (S845...and voltage- gated ion channels. These include the NMDA-type glutamate receptors (Fienberg et al., 1998) and the AMPA -type glutamate receptors (Yan

  9. Antagonists of the TMEM16A Calcium-Activated Chloride Channel Modulate Airway Smooth Muscle Tone and Intracellular Calcium

    PubMed Central

    Danielsson, Jennifer; Perez-Zoghbi, Jose; Bernstein, Kyra; Barajas, Matthew B.; Zhang, Yi; Kumar, Satish; Sharma, Pawan K.; Gallos, George; Emala, Charles W.

    2015-01-01

    Background Perioperative bronchospasm refractory to β-agonists continues to challenge anesthesiologists and intensivists. The TMEM16A calcium-activated chloride channel modulates airway smooth muscle (ASM) contraction. We hypothesized that TMEM16A antagonists would relax ASM contraction by modulating membrane potential and calcium flux. Methods Human ASM, guinea pig tracheal rings or mouse peripheral airways were contracted with acetylcholine (Ach) or leukotriene D4 (LTD4) and then treated with the TMEM16A antagonists: benzbromarone, T16Ainh-A01, MONNA or B25. In separate studies, guinea pig tracheal rings were contracted with Ach and then exposed to increasing concentrations of isoproterenol (0.01nM-10μM) ± benzbromarone. Plasma membrane potential and intracellular calcium concentrations were measured in human ASM cells. Results Benzbromarone was the most potent TMEM16A antagonist tested for relaxing an Ach-induced contraction in guinea pig tracheal rings (n=6). Further studies were done to investigate benzbromarone’s clinical utility. In human ASM, benzbromarone relaxed either an acetylcholine- or LTD4-induced contraction (n=8). Benzbromarone was also effective in relaxing peripheral airways (n=9) and potentiating relaxation by β-agonists (n=5–10). In cellular mechanistic studies, benzbromarone hyperpolarized human ASM cells (n=9–12) and attenuated intracellular calcium flux from both the plasma membrane and sarcoplasmic reticulum (n=6–12). Conclusions TMEM16A antagonists work synergistically with β-agonists and through a novel pathway of interrupting ion flux both at the plasma membrane and sarcoplasmic reticulum to acutely relax human airway smooth muscle. PMID:26181339

  10. Mitofusin 2 decreases intracellular lipids in macrophages by regulating peroxisome proliferator-activated receptor-γ

    SciTech Connect

    Liu, Chun; Ge, Beihai; He, Chao; Zhang, Yi; Liu, Xiaowen; Liu, Kejian; Qian, Cuiping; Zhang, Yu; Peng, Wenzhong; Guo, Xiaomei

    2014-07-18

    Highlights: • Mfn2 decreases cellular lipid accumulation by activating cholesterol transporters. • PPARγ is involved in the Mfn2-mediated increase of cholesterol transporter expressions. • Inactivation of ERK1/2 and p38 is involved in Mfn2-induced PPARγ expression. - Abstract: Mitofusin 2 (Mfn2) inhibits atherosclerotic plaque formation, but the underlying mechanism remains elusive. This study aims to reveal how Mfn2 functions in the atherosclerosis. Mfn2 expression was found to be significantly reduced in arterial atherosclerotic lesions of both mice and human compared with healthy counterparts. Here, we observed that Mfn2 increased cellular cholesterol transporter expression in macrophages by upregulating peroxisome proliferator-activated receptor-γ, an effect achieved at least partially by inhibiting extracellular signal-regulated kinase1/2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPKs) pathway. These findings provide insights into potential mechanisms of Mfn2-mediated alterations in cholesterol transporter expression, which may have significant implications for the treatment of atherosclerotic heart disease.

  11. Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.

    PubMed

    Körschgen, Hagen; Kuske, Michael; Karmilin, Konstantin; Yiallouros, Irene; Balbach, Melanie; Floehr, Julia; Wachten, Dagmar; Jahnen-Dechent, Willi; Stöcker, Walter

    2017-09-01

    How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like

  12. Improved in vitro anti-tumoral activity, intracellular uptake and apoptotic induction of gemcitabine-loaded pegylated unilamellar liposomes.

    PubMed

    Celia, Christian; Calvagno, Maria Grazia; Paolino, Donatella; Bulotta, Stefania; Ventura, Cinzia Anna; Russo, Diego; Fresta, Massimo

    2008-04-01

    Anaplastic thyroid carcinoma is one of the most aggressive and lethal solid carcinomas affecting humans. A major limit of the chemotherapeutic agents is represented by their low therapeutic index. In this work, we investigated the possibility of improving the anti-tumoral activity of gemcitabine by using pegylated unilamellar liposomes. Liposomes were made up of 1,2-dipalmitoyl-sn-glycero-3-phospocholine monohydrate/cholesterol/N-(carbonyl-methoxypolyethylene glycol-2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (6:3:1 molar ratio) and they were prepared with a pH gradient to improve the gemcitabine loading capacity. The anti-tumoral efficacy of the liposomal formulation was tested in vitro on human anaplastic thyroid carcinoma cells (ARO) in culture, comparing the effects with those of the free drug. Gemcitabine-loaded unilamellar liposomes had a mean size approximately 200 nm with a zeta potential approximately -2 mV. The liposomal carrier noticeably improved the anti-tumoral activity of gemcitabine against ARO cells in terms of both dose-dependent cytotoxic effect and of drug exposition effect. Namely, gemcitabine-loaded liposomes showed a cytotoxic effect (58.2% increase of cell mortality at 1 microM with respect to free drug) after 12 h incubation, while the free drug showed a significant activity only after 72 h incubation. Moreover, a significant effect on the cell mortality appeared at 0.1 microM and 100% mortality was detected at a concentration of 1 microM of gemcitabine-loaded liposomes, while the free drug elicited the same effect at a concentration of 100 microM. The improved anti-tumoral activity of gemcitabine determined by the liposomal carrier was due to a greater intracellular uptake. The intracellular gemcitabine levels as a function of time showed a sinusoidal profile with peaks after 2 h, 6 h and 11 h, related to the cellular cycle of ARO. PARP cleavage and DNA fragmentation analysis provided clear evidence of the apoptosis induction in

  13. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

  14. Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box against Intracellular Leishmania major Amastigotes

    PubMed Central

    Khraiwesh, Mozna; Leed, Susan; Roncal, Norma; Johnson, Jacob; Sciotti, Richard; Smith, Philip; Read, Lisa; Paris, Robert; Hudson, Thomas; Hickman, Mark; Grogl, Max

    2016-01-01

    Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries. In our study, we developed a Leishmania major intracellular amastigote assay that uses the activity of luciferase as a measure of parasite proliferation and used this assay to screen a collection of 400 compounds obtained from Medicines for Malaria Venture (MMV) for their antileishmanial activity. Our results showed that 14 compounds identified by MMV as antimalarial drugs have antileishmanial activity and can potentially be optimized for CL drug development. PMID:26503273

  15. Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box Against Intracellular Leishmania major Amastigotes.

    PubMed

    Khraiwesh, Mozna; Leed, Susan; Roncal, Norma; Johnson, Jacob; Sciotti, Richard; Smith, Philip; Read, Lisa; Paris, Robert; Hudson, Thomas; Hickman, Mark; Grogl, Max

    2016-02-01

    Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries. In our study, we developed a Leishmania major intracellular amastigote assay that uses the activity of luciferase as a measure of parasite proliferation and used this assay to screen a collection of 400 compounds obtained from Medicines for Malaria Venture (MMV) for their antileishmanial activity. Our results showed that 14 compounds identified by MMV as antimalarial drugs have antileishmanial activity and can potentially be optimized for CL drug development. © The American Society of Tropical Medicine and Hygiene.

  16. Intracellular distribution of fatty alcohol oxidase activity in Mucor circinelloides YR-1 isolated from petroleum contaminated soils.

    PubMed

    Silva-Jiménez, Hortencia; Zazueta-Novoa, Vanesa; Durón-Castellanos, Arelí; Rodríguez-Robelo, Carmen; Leal-Morales, Carlos A; Zazueta-Sandoval, Roberto

    2009-11-01

    In previous studies, Mucor circinelloides YR-1 isolated from petroleum-contaminated soils grown in decane as sole carbon source, showed fatty alcohol oxidase (FAO) activities in either particulate or soluble fractions from a cell-free extract. One is associated to internal membranes (mFAO) and the other one is soluble (sFAO). Both activities appear to be located in the cells in specific compartments other than peroxisomes. Results suggested that mFAO could be located on the inner face of the membrane of these compartments, and sFAO could be in the lumen of the specific compartments. This study reports on the intracellular distribution of FAO activity and the purification of sFAOs and mFAO after several different procedures for release from the membranous fraction using the mixed membrane fraction (MMF) after cellular homogenization as enzymatic source. Results with the purified mFAO show, by molecular weight criteria, that the enzyme has only one type of subunit with molecular mass of 46 kDa, with two isoelectric point components: 6.0 and 6.3. We found that mFAO is strongly associated to the MMF, possibly in a transitory fashion. Using non-denaturating gels, we suggest that sFAO and mFAO have the same subunits in their native structures, and, due to their native molecular weight of approximately 350 kDa, each could be natively structured as an octameric complex.

  17. Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

    PubMed Central

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

    2016-01-01

    Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis. PMID:26726017

  18. Regulation of spine and synapse formation by activity-dependent intracellular signaling pathways

    PubMed Central

    Saneyoshi, Takeo; Fortin, Dale A; Soderling, Thomas R

    2010-01-01

    Formation of the human brain during embryonic and postnatal development is an extraordinarily complex process resulting at maturity in billions of neurons with trillions of specialized connections called synapses. These synapses, composed of a varicosity or bouton from a presynaptic neuron that communicates with a dendritic spine of the postsynaptic neuron, comprise the neural network that is essential for complex behavioral phenomena and cognition. Inappropriate synapse formation or structure is thought to underlie several developmental neuropathologies. Even in the mature CNS, alterations in synapse structure and function continues to be a very dynamic process that is foundational to learning and memory as well as other adaptive abilities of the brain. This synaptic plasticity in mature neurons, which is often triggered by certain patterns of neural activity, is again multifaceted and involves post-translational modifications (e.g. phosphorylation) and subcellular relocalization or trafficking (endocytosis/exocytosis) of existing synaptic proteins, initiation of protein synthesis from existing mRNAs localized in dendrites or spines, and triggering of new gene transcription in the nucleus. These various cellular processes support varying temporal components of synaptic plasticity that begin within 1–2 min but can persist for hours to days. This review will give a critical assessment of activity-dependent molecular modulations of synapses reported over the past couple years. Owing to space limitations, it will focus on mammalian excitatory (i.e. glutamatergic) synapses and will not consider several activity-independent signaling pathways (e.g. ephrinB receptor) that also modulate spine and synapse formation [1,2]. PMID:19896363

  19. Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity

    NASA Technical Reports Server (NTRS)

    Janeczko, Richard A.; Etlinger, Joseph D.

    1984-01-01

    The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures are studied. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport. Similar size effects on protein metabolism and amino acid uptake in serum-free media were observed in parallel studies with insulin, although insulin levels well in excess of the normal physiological range are required to produce significant effects. It is suggested that there is a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues.

  20. Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity

    NASA Technical Reports Server (NTRS)

    Janeczko, Richard A.; Etlinger, Joseph D.

    1984-01-01

    The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures are studied. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport. Similar size effects on protein metabolism and amino acid uptake in serum-free media were observed in parallel studies with insulin, although insulin levels well in excess of the normal physiological range are required to produce significant effects. It is suggested that there is a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues.

  1. Active Suppression of Instabilities in Engine Combustors

    NASA Technical Reports Server (NTRS)

    Kopasakis, George

    2004-01-01

    A method of feedback control has been proposed as a means of suppressing thermo-acoustic instabilities in a liquid- fueled combustor of a type used in an aircraft engine. The basic principle of the method is one of (1) sensing combustor pressure oscillations associated with instabilities and (2) modulating the rate of flow of fuel to the combustor with a control phase that is chosen adaptively so that the pressure oscillations caused by the modulation oppose the sensed pressure oscillations. The need for this method arises because of the planned introduction of advanced, lean-burning aircraft gas turbine engines, which promise to operate with higher efficiencies and to emit smaller quantities of nitrogen oxides, relative to those of present aircraft engines. Unfortunately, the advanced engines are more susceptible to thermoacoustic instabilities. These instabilities are hard to control because they include large dead-time phase shifts, wide-band noise characterized by amplitudes that are large relative to those of the instabilities, exponential growth of the instabilities, random net phase walks, and amplitude fluctuations. In this method (see figure), the output of a combustion-pressure sensor would be wide-band-pass filtered and then further processed to generate a control signal that would be applied to a fast-actuation valve to modulate the flow of fuel. Initially, the controller would rapidly take large phase steps in order to home in, within a fraction of a second, to a favorable phase region within which the instability would be reduced. Then the controller would restrict itself to operate within this phase region and would further restrict itself to operate within a region of stability, as long as the power in the instability signal was decreasing. In the phase-shifting scheme of this method, the phase of the control vector would be made to continuously bounce back and forth from one boundary of an effective stability region to the other. Computationally

  2. MANAGING ENGINEERING ACTIVITIES FOR THE PLATEAU REMEDIATION CONTRACT - HANFORD

    SciTech Connect

    KRONVALL CM

    2011-01-14

    In 2008, the primary Hanford clean-up contract transitioned to the CH2MHill Plateau Remediation Company (CHPRC). Prior to transition, Engineering resources assigned to remediation/Decontamination and Decommissioning (D&D) activities were a part of a centralized engineering organization and matrixed to the performing projects. Following transition, these resources were reassigned directly to the performing project, with a loose matrix through a smaller Central Engineering (CE) organization. The smaller (10 FTE) central organization has retained responsibility for the overall technical quality of engineering for the CHPRC, but no longer performs staffing and personnel functions. As the organization has matured, there are lessons learned that can be shared with other organizations going through or contemplating performing a similar change. Benefits that have been seen from the CHPRC CE organization structure include the following: (1) Staff are closely aligned with the 'Project/facility' that they are assigned to support; (2) Engineering priorities are managed to be consistent with the 'Project/facility' priorities; (3) Individual Engineering managers are accountable for identifying staffing needs and the filling of staffing positions; (4) Budget priorities are managed within the local organization structure; (5) Rather than being considered a 'functional' organization, engineering is considered a part of a line, direct funded organization; (6) The central engineering organization is able to provide 'overview' activities and maintain independence from the engineering organizations in the field; and (7) The central engineering organization is able to maintain a stable of specialized experts that are able to provide independent reviews of field projects and day-to-day activities.

  3. Experimental strategies towards increasing intracellular mitochondrial activity in oocytes: A systematic review.

    PubMed

    Darbandi, Sara; Darbandi, Mahsa; Khorshid, Hamid Reza Khorram; Sadeghi, Mohammad Reza; Al-Hasani, Safaa; Agarwal, Ashok; Shirazi, Abolfazl; Heidari, Mahnaz; Akhondi, Mohammad Mehdi

    2016-09-01

    The mitochondrial complement is critical in sustaining the earliest stages of life. To improve the Assisted Reproductive Technology (ART), current methods of interest were evaluated for increasing the activity and copy number of mitochondria in the oocyte cell. This covered the researches from 1966 to September 2015. The results provided ten methods that can be studied individually or simultaneously. Though the use of these techniques generated great concern about heteroplasmy observation in humans, it seems that with study on these suggested methods there is real hope for effective treatments of old oocyte or oocytes containing mitochondrial problems in the near future. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  4. Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting.

    PubMed

    Zou, Jiawei; Huang, Xin; Wu, Lei; Chen, Gangyi; Dong, Juan; Cui, Xin; Tang, Zhuo

    2015-12-01

    Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36% brighter than Spinach when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

  5. Substantial depletion of the intracellular Ca2+ stores is required for macroscopic activation of the Ca2+ release-activated Ca2+ current in rat basophilic leukaemia cells

    PubMed Central

    Fierro, Leonardo; Parekh, Anant B

    2000-01-01

    Tight-seal whole-cell patch clamp experiments were performed to examine the ability of different intracellular Ca2+ mobilising agents to activate the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL-1) cells under conditions of weak cytoplasmic Ca2+ buffering. Dialysis with a maximal concentration of inositol 1,4,5-trisphosphate (IP3) routinely failed to activate macroscopic ICRAC in low buffer (0.1 mM EGTA, BAPTA or dimethyl BAPTA), whereas it activated the current to its maximal extent in high buffer (10 mM EGTA). Dialysis with a poorly metabolisable analogue of IP3, with ionomycin, or with IP3 and ionomycin all failed to generate macroscopic ICRAC in low Ca2+ buffering conditions. Dialysis with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump blocker thapsigargin was able to activate ICRAC even in the presence of low cytoplasmic Ca2+ buffering, albeit at a slow rate. Exposure to IP3 together with the SERCA blockers thapsigargin, thapsigargicin or cyclopiazonic acid rapidly activated ICRAC in low buffer. Following activation of ICRAC by intracellular dialysis with IP3 and thapsigargin in low buffer, the current was very selective for Ca2+ (apparent KD of 1 mM). Sr2+ and Ba2+ were less effective charge carriers and Na+ was not conducted to any appreciable extent. The ionic selectivity of ICRAC was very similar in low or high intracellular Ca2+ buffer. Fast Ca2+-dependent inactivation of ICRAC occurred at a similar rate and to a similar extent in low or high Ca2+ buffer. Ca2+-dependent inactivation is not the reason why macroscopic ICRAC cannot be seen under conditions of low cytoplasmic Ca2+ buffering. ICRAC could be activated by combining IP3 with thapsigargin, even in the presence of 100 μM Ca2+ and the absence of any exogenous Ca2+ chelator, where ATP and glutamate represented the only Ca2+ buffers in the pipette solution. Our results suggest that a threshold exists within the IP3-sensitive Ca2+ store, below which

  6. A-FABP mediates adaptive thermogenesis by promoting intracellular activation of thyroid hormones in brown adipocytes

    PubMed Central

    Shu, Lingling; Hoo, Ruby L. C.; Wu, Xiaoping; Pan, Yong; Lee, Ida P. C.; Cheong, Lai Yee; Bornstein, Stefan R; Rong, Xianglu; Guo, Jiao; Xu, Aimin

    2017-01-01

    The adipokine adipocyte fatty acid-binding protein (A-FABP) has been implicated in obesity-related cardio-metabolic complications. Here we show that A-FABP increases thermogenesis by promoting the conversion of T4 to T3 in brown adipocytes. We find that A-FABP levels are increased in both white (WAT) and brown (BAT) adipose tissues and the bloodstream in response to thermogenic stimuli. A-FABP knockout mice have reduced thermogenesis and whole-body energy expenditure after cold stress or after feeding a high-fat diet, which can be reversed by infusion of recombinant A-FABP. Mechanistically, A-FABP induces the expression of type-II iodothyronine deiodinase in BAT via inhibition of the nuclear receptor liver X receptor α, thereby leading to the conversion of thyroid hormone from its inactive form T4 to active T3. The thermogenic responses to T4 are abrogated in A-FABP KO mice, but enhanced by A-FABP. Thus, A-FABP acts as a physiological stimulator of BAT-mediated adaptive thermogenesis. PMID:28128199

  7. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

    SciTech Connect

    Peterson, K.M.; Alderete, J.F.

    1984-08-01

    Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

  8. Basic Tetrapeptides as Potent Intracellular Inhibitors of Type A Botulinum Neurotoxin Protease Activity*

    PubMed Central

    Hale, Martha; Oyler, George; Swaminathan, Subramanyam; Ahmed, S. Ashraf

    2011-01-01

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development. PMID:20961849

  9. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    SciTech Connect

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  10. AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae.

    PubMed

    Klöckner, Anna; Otten, Christian; Derouaux, Adeline; Vollmer, Waldemar; Bühl, Henrike; De Benedetti, Stefania; Münch, Daniela; Josten, Michaele; Mölleken, Katja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-06-23

    Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation.

  11. Thalamic Activation Modulates the Responses of Neurons in Rat Primary Auditory Cortex: An In Vivo Intracellular Recording Study

    PubMed Central

    Han, Lei; Zhang, Yonghai; Lou, Yunxiao; Xiong, Ying

    2012-01-01

    Auditory cortical plasticity can be induced through various approaches. The medial geniculate body (MGB) of the auditory thalamus gates the ascending auditory inputs to the cortex. The thalamocortical system has been proposed to play a critical role in the responses of the auditory cortex (AC). In the present study, we investigated the cellular mechanism of the cortical activity, adopting an in vivo intracellular recording technique, recording from the primary auditory cortex (AI) while presenting an acoustic stimulus to the rat and electrically stimulating its MGB. We found that low-frequency stimuli enhanced the amplitudes of sound-evoked excitatory postsynaptic potentials (EPSPs) in AI neurons, whereas high-frequency stimuli depressed these auditory responses. The degree of this modulation depended on the intensities of the train stimuli as well as the intervals between the electrical stimulations and their paired sound stimulations. These findings may have implications regarding the basic mechanisms of MGB activation of auditory cortical plasticity and cortical signal processing. PMID:22514672

  12. Flavonoids in Juglans regia L. leaves and evaluation of in vitro antioxidant activity via intracellular and chemical methods.

    PubMed

    Zhao, Ming-Hui; Jiang, Zi-Tao; Liu, Tao; Li, Rong

    2014-01-01

    Flavonoids are rich in Juglans regia L. leaves. They have potent antioxidant properties, which have been related to regulating immune function and enhancing anticancer activity. Herein, qualitative and quantitative determination of flavonoids from J. regia leaves was carried out using high performance liquid chromatography coupled with tandem mass spectrometry with electrospray ionization and negative ion detection (HPLC-ESI-MS/MS) by comparison of the retention times and mass spectral fragments with standard substances or related literatures. Seventeen compounds were identified and major components are quercetin-3-O-rhamnoside (453.11 μg/g, dry weight), quercetin-3-O-arabinoside (73.91 μg/g), quercetin-3-O-xyloside (70.04 μg/g), kaempferol-O-pentoside derivative (49.04 μg/g), quercetin-3-O-galactoside (48.61 μg/g), and kaempferol-O-pentoside (48.46 μg/g). The in vitro intracellular antioxidation indicated that flavonoids from J. regia leaves could reduce the reactive oxygen species (ROS) level in RAW264.7 cells and showed good radical scavenging activities. These results proved to be more related to the flavonoids that could be considered in the design of new formulations of dietary supplements or functional foods.

  13. Flavonoids in Juglans regia L. Leaves and Evaluation of In Vitro Antioxidant Activity via Intracellular and Chemical Methods

    PubMed Central

    Zhao, Ming-Hui; Liu, Tao; Li, Rong

    2014-01-01

    Flavonoids are rich in Juglans regia L. leaves. They have potent antioxidant properties, which have been related to regulating immune function and enhancing anticancer activity. Herein, qualitative and quantitative determination of flavonoids from J. regia leaves was carried out using high performance liquid chromatography coupled with tandem mass spectrometry with electrospray ionization and negative ion detection (HPLC-ESI-MS/MS) by comparison of the retention times and mass spectral fragments with standard substances or related literatures. Seventeen compounds were identified and major components are quercetin-3-O-rhamnoside (453.11 μg/g, dry weight), quercetin-3-O-arabinoside (73.91 μg/g), quercetin-3-O-xyloside (70.04 μg/g), kaempferol-O-pentoside derivative (49.04 μg/g), quercetin-3-O-galactoside (48.61 μg/g), and kaempferol-O-pentoside (48.46 μg/g). The in vitro intracellular antioxidation indicated that flavonoids from J. regia leaves could reduce the reactive oxygen species (ROS) level in RAW264.7 cells and showed good radical scavenging activities. These results proved to be more related to the flavonoids that could be considered in the design of new formulations of dietary supplements or functional foods. PMID:25133218

  14. AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae

    PubMed Central

    Klöckner, Anna; Otten, Christian; Derouaux, Adeline; Vollmer, Waldemar; Bühl, Henrike; De Benedetti, Stefania; Münch, Daniela; Josten, Michaele; Mölleken, Katja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-01-01

    Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation. PMID:24953137

  15. Aqueous solution behaviour and membrane disruptive activity of pH-responsive PEGylated pseudo-peptides and their intracellular distribution.

    PubMed

    Chen, Rongjun; Yue, Zhilian; Eccleston, Mark E; Slater, Nigel K H

    2008-11-01

    The effect of PEGylation on the aqueous solution properties and cell membrane disruptive activity of a pH-responsive pseudo-peptide, poly(l-lysine iso-phthalamide), has been investigated by dynamic light scattering, haemolysis and lactate dehydrogenase (LDH) assays. Intracellular trafficking of the polymers has been examined using confocal and fluorescence microscopy. With increasing degree of PEGylation, the modified polymers can form stabilised compact structures with reduced mean hydrodynamic diameters. Poly(l-lysine iso-phthalamide) with a low degree of PEGylation (17.4 wt%) retained pH-dependent solution behaviour and showed enhanced kinetic membrane disruptive activity compared to the parent polymer. It facilitated trafficking of endocytosed materials into the cytoplasm of HeLa cells. At levels of PEGylation in excess of 25.6 wt%, the modified polymers displayed a single particle size distribution unresponsive to pH, as well as a decrease in cell membrane lytic ability. The mechanism involved in membrane destabilisation was also investigated, and the potential applications of these modified polymers in drug delivery were discussed.

  16. High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis.

    PubMed

    Leite, F C B; Leite, D V da R; Pereira, L F; de Barros Pita, W; de Morais, M A

    2016-09-01

    Dekkera bruxellensis hit the spotlight in the past decade mostly due to its rather high ability to adapt to several different fermentation processes. This yeast relies on different genetic and physiological aspects to achieve and preserve its high industrial fitness and some of these traits are shared with Saccharomyces cerevisiae. We have previously described that D. bruxellensis is unable to make use of accumulating trehalose as a strategy for cell adaptation and survival in the industrial scenario, as opposed to S. cerevisiae. Since trehalose is often involved in mechanisms related to cell protection, we aimed to investigate both cause and effect of the absence of this metabolite in the cell adaptive capacity in the industrial environment. Our results indicate that the major cause for the nonaccumulation of trehalose is the high constitutive activity of neutral trehalase. Therefore, the rate of trehalose degradation could be higher than its rate of synthesis, preventing accumulation. Altogether, our data elucidate the mechanisms involved in the lack of trehalose accumulation in D. bruxellensis as well as evaluates the implications of this feature. Dekkera bruxellensis can successfully take advantage of its peculiar physiological and genetic traits in order to adapt and survive in fermentation processes. So far, tolerance to stress has been credited to trehalose synthesis. The data presented in this work provided information on the underlying mechanism that prevents trehalose accumulation and corroborated the recent information that trehalose itself is not implicated in yeast stress tolerance. Second, it showed that D. bruxellensis responds differently to Saccharomyces cerevisiae to excess of sugar, which may explain its preference for respiration (oxidative metabolism) over fermentation (reductive metabolism) even at limited oxygen supply. These findings help to understand the drop on ethanol production in processes overtaken by this yeast. © 2016 The

  17. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  18. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  19. Situational Interest in Engineering Design Activities

    NASA Astrophysics Data System (ADS)

    Bonderup Dohn, Niels

    2013-08-01

    The aim of the present mixed-method study was to investigate task-based situational interest of sixth grade students (n = 46), between 12 and 14 years old, during an eight-week engineering design programme in a Science & Technology-class. Students' interests were investigated by means of a descriptive interpretative analysis of qualitative data from classroom observations and informal interviews. The analysis was complemented by a self-report survey to validate findings and determine prevalence. The analysis revealed four main sources of interest: designing inventions, trial-and-error experimentation, achieved functionality of invention, and collaboration. These sources differ in terms of stimuli factors, such as novelty, autonomy (choice), social involvement, self-generation of interest, and task goal orientation. The study shows that design tasks stimulated interest, but only to the extent that students were able to self-regulate their learning strategies.

  20. International Collaboration Activities on Engineered Barrier Systems

    SciTech Connect

    Jove-Colon, Carlos F.

    2016-08-31

    The Used Fuel Disposition Campaign (UFDC) within the DOE Fuel Cycle Technologies (FCT) program has been engaging in international collaborations between repository R&D programs for high-level waste (HLW) disposal to leverage on gathered knowledge and laboratory/field data of near- and far-field processes from experiments at underground research laboratories (URL). Heater test experiments at URLs provide a unique opportunity to mimetically study the thermal effects of heat-generating nuclear waste in subsurface repository environments. Various configurations of these experiments have been carried out at various URLs according to the disposal design concepts of the hosting country repository program. The FEBEX (Full-scale Engineered Barrier Experiment in Crystalline Host Rock) project is a large-scale heater test experiment originated by the Spanish radioactive waste management agency (Empresa Nacional de Residuos Radiactivos S.A. – ENRESA) at the Grimsel Test Site (GTS) URL in Switzerland. The project was subsequently managed by CIEMAT. FEBEX-DP is a concerted effort of various international partners working on the evaluation of sensor data and characterization of samples obtained during the course of this field test and subsequent dismantling. The main purpose of these field-scale experiments is to evaluate feasibility for creation of an engineered barrier system (EBS) with a horizontal configuration according to the Spanish concept of deep geological disposal of high-level radioactive waste in crystalline rock. Another key aspect of this project is to improve the knowledge of coupled processes such as thermal-hydro-mechanical (THM) and thermal-hydro-chemical (THC) operating in the near-field environment. The focus of these is on model development and validation of predictions through model implementation in computational tools to simulate coupled THM and THC processes.

  1. Intracellular ROS protection efficiency and free radical-scavenging activity of quercetin and quercetin-encapsulated liposomes.

    PubMed

    Rezaei-Sadabady, Rogaie; Eidi, Akram; Zarghami, Nosratollah; Barzegar, Abolfazl

    2016-01-01

    Quercetin (3,5,7,3',4'-pentahydroxyflavone) is a natural bio-flavonoid originating from fruits, vegetables, seeds, berries, and tea. The antioxidant activity of quercetin and its protective effects against cardiovascular disorders, anti-cancer, anti-inflammatory, and anti-viral activities have been extensively documented; however, the clinical request of quercetin in cancer treatment is significantly limited due to its very poor delivery features. In order to increase the hydrophilicity and drug delivery capability, we encapsulated quercetin into liposomes. Our data indicated that liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be used as an effective antioxidant for ROS protection within the polar cytoplasm, and the nano-sized quercetin encapsulated by liposomes enhanced the cellular uptake (cancer cell human MCF_7). Quercetin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of quercetin in polar solvents by a comparative study using reduction of ferric iron in aqueous medium, intracellular ROS/toxicity assays, and reducing DPPH assays. Cell viability and ROS assays demonstrated that quercetin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and deadly belongings of cumene hydroperoxide. The purpose of this study was to determine whether a liposomal formulation of quercetin can suggestively improve its solubility and bioavailability and can be a possible request in the treatment of tumor. The authors encapsulated quercetin in a liposomal delivery system. They studied the in vitro effects of this compound on proliferation using human MCF-7 carcinoma cells. The activity of liposomal quercetin was equal to or better than that of free quercetin at equimolar concentrations. Our data indicated that liposomal quercetin can significantly improve the

  2. In Situ Ratiometric Quantitative Tracing of Intracellular Leucine Aminopeptidase Activity via an Activatable Near-Infrared Fluorescent Probe.

    PubMed

    Gu, Kaizhi; Liu, Yajing; Guo, Zhiqian; Lian, Cheng; Yan, Chenxu; Shi, Ping; Tian, He; Zhu, Wei-Hong

    2016-10-03

    Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM-Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM-Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with "build-in calibration" as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM-Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM-Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM-Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor.

  3. The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.

    PubMed

    Gamble, Michael; Künze, Georg; Brancale, Andrea; Wilson, Keith S; Jones, D Dafydd

    2012-01-01

    The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions.

  4. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility.

    PubMed

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2016-09-27

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  5. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors.

  6. Intracellular delivery and ultrasonic activation of folate receptor-targeted phase-change contrast agents in breast cancer cells in vitro.

    PubMed

    Marshalek, Joseph P; Sheeran, Paul S; Ingram, Pier; Dayton, Paul A; Witte, Russell S; Matsunaga, Terry O

    2016-12-10

    Breast cancer is a diverse and complex disease that remains one of the leading causes of death among women. Novel, outside-of-the-box imaging and treatment methods are needed to supplement currently available technologies. In this study, we present evidence for the intracellular delivery and ultrasound-stimulated activation of folate receptor (FR)-targeted phase-change contrast agents (PCCAs) in MDA-MB-231 and MCF-7 breast cancer cells in vitro. PCCAs are lipid-coated, perfluorocarbon-filled particles formulated as nanoscale liquid droplets capable of vaporization into gaseous microbubbles for imaging or therapy. Cells were incubated with 1:1 decafluorobutane (DFB)/octafluoropropane (OFP) PCCAs for 1h, imaged via confocal microscopy, exposed to ultrasound (9MHz, MI=1.0 or 1.5), and imaged again after insonation. FR-targeted PCCAs were observed intracellularly in both cell lines, but uptake was significantly greater (p<0.001) in MDA-MB-231 cells (93.0% internalization at MI=1.0, 79.5% at MI=1.5) than MCF-7 cells (42.4% internalization at MI=1.0, 35.7% at MI=1.5). Folate incorporation increased the frequency of intracellular PCCA detection 45-fold for MDA-MB-231 cells and 7-fold for MCF-7 cells, relative to untargeted PCCAs. Intracellularly activated PCCAs ranged from 500nm to 6μm (IQR=800nm-1.5μm) with a mean diameter of 1.15±0.59 (SD) microns. The work presented herein demonstrates the feasibility of PCCA intracellular delivery and activation using breast cancer cells, illuminating a new platform toward intracellular imaging or therapeutic delivery with ultrasound.

  7. Vehicle Engineering Development Activities at the Marshall Space Flight Center

    NASA Technical Reports Server (NTRS)

    Fisher, Mark F.; Champion, Robert H., Jr.

    1999-01-01

    New initiatives in the Space Transportation Directorate at the Marshall Space Flight Center include an emphasis on Vehicle Engineering to enhance the strong commitment to the Directorate's projects in the development of flight hardware and flight demonstrators for the advancement of space transportation technology. This emphasis can be seen in the activities of a newly formed organization in the Transportation Directorate, The Vehicle Subsystems Engineering Group. The functions and type of activities that this group works on are described. The current projects of this group are outlined including a brief description of the status and type of work that the group is performing. A summary section is included to describe future activities.

  8. Na-KATPase activity and intracellular ion concentrations in the lactating guinea pig mammary gland. Studies on Na-K activated adenosine triphosphatase, XXXVI.

    PubMed

    Vreeswijk, J H; de Pont, J J; Bonting, S L

    1975-01-01

    The intracellular sodium, potassium and chloride concentrations in slices of lactating guinea pig mammary gland have been determined by chemical analysis and the use of appropriate values for extracellular space. These ion concentrations after 1 hr incubation at 37 degrees C in a Krebs-Ringer bicarbonate solution are 45mM Na+, 138 mM K+ and 44 mM Cl-, which values are in agreement with those found in fresh mammary gland slices. Inhibition of the NaK activated ATPase cation pump system of the tissue by 10(-4)M ouabain, anoxia or cooling to 0 degrees C Causes a gain of Na+ and an equimolar loss of K+ without a significant change in chloride concentration. The effect of cooling (0 degrees C) is reversible by reincubation at 37 degrees C. Water content of the tissue (76.5% of wet weight) and extracellular space (40.5%) do not change under these conditions. The results permit the conclusion that the NaK activated ATPase system is responsible for the maintenance of the intracellular Na+ and K+ concentrations, but do not support the presence of a chloride pump.

  9. Exploring Careers in Science and Engineering. Second Edition. [Student Activities.

    ERIC Educational Resources Information Center

    Research Triangle Inst., Research Triangle Park, NC.

    This program (which consists of 12 activities) is aimed at increasing the career relevance of science education for all students in grades 4 through 9, while at the same time particularly encouraging female and minority students to consider careers in science and engineering. Major areas addressed in the activities are: (1) students' images of…

  10. Transporting Radioactive Waste: An Engineering Activity. Grades 5-12.

    ERIC Educational Resources Information Center

    HAZWRAP, The Hazardous Waste Remedial Actions Program.

    This brochure contains an engineering activity for upper elementary, middle school, and high school students that examines the transportation of radioactive waste. The activity is designed to inform students about the existence of radioactive waste and its transportation to disposal sites. Students experiment with methods to contain the waste and…

  11. Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes*

    PubMed Central

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, KeWei

    2012-01-01

    α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders. PMID:22679014

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

    PubMed

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

    2015-11-01

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

  13. Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1991-01-01

    We have been exploring the role of iron in the pathogenesis of the intracellular bacterial pathogen Legionella pneumophila. In previous studies, we have demonstrated that L. pneumophila intracellular multiplication in human monocytes is iron dependent and that IFN gamma-activated monocytes inhibit L. pneumophila intracellular multiplication by limiting the availability of iron. In this study, we have investigated the effect on L. pneumophila intracellular multiplication of lactoferrin, an iron-binding protein which is internalized via specific receptors on monocytes, and of nonphysiologic iron chelates which enter monocytes by a receptor-independent route. Apolactoferrin completely inhibited L. pneumophila multiplication in nonactivated monocytes, and enhanced the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication. In contrast, iron-saturated lactoferrin had no effect on the already rapid rate of L. pneumophila multiplication in nonactivated monocytes. Moreover, it reversed the capacity of activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron from the lactoferrin-lactoferrin receptor pathway. The capacity of iron-lactoferrin to reverse monocyte activation was dependent upon its percent iron saturation and not just its total iron content. Similarly, the nonphysiologic iron chelates ferric nitrilotriacetate and ferric ammonium citrate completely reverse and ferric pyrophosphate partially reversed the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron derived from nonphysiologic iron chelates internalized by monocytes independently of the transferrin and lactoferrin endocytic pathways. This study suggests that at sites of inflammation, lactoferrin may inhibit or promote L. pneumophila intracellular multiplication in mononuclear phagocytes depending upon

  14. A triple-barreled microelectrode for simultaneous measurements of intracellular Na+ and K+ activities and membrane potential in biological cells.

    PubMed

    Fujimoto, M; Honda, M

    1980-01-01

    A triple-barreled Na+, K+-selective microelectrode was constructed with liquid ion exchangers for Na+ (monensin) and K+ (Corning #477317) to measure the intracellular Na+ and K+ activities ((Na)i and (K)i) of a single cell and its membrane potential (EM), simultaneously. The tip of the triple-barreled assembly was made less than 0.6 micron in outside diameter. Prior to in vivo measurements, some physiochemical properties of microelectrodes were examined in vitro for the slope constant, selectivity coefficient, electrical resistance, and pH effect, as well as measurements of the activity coefficient on ions in blood serum and Ringer solution. Carrying out direct micropunctures on single cells of the sartorius muscle and renal proximal tubule of bullfrogs in vivo, we obtained the following results: (1) In sartorius muscle, the average (Na)i was 14.8 mEq/liter, the (K)i 64.5 mEq/liter, and the EM -86.2 mV. (2) In proximal tubule cells, the average (Na)i, (K)i and EM were 16.8, 63.0 mEq/liter and -65.9 mV, respectively. (3) There were significant correlations in the proximal tubule between (K)i and EM, and inversely between (Na)i and EM, and between (Na)i and (K)i. These facts may somehow be related to both the activity of Na+-K+ exchange pump and the osmotic equilibrium of water across the membrane. Further, several problems inherent in the multibarreled microelectrode were discussed from the practical point of view.

  15. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

  16. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing.

    PubMed

    Erickson, Hans K; Park, Peter U; Widdison, Wayne C; Kovtun, Yelena V; Garrett, Lisa M; Hoffman, Karen; Lutz, Robert J; Goldmacher, Victor S; Blättler, Walter A

    2006-04-15

    Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen-specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, we used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, we examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.

  17. A novel DNA tetrahedron-hairpin probe for in situ"off-on" fluorescence imaging of intracellular telomerase activity.

    PubMed

    Feng, Qiu-Mei; Zhu, Meng-Jiao; Zhang, Ting-Ting; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-21

    A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.

  18. Altered intracellular distribution of PrPC and impairment of proteasome activity in tau overexpressing cortical neurons.

    PubMed

    Canu, Nadia; Filesi, Ilaria; Pristerà, Andrea; Ciotti, Maria Teresa; Biocca, Silvia

    2011-01-01

    The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-regulation of the proteasome system, suggesting a possible mechanism that contributes to intracellular PrP(C) accumulation. These findings open a new perspective for the possible crosstalk between tau and prion proteins in the pathogenesis of tau induced-neurodegeneration.

  19. Inflammation and exercise: Inhibition of monocytic intracellular TNF production by acute exercise via β2-adrenergic activation.

    PubMed

    Dimitrov, Stoyan; Hulteng, Elaine; Hong, Suzi

    2017-03-01

    Regular exercise is shown to exert anti-inflammatory effects, yet the effects of acute exercise on cellular inflammatory responses and its mechanisms remain unclear. We tested the hypothesis that sympathoadrenergic activation during a single bout of exercise has a suppressive effect on monocytic cytokine production mediated by β2 adrenergic receptors (AR). We investigated the effects of 20-min moderate (65-70% VO2 peak) exercise-induced catecholamine production on LPS-stimulated TNF production by monocytes in 47 healthy volunteers and determined AR subtypes involved. We also examined the effects of β-agonist isoproterenol and endogenous β- and α-agonists epinephrine and norepinephrine, and receptor-subtype-specific β- and α-antagonists on TNF production in a series of in vitro investigations. LPS-stimulated TNF production by peripheral blood monocytes was determined intracellularly by flow cytometry, using an intracellular protein transport inhibitor. Percent TNF-producing monocytes and per-cell TNF production with and without LPS was suppressed by exercise with moderate to large effects, which was reversed by a β2-AR antagonist in spite that plasma TNF levels did not change. This inhibitory response in TNF production by exercise was mirrored by β-AR agonists in an agonist-specific and dose-dependent manner in vitro: similar isoproterenol (EC50=2.1-4.7×10(-10)M) and epinephrine (EC50=4.4-10×10(-10)M) potency and higher norepinephrine concentrations (EC50=2.6-4.3×10(-8)M) needed for the effects. Importantly, epinephrine levels observed during acute exercise in vivo significantly inhibited TNF production in vitro. The inhibitory effect of the AR agonists was abolished by β2-, but not by β1- or α-AR blockers. We conclude that the downregulation of monocytic TNF production during acute exercise is mediated by elevated epinephrine levels through β2-ARs. Decreased inflammatory responses during acute exercise may protect against chronic conditions with low

  20. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

  1. Pituitary Adenylate Cyclase-Activating Polypeptide Induces the Voltage-Independent Activation of Inward Membrane Currents and Elevation of Intracellular Calcium in HIT-T15 Insulinoma Cells*

    PubMed Central

    LEECH, COLIN A.; HOLZ, GEORGE G.; HABENER, JOEL F.

    2010-01-01

    The secretion of insulin by pancreatic β-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockable by La3+, and which exhibits a reversal potential of about −28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at −70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic β-cells during the transition period from fasting to feeding. PMID:7895663

  2. Enhancing learning in geosciences and water engineering via lab activities

    NASA Astrophysics Data System (ADS)

    Valyrakis, Manousos; Cheng, Ming

    2016-04-01

    This study focuses on the utilisation of lab based activities to enhance the learning experience of engineering students studying Water Engineering and Geosciences. In particular, the use of modern highly visual and tangible presentation techniques within an appropriate laboratory based space are used to introduce undergraduate students to advanced engineering concepts. A specific lab activity, namely "Flood-City", is presented as a case study to enhance the active engagement rate, improve the learning experience of the students and better achieve the intended learning objectives of the course within a broad context of the engineering and geosciences curriculum. Such activities, have been used over the last few years from the Water Engineering group @ Glasgow, with success for outreach purposes (e.g. Glasgow Science Festival and demos at the Glasgow Science Centre and Kelvingrove museum). The activity involves a specific setup of the demonstration flume in a sand-box configuration, with elements and activities designed so as to gamely the overall learning activity. Social media platforms can also be used effectively to the same goals, particularly in cases were the students already engage in these online media. To assess the effectiveness of this activity a purpose designed questionnaire is offered to the students. Specifically, the questionnaire covers several aspects that may affect student learning, performance and satisfaction, such as students' motivation, factors to effective learning (also assessed by follow-up quizzes), and methods of communication and assessment. The results, analysed to assess the effectiveness of the learning activity as the students perceive it, offer a promising potential for the use of such activities in outreach and learning.

  3. ENGINEERING BULLETIN: GRANULAR ACTIVATED CARBON TREATMENT

    EPA Science Inventory

    Granular activated carbon (GAC) treatment is a physicochemical process that removes a wide variety of contaminants by adsorbing them from liquid and gas streams [1, p. 6-3]. This treatment is most commonly used to separate organic contaminants from water or air; however, it can b...

  4. Bioglass Activated Skin Tissue Engineering Constructs for Wound Healing.

    PubMed

    Yu, Hongfei; Peng, Jinliang; Xu, Yuhong; Chang, Jiang; Li, Haiyan

    2016-01-13

    Wound healing is a complicated process, and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts, and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied, and the bioactive skin tissue engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I, and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed, and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which would finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and extracellular matrix proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.

  5. Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum.

    PubMed

    Kim, Minkyung; Hennig, Grant W; Smith, Terence K; Perrino, Brian A

    2008-02-01

    Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.

  6. Methylmercury-induced toxicity is mediated by enhanced intracellular calcium through activation of phosphatidylcholine-specific phospholipase C

    SciTech Connect

    Kang, Mi Sun; Jeong, Ju Yeon; Seo, Ji Heui; Jeon, Hyung Jun; Jung, Kwang Mook; Chin, Mi-Reyoung; Moon, Chang-Kiu; Bonventre, Joseph V.; Jung, Sung Yun; Kim, Dae Kyong . E-mail: proteinlab@hanmail.net

    2006-10-15

    Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca{sup 2+} levels ([Ca{sup 2+}]{sub i}). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A{sub 2} (cPLA{sub 2}) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca{sup 2+}]{sub i} and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca{sup 2+} in the culture media, D609 completely prevented cell death with parallel decrease in [Ca{sup 2+}]{sub i}. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca{sup 2+}]{sub i} through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA{sub 2}, A-SMase, and PKC, or to the generation of ROS.

  7. Sumoylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the erbb4 receptor tyrosine kinase.

    PubMed

    Knittle, Anna Maria; Helkkula, Maria; Johnson, Mark S; Sundvall, Maria; Elenius, Klaus

    2017-10-03

    Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2 and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site neither compromised ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  8. Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1.

    PubMed

    Al-Samir, Samer; Papadopoulos, Symeon; Scheibe, Renate J; Meißner, Joachim D; Cartron, Jean-Pierre; Sly, William S; Alper, Seth L; Gros, Gerolf; Endeward, Volker

    2013-10-15

    We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3(-) permeability (P(HCO3(-))) sensitive to inhibition by 4,4-Diisothiocyano-2,2-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular P(HCO3(-)) and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional

  9. [Transcription activator-like effectors(TALEs)based genome engineering].

    PubMed

    Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

    2013-10-01

    Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

  10. Identifying the ionically bound cell wall and intracellular glycoside hydrolases in late growth stage Arabidopsis stems: implications for the genetic engineering of bioenergy crops.

    PubMed

    Wei, Hui; Brunecky, Roman; Donohoe, Bryon S; Ding, Shi-You; Ciesielski, Peter N; Yang, Shihui; Tucker, Melvin P; Himmel, Michael E

    2015-01-01

    Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.

  11. Identifying the ionically bound cell wall and intracellular glycoside hydrolases in late growth stage Arabidopsis stems: Implications for the genetic engineering of bioenergy crops

    DOE PAGES

    Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.; ...

    2015-05-13

    Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, for this study, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has ledmore » to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. Finally, the implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.« less

  12. Identifying the ionically bound cell wall and intracellular glycoside hydrolases in late growth stage Arabidopsis stems: Implications for the genetic engineering of bioenergy crops

    SciTech Connect

    Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.; Ding, Shi -You; Ciesielski, Peter N.; Yang, Shihui; Tucker, Melvin P.; Himmel, Michael E.

    2015-05-13

    Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, for this study, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. Finally, the implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.

  13. Identifying the ionically bound cell wall and intracellular glycoside hydrolases in late growth stage Arabidopsis stems: implications for the genetic engineering of bioenergy crops

    PubMed Central

    Wei, Hui; Brunecky, Roman; Donohoe, Bryon S.; Ding, Shi-You; Ciesielski, Peter N.; Yang, Shihui; Tucker, Melvin P.; Himmel, Michael E.

    2015-01-01

    Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, β-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (β-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed. PMID:26029221

  14. In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide

    PubMed Central

    Neamati, Nouri; Shekhtman, Alexander; Camarero, Julio A.

    2013-01-01

    The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions. PMID:23848581

  15. Desnitro-imidacloprid activates the extracellular signal-regulated kinase cascade via the nicotinic receptor and intracellular calcium mobilization in N1E-115 cells.

    PubMed

    Tomizawa, Motohiro; Casida, John E

    2002-11-01

    Imidacloprid (IMI) is the principal neonicotinoid (the only major new class of synthetic insecticides of the past three decades). The excellent safety profile of IMI is not shared with a metabolite, desnitro-IMI (DNIMI), which displays high toxicity to mammals associated with agonist action at the alpha4beta2 nicotinic acetylcholine receptor (nAChR) in brain. This study examines the hypothesis that IMI, DNIMI, and (-)-nicotine activate the extracellular signal-regulated kinase (ERK) cascade via primary interaction with the alpha4beta2 nAChR in mouse neuroblastoma N1E-115 cells. These three nicotinic agonists induce phosphorylation of ERK (p44/p42) in a concentration-dependent manner with an optimal incubation period of 30 min. DNIMI (1 microM)-induced ERK activation is blocked by nicotinic antagonist mecamylamine but not by alpha-bungarotoxin and muscarinic antagonist atropine. This activation is prevented by intracellular Ca(2+) chelator BAPTA-AM but not by removal of external Ca(2+) using EGTA and Ca(2+)-free medium. 2-Aminoethoxy-diphenylborate, a blocker for inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from intracellular stores, inhibits DNIMI-induced ERK activation but a high level of ryanodine (to block ryanodine receptor-mediated Ca(2+) release) does not. The inhibitor U-73122 for phospholipase C (to suppress IP(3) production) prevents ERK activation evoked by DNIMI. Inhibitors for protein kinase C (PKC) (GF109203X) and ERK kinase (PD98059) block this activation whereas an inhibitor (H-89) for cyclic AMP-dependent protein kinase does not. Thus, neonicotinoids activate the ERK cascade triggered by primary action at the alpha4beta2 nAChR with an involvement of intracellular Ca(2+) mobilization possibly mediated by IP(3). It is further suggested that intracellular Ca(2+) activates a sequential pathway from PKC to ERK.

  16. ß-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

    PubMed Central

    Li, Jiao; Imtiaz, Mohammad S.; Beard, Nicole A.; Dulhunty, Angela F.; Thorne, Rick; vanHelden, Dirk F.; Laver, Derek R.

    2013-01-01

    Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] <1 µM, ß-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter. PMID:23533585

  17. Active Combustion Control for Aircraft Gas Turbine Engines

    NASA Technical Reports Server (NTRS)

    DeLaat, John C.; Breisacher, Kevin J.; Saus, Joseph R.; Paxson, Daniel E.

    2000-01-01

    Lean-burning combustors are susceptible to combustion instabilities. Additionally, due to non-uniformities in the fuel-air mixing and in the combustion process, there typically exist hot areas in the combustor exit plane. These hot areas limit the operating temperature at the turbine inlet and thus constrain performance and efficiency. Finally, it is necessary to optimize the fuel-air ratio and flame temperature throughout the combustor to minimize the production of pollutants. In recent years, there has been considerable activity addressing Active Combustion Control. NASA Glenn Research Center's Active Combustion Control Technology effort aims to demonstrate active control in a realistic environment relevant to aircraft engines. Analysis and experiments are tied to aircraft gas turbine combustors. Considerable progress has been shown in demonstrating technologies for Combustion Instability Control, Pattern Factor Control, and Emissions Minimizing Control. Future plans are to advance the maturity of active combustion control technology to eventual demonstration in an engine environment.

  18. Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation.

    PubMed

    Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

    2013-09-01

    The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. An Educational Program of Engineering Ethics and Its Dissemination Activity

    NASA Astrophysics Data System (ADS)

    Muramatsu, Ryujiro; Nagashima, Shigeo

    Education on ethics for corporate employees, especially for engineers, seems to become increasingly important for most of companies in Japan, because some affairs or scandals caused by ethical problem in many companies were likely to subject them to operational disadvantages. Even in Hitachi, Ltd., we have worked on education of engineering ethics for two years. In this paper, we describe some activities of committees on engineering ethics, an e-learning training course which is usable on our intranet e-learning system, and a short-term in-house training course operated regularly in our training institute. And we also refer to its dissemination activities to employees in each division and some subsidiaries.

  20. Developmental axon stretch stimulates neuron growth while maintaining normal electrical activity, intracellular calcium flux, and somatic morphology

    PubMed Central

    Loverde, Joseph R.; Pfister, Bryan J.

    2015-01-01

    Elongation of nerve fibers intuitively occurs throughout mammalian development, and is synchronized with expansion of the growing body. While most tissue systems enlarge through mitosis and differentiation, elongation of nerve fibers is remarkably unique. The emerging paradigm suggests that axons undergo stretch as contiguous tissues enlarge between the proximal and distal segments of spanning nerve fibers. While stretch is distinct from growth, tension is a known stimulus which regulates the growth of axons. Here, we hypothesized that the axon stretch-growth process may be a natural form of injury, whereby regenerative processes fortify elongating axons in order to prevent disconnection. Harnessing the live imaging capability of our axon stretch-growth bioreactors, we assessed neurons both during and following stretch for biomarkers associated with injury. Utilizing whole-cell patch clamp recording, we found no evidence of changes in spontaneous action potential activity or degradation of elicited action potentials during real-time axon stretch at strains of up to 18% applied over 5 min. Unlike traumatic axonal injury, functional calcium imaging of the soma revealed no shifts in free intracellular calcium during axon stretch. Finally, the cross-sectional areas of nuclei and cytoplasms were normal, with no evidence of chromatolysis following week-long stretch-growth limited to the lower of 25% strain or 3 mm total daily stretch. The neuronal growth cascade coupled to stretch was concluded to be independent of the changes in membrane potential, action potential generation, or calcium flux associated with traumatic injury. While axon stretch-growth is likely to share overlap with regenerative processes, we conclude that developmental stretch is a distinct stimulus from traumatic axon injury. PMID:26379492

  1. Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells.

    PubMed

    Nicola, Pieris A; Taylor, Caroline J; Wang, Shanshan; Barrand, Margery A; Hladky, Stephen B

    2008-08-01

    Transport activities involved in intracellular pH (pH(i)) recovery after acid or alkali challenge were investigated in cultured rat brain microvascular endothelial cells by monitoring pH(i) using a pH-sensitive dye. Following relatively small acid loads with pH(i) approximately 6.5, HCO(-)(3) influx accounted for most of the acid extrusion from the cell with both Cl(-)-independent and Cl(-)-dependent, Na(+)-dependent transporters involved. The Cl(-)-independent component has the same properties as the NBC-like transporter previously shown to account for most of the acid extrusion near the resting pH(i). Following large acid loads with pH(i) < 6.5, most of the acid extrusion was mediated by Na(+)/H(+) exchange, the rate of which was steeply dependent on pH(i). Concanamycin A, an inhibitor of V-type ATPase, had no effect on the rates of acid extrusion. Following an alkali challenge, the major component of the acid loading leading to recovery of pH(i) occurred by Cl(-)/HCO(-)(3) exchange. This exchange had the same properties as the AE-like transporter previously identified as a major acid loader near resting pH(i). These acid-loading and acid-extruding transport mechanisms together with the Na(+), K(+), ATPase may be sufficient to account not only for pH(i) regulation in brain endothelial cells but also for the net secretion of HCO(-)(3) across the blood-brain barrier.

  2. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

    PubMed Central

    Hostettler, Isabel; Müller, Joachim; Stephens, Chad E.; Haynes, Richard; Hemphill, Andrew

    2014-01-01

    Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48–72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development. PMID:25516828

  3. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

    PubMed

    Hostettler, Isabel; Müller, Joachim; Stephens, Chad E; Haynes, Richard; Hemphill, Andrew

    2014-12-01

    Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

  4. Quantitative analysis of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) activities against intracellular Staphylococcus aureus in mouse J774 macrophages.

    PubMed

    Seral, Cristina; Van Bambeke, Françoise; Tulkens, Paul M

    2003-07-01

    Using J774 macrophages, the intracellular activities of gentamicin, azithromycin, telithromycin, ciprofloxacin, moxifloxacin, and oritavancin (LY333328) against Staphylococcus aureus (strain ATCC 25923) have been quantitatively assessed in a 24-h model. S. aureus was positively localized in phagolysosomes by confocal and electron microscopy, and extracellular growth was prevented with 0.5 mg of gentamicin/liter (1x MIC) in controls. When tested at extracellular concentrations equivalent to their maximum concentrations in human serum, all antibiotics except azithromycin caused a significant reduction of the postphagocytosis inoculum within 24 h, albeit to markedly different extents (telithromycin [2 mg/liter], 0.60 log; ciprofloxacin [4.3 mg/liter], 0.81 log; gentamicin [18 mg/liter], 1.21 log; moxifloxacin [4 mg/liter], 1.51 log; oritavancin [25 mg/liter], 3.49 log). Intracellular activities were not systematically related to drug accumulation (apparent cellular-to-extracellular concentration ratios in infected cells: ciprofloxacin, 3.2; gentamicin, 6.8; telithromycin, 8.7; moxifloxacin, 13.4; azithromycin, 50; oritavancin, 348). Intracellular activity was not directly correlated to extracellular activity as measured in broth. Conditions of pH 5 (i.e., mimicking that of phagolysosomes) markedly reduced the activity of gentamicin, azithromycin, and telithromycin (>or=32 x) and fairly extensively reduced that of ciprofloxacin and moxifloxacin (>or=4 x) but did not affect oritavancin activity. We conclude that the cellular accumulation of antibiotics is not the only parameter to take into account for intracellular activity but that local environmental conditions (such as pH) and other factors can also prove critical.

  5. Accelerator mass spectrometry measurement of intracellular concentrations of active drug metabolites in human target cells in vivo.

    PubMed

    Chen, J; Garner, R C; Lee, L S; Seymour, M; Fuchs, E J; Hubbard, W C; Parsons, T L; Pakes, G E; Fletcher, C V; Flexner, C

    2010-12-01

    Accelerator mass spectrometry (AMS) is an ultrasensitive technique to detect radiolabeled compounds. We administered a microdose (100 µg) of (14)C-labeled zidovudine (ZDV) with or without a standard unlabeled dose (300 mg) to healthy volunteers. Intracellular ZDV-triphosphate (ZDV-TP) concentration was measured using AMS and liquid chromatography-tandem mass spectrometry (LC/MS/MS). AMS analysis yielded excellent concordance with LC/MS/MS and was 30,000-fold more sensitive. The kinetics of intracellular ZDV-TP formation changed several-fold over the dose range studied (100 µg-300 mg). AMS holds promise as a tool for quantifying intracellular drug metabolites and other biomediators in vivo.

  6. Cellular Quantitative Structure–Activity Relationship (Cell-QSAR): Conceptual Dissection of Receptor Binding and Intracellular Disposition in Antifilarial Activities of Selwood Antimycins

    PubMed Central

    2012-01-01

    We present the cellular quantitative structure–activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug’s lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition. PMID:22468611

  7. Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

    PubMed Central

    Hermann, M; Jaconi, M E; Dahlgren, C; Waldvogel, F A; Stendahl, O; Lew, D P

    1990-01-01

    The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections. Images PMID:2394841

  8. Applications of active adaptive noise control to jet engines

    NASA Technical Reports Server (NTRS)

    Shoureshi, Rahmat; Brackney, Larry

    1993-01-01

    During phase 2 research on the application of active noise control to jet engines, the development of multiple-input/multiple-output (MIMO) active adaptive noise control algorithms and acoustic/controls models for turbofan engines were considered. Specific goals for this research phase included: (1) implementation of a MIMO adaptive minimum variance active noise controller; and (2) turbofan engine model development. A minimum variance control law for adaptive active noise control has been developed, simulated, and implemented for single-input/single-output (SISO) systems. Since acoustic systems tend to be distributed, multiple sensors, and actuators are more appropriate. As such, the SISO minimum variance controller was extended to the MIMO case. Simulation and experimental results are presented. A state-space model of a simplified gas turbine engine is developed using the bond graph technique. The model retains important system behavior, yet is of low enough order to be useful for controller design. Expansion of the model to include multiple stages and spools is also discussed.

  9. Army Corps of Engineers: Water Resource Authorizations, Appropriations, and Activities

    DTIC Science & Technology

    2016-02-09

    Engineers undertakes activities to maintain navigable channels, reduce flood and storm damage, and restore aquatic ecosystems . Congress directs the Corps...aquatic ecosystems . 1 The agency attracts congressional attention because its projects can have significant local and regional economic benefits and...reduction, navigation, and aquatic ecosystem restoration. Over the last decade, enacted annual Corps civil works appropriations (excluding supplemental

  10. Active control of thermoacoustic amplification in an annular engine

    NASA Astrophysics Data System (ADS)

    Desjouy, C.; Penelet, G.; Lotton, P.

    2010-12-01

    In this paper, a new method is proposed to control the thermoacoustic amplification in thermoacoustic engines. This method, based on the active control of the spatial distribution of the acoustic field by means of auxiliary acoustic sources, is applied here to an annular thermoacoustic engine. Two auxiliary acoustic sources are used to tune the spatial distribution of the sound field in the engine in such a way that the thermal-to-acoustic energy conversion occurring into the thermoacoustic core is maximized. An experimental study of this device is proposed, which should be considered as a proof-of-concept study, aiming at demonstrating that the addition of auxiliary acoustic sources can be used advantageously to improve the efficiency of thermoacoustic engines. The overall device is characterized below and above the onset of thermoacoustic instability. It is demonstrated that below the onset of thermoacoustic instability, there exists an optimum phase shift between the auxiliary sources which maximizes the acoustic power available in the annular waveguide. When the device is operated above the onset of thermoacoustic instability, it is demonstrated that the appropriate tuning of the two auxiliary sources enables to improve significantly the acoustic work produced into the engine (compared to the case without active control), that the additional output acoustic power is significantly larger than the input electric power supplied to the acoustic sources, and that the overall efficiency of the engine is thus significantly increased. A discussion about the applicability of this new method for the improvement of actual, high power thermoacoustic engines is also provided.

  11. Intracellular, biofilm-inhibitory and membrane-damaging activities of nimbolide isolated from Azadirachta indica A. Juss (Meliaceae) against meticillin-resistant Staphylococcus aureus.

    PubMed

    Sarkar, Prodipta; Acharyya, Saurabh; Banerjee, Anirban; Patra, Amarendra; Thankamani, Karthika; Koley, Hemanta; Bag, Prasanta K

    2016-10-01

    Staphylococcus aureus is a leading aetiologic agent of nosocomial- and community-acquired infectious diseases worldwide. The public health concern regarding staphylococcal infections is inflated by the increasing occurrence of multidrug-resistant strains, e.g. multidrug- and meticillin-resistant S.aureus (MDR MRSA). This study was designed to evaluate the intracellular killing, membrane-damaging and biofilm-inhibitory activities of nimbolide isolated from Azadirachta indica against MDR MRSA. In vitro antibacterial activity of nimbolide was determined by performing MIC, minimal bactericidal concentration (MBC) and time-kill kinetic studies. Bacterial membrane-damaging activity was determined by membrane perturbation and scanning electron microscopy (SEM) examination. Biofilm-inhibitory activities were determined by SEM. Cellular drug accumulation and assessments of intracellular activities were performed using Vero cell culture. SEM revealed that nimbolide caused significant membrane damage and lysis of the S. aureus cells. The biofilm structure was disrupted, and the biofilm formation was greatly reduced in the presence of nimbolide as examined by SEM. The level of accumulation of nimbolide in Vero cells incubated for 24 h is relatively higher than that of ciprofloxacin and nalidixic acid (Cc/Ce for nimbolide > ciprofloxacin and nalidixic acid). The viable number of intracellular S. aureus was decreased [reduction of ~2 log10 c.f.u. (mg Vero cell protein)-1] in a time-dependent manner in the presence of nimbolide (4× MBC) that was comparable to that of tetracycline and nalidixic acid. The significant intracellular, biofilm-inhibitory and bacterial membrane-damaging activities of nimbolide demonstrated here suggested that it has potential as an effective antibacterial agent for the treatment of severe infections caused by MDR MRSA.

  12. Thrombin receptors activate G(o) proteins in endothelial cells to regulate intracellular calcium and cell shape changes.

    PubMed

    Vanhauwe, Jurgen F; Thomas, Tarita O; Minshall, Richard D; Tiruppathi, Chinnaswamy; Li, Anli; Gilchrist, Annette; Yoon, Eun-ja; Malik, Asrar B; Hamm, Heidi E

    2002-09-13

    Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but

  13. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

    PubMed Central

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard

    2015-01-01

    Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. 13C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using 3H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. PMID:25956769

  14. Magnetic fields in the central engines of active galactic nuclei

    NASA Technical Reports Server (NTRS)

    Begelman, Mitchell C.

    1989-01-01

    Important physical processes which may occur in the central engines of active galactic nuclei and which rely on the presence of a strong magnetic field are discussed. These processes include those involved in the plasma physics of hot tenuous accretion flows, the production of nonthermal continuum radiation, and the radiative manifestation of hydromagnetic jet production. The main arguments which support the hypothesis that supermassive black holes are the prime movers in the central engines are reviewed, and some major deduction regarding the physical state of the accreting gas are pointed out.

  15. PlyC, a bacteriophage endolysin that is internalized by epithelial cells and retains bacteriolytic activity against intracellular streptococci

    USDA-ARS?s Scientific Manuscript database

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial...

  16. Direct protein–protein interaction between the intracellular domain of TRA-2 and the transcription factor TRA-1A modulates feminizing activity in C. elegans

    PubMed Central

    Lum, David H.; Kuwabara, Patricia E.; Zarkower, David; Spence, Andrew M.

    2000-01-01

    In the nematode Caenorhabditis elegans, the zinc finger transcriptional regulator TRA-1A directs XX somatic cells to adopt female fates. The membrane protein TRA-2A indirectly activates TRA-1A by binding and inhibiting a masculinizing protein, FEM-3. Here we report that a part of the intracellular domain of TRA-2A, distinct from the FEM-3 binding region, directly binds TRA-1A. Overproduction of this TRA-1A-binding region has tra-1-dependent feminizing activity in somatic tissues, indicating that the interaction enhances TRA-1A activity. Consistent with this hypothesis, we show that tra-2(mx) mutations, which weakly masculinize somatic tissues, disrupt the TRA-2/TRA-1A interaction. Paradoxically, tra-2(mx) mutations feminize the XX germ line, as do tra-1 mutations mapping to the TRA-2 binding domain. We propose that these mutations render tra-2 insensitive to a negative regulator in the XX germ line, and we speculate that this regulator targets the TRA-2/TRA-1 complex. The intracellular domain of TRA-2A is likely to be produced as a soluble protein in vivo through proteolytic cleavage of TRA-2A or through translation of an XX germ line-specific mRNA. We further show that tagged derivatives of the intracellular domain of TRA-2 localize to the nucleus, supporting the hypothesis that this domain is capable of modulating TRA-1A activity in a manner reminiscent of Notch and Su(H). PMID:11124807

  17. Intracellular routing of cytotoxic pancreatic-type ribonucleases.

    PubMed

    Benito, Antoni; Vilanova, Maria; Ribó, Marc

    2008-06-01

    In addition to their ribonucleolytic activity, several ribonucleases (RNases) play important roles in other specific biological activities, such as dendritic cell activation, certain pollen-induced allergies, blood vessel formation and defense against parasitic or microbial infections. Among these diverse actions, cytotoxic activity, which relies in most cases on ribonucleolytic activity, has attracted a considerable attention because of the potential for using RNases as therapeutic agents for the treatment of different malignancies. In addition to use naturally existing RNases, major efforts have been made in the development of engineered variants, which display more potent cytotoxic activity and greater selectivity for malignant cells. This review focuses on the molecular and cellular aspects of the internalization, intracellular trafficking and final sorting of cytotoxic RNases. Knowledge about the strategies used by these promising toxins provides us with essential information about the mechanisms that can be used to gain access to different subcellular compartments and intracellular sorting.

  18. Intracellular ion activities in frog skin in relation to external sodium and effects of amiloride and/or ouabain.

    PubMed Central

    Harvey, B J; Kernan, R P

    1984-01-01

    Intracellular activities of sodium, potassium and chloride ions, aiNa, aiK, and aiCl were measured with ion-selective single-, double- and triple-barrelled micro-electrodes in skin and isolated epithelia of Rana temporaria bathed on both sides with normal or modified physiological saline. Apical and basolateral membrane potentials, psi ac and psi cs and resistance Ra and Rb respectively were also measured and from the latter the fractional resistance of the apical membrane, F(Ra) and voltage divider ratio, delta psi ac/delta psi cs were measured as criteria of satisfactory membrane penetration by the micro-electrodes. Under control conditions, aiNa was 12.3 +/- 0.8 mM, aiK was 70.3 +/- 22 mM and aiCl was 20.3 +/- 1.6 mM with psi ac averaging -38.0 +/- 3.2 mV. When 10(-4) M-amiloride was added to the apical bathing fluid aiNa fell within 10 min to 1.18 +/- 0.1 mM and aiCl to 5.2 +/- 0.9 mM, while aiK increased to 86.2 +/- 3.8 mM as measured from the basolateral border of isolated epithelia. The sodium transport pool of the skin was measured from the fall in aiNa in the presence of amiloride and could be expressed as 33 X 10(-9) mol cm-2 of epithelium. The mean rate of fall of aiNa under these conditions corresponded to an efflux rate at the basolateral border of 30.1 X 10(-9) mol cm-2 min-1 (48 microA cm-2) giving a half-time for turnover of the sodium transport pool of 33 s. Reduction of sodium concentration in the apical fluid from the normal 79 mM-Na to 10, 1 and 0.1 mM caused aiNa to fall in stages to 2 mM. Because psi ac increased in negativity to -101 mV in the process, this driving force for passive sodium accumulation, more than offset the increased sodium gradient opposing sodium influx across the apical border. PMID:6610743

  19. Intracellular uptake of an antitumor-active azole-bridged dinuclear platinum(II) complex in cisplatin-resistant tumor cells.

    PubMed

    Kishimoto, Shuichi; Yasuda, Megumi; Suzuki, Ryosuke; Fukushima, Shoji

    2016-12-01

    A cationic azolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH3)2}2(μ-OH)(μ-methyl-pyrazolate)](2+) (4M-PzPt), was developed to overcome resistance to cisplatin (CDDP). This study aimed to assess the cytotoxicity of 4M-PzPt against a CDDP-resistant cell line, H4-II-E/CDDP, and compare the intracellular accumulation of CDDP and 4M-PzPt. H4-II-E and H4-II-E/CDDP displayed similar sensitivity to 4M-PzPt; however, the sensitivity of H4-II-E/CDDP to CDDP was approximately 19-fold lower than that of H4-II-E. The difference in the sensitivity to both platinum complexes corresponded with the difference in the amount of intracellular platinum accumulation after exposure to CDDP or 4M-PzPt in both cell lines. In H4-II-E, HepG2, and HuH-7 cells, the intracellular uptake of CDDP and 4M-PzPt occurred via active transport and passive transport. Results of co-exposure with the transport inhibitors ouabain, tetraethylammonium, and cimetidine indicated that the intracellular uptake of CDDP was dependent on Na(+)/K(+)-ATPase and that of 4M-PzPt was dependent on organic cation transporters (OCTs), probably OCT1. This study suggested that 4M-PzPt could inhibit the growth of a CDDP-resistant tumor via an intracellular uptake mechanism different from that of CDDP.

  20. Contrasting Effects of Acidic pH on the Extracellular and Intracellular Activities of the Anti-Gram-Positive Fluoroquinolones Moxifloxacin and Delafloxacin against Staphylococcus aureus ▿ †

    PubMed Central

    Lemaire, Sandrine; Tulkens, Paul M.; Van Bambeke, Françoise

    2011-01-01

    In contrast to currently marketed fluoroquinolones, which are zwitterionic, delafloxacin is an investigational fluoroquinolone with an anionic character that is highly active against Gram-positive bacteria. We have examined the effect of acidic pH on its accumulation in Staphylococcus aureus and in human THP-1 cells, in parallel with its activity against extracellular and intracellular S. aureus. Moxifloxacin was used as a comparator. Delafloxacin showed MICs 3 to 5 log2 dilutions lower than those of moxifloxacin for a collection of 35 strains with relevant resistance mechanisms and also proved to be 10-fold more potent against intracellular S. aureus ATCC 25923. In medium at pH 5.5, this difference was further enhanced, with the MIC decreasing by 5 log2 dilutions. In infected cells incubated in acidic medium, the relative potency was 10-fold higher than that at neutral pH and the maximal relative efficacy reached a bactericidal effect at 24 h. These results can be explained by a 10-fold increase in delafloxacin accumulation in both bacteria and cells at acidic pH, making delafloxacin one of the most efficient drugs tested in this model. Opposite effects were seen for moxifloxacin with respect to both activity and accumulation. As reported for zwitterionic fluoroquinolones, delafloxacin was found associated with the soluble fraction in homogenates of eukaryotic cells. Taken together, these properties may confer to delafloxacin an advantage for the eradication of S. aureus in acidic environments, including intracellular infections. PMID:21135179

  1. Quercetin and Ascorbic Acid Suppress Fructose-Induced NLRP3 Inflammasome Activation by Blocking Intracellular Shuttling of TXNIP in Human Macrophage Cell Lines.

    PubMed

    Choe, Jung-Yoon; Kim, Seong-Kyu

    2017-03-22

    The aim of this study was to identify the role of thioredoxin-interacting protein (TXNIP) and its interaction with antioxidants in the activation of the fructose-induced NOD-like receptor protein 3 (NLRP3) inflammasome in human macrophages. The study was performed with U937 and THP-1 macrophage cell lines. Total reactive oxygen species (ROS) were measured by flow cytometry. Interleukin-1β (IL-1β), IL-18, NLRP3, TXNIP, and caspase-1 protein expression was detected using western blotting. Quantitative real-time polymerase chain reaction was used to detect IL-1β, IL-18, and caspase-1 gene expression. Intracellular shuttling of TXNIP was assessed by immunofluorescent staining with MitoTracker Red. Increased production of ROS and expression of IL-1β, IL-18, and caspase-1 genes and proteins were observed in U937 and THP-1 cells incubated with fructose and were effectively inhibited by quercetin and ascorbic acid. Intracellular shuttling of TXNIP from the nucleus into the mitochondria was detected under stimulation with fructose, which was also attenuated by antioxidants quercetin and ascorbic acid but not butylated hydroxyanisole. Treatment of macrophages with fructose promoted the association between TXNIP and NLRP3 in the cytosol, sequentially resulting in the activation of the NLRP3 inflammasome. This study revealed that intracellular TXNIP protein is a critical regulator of activation of the fructose-induced NLRP3 inflammasome, which can be effectively blocked by the antioxidants quercetin and ascorbic acid.

  2. [Intracellular cAMP involvement in the synchronized activity of noradrenaline in response to evoked release of the transmitter quanta in the frog synapses].

    PubMed

    Bukharaeva, E A; Samigullin, D V; Nikol'skiĭ, E E; Vyskocil, F

    2000-04-01

    An analogue of cyclic AMP (db-cAMP) penetrating into the frog neuromuscular junction's cell, as well as the adenylyl cyclase activator forskolin, and inhibitor of nucleotide-depending phosphodiesterase isobutilmethylxantine alter the kinetics of the quanta secretion resulting in synchronizing of the process of the transmitter release. Following a db-cAMP preliminary action, no such synchronizing of the transmitter release occurred. Action of noradrenaline on the time course of the secretion seems to be realised through activation of presynaptic beta-adrenoreceptors, augmentation of the adenylyl cyclase activity, and the rise of the intracellular cAMP.

  3. Active automotive engine vibration isolation using feedback control

    NASA Astrophysics Data System (ADS)

    Olsson, Claes

    2006-06-01

    Large frequency band feedback active automotive engine vibration isolation is considered. A MIMO (multi-input multi-output) controller design for an active engine suspension system has been performed making use of a virtual development environment for design, analysis, and co-simulation based closed-loop verification. Utilising relevant control object dynamic modelling, this design strategy provides a powerful opportunity to deal with various plant dynamics, such as structural flexibility and nonlinear characteristics where the main objective is to approach the actual physical characteristics for design and verification in early design phases where no prototypes are yet physically available. H2 loop shaping technique proves to be powerful when achieving the desired frequency dependent loop gain while ensuring closed-loop stability. However, to achieve closed-loop stability two kinds of nonlinearities have to be taken into account. Those are nonlinear material characteristics of the engine mounts and large angular engine displacements. It is demonstrated how the adopted design strategy facilitates the investigation of the latter nonlinearity's impact on closed-loop characteristics. To deal with the nonlinearities, gain scheduling has been used.

  4. Engineered N-acetylhexosamine-active enzymes in glycoscience.

    PubMed

    Slámová, Kristýna; Bojarová, Pavla

    2017-08-01

    In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, β-N-acetylhexosaminidases, and endo-β-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good

  5. Calcium-activated potassium channels in the luminal membrane of Amphiuma diluting segment: voltage-dependent block by intracellular Na+ upon depolarisation.

    PubMed

    Kawahara, K; Hunter, M; Giebisch, G

    1990-06-01

    Calcium-activated potassium channels in the luminal membrane of Amphiuma diluting segment were studied using the patch-clamp technique in both the cell-attached and inside-out configurations. The open probability (Po) of the channel is sensitive to both membrane potential and cytoplasmic calcium activity; depolarizing potentials and high calcium concentrations leading to an increased Po. In the cell-attached condition, channel openings were observed between pipette potentials of -100 and -240 mV. As the driving force for potassium exit from the cell into the pipette is increased the single channel currents show a biphasic response. First, the currents increase as expected; however, the single channel currents diminish in magnitude at pipette potentials more negative than -120 mV. We propose that this reduction is due to rapid blockade of the potassium channel by intracellular sodium. This proposal is supported by two facts: (a) using inside-out patches it was possible to reduce the single channel currents in a concentration- and voltage-dependent manner, similar to that observed in the cell-attached condition, by raising the sodium concentration of the fluid bathing the cytoplasmic face of the patch; (b) pretreatment of tubules with the loop-acting diuretic furosemide (10(-5) M), an agent known to decrease the intracellular sodium activity, caused an attenuation of the reduction in single channel current seen under control conditions. Given the very low Po of the channels at the resting membrane potential and the sensitivity of the channels to intracellular sodium, it is unlikely that blockade of these channels by intracellular sodium would lead to a physiological regulation of the apical K conductance.

  6. Melanin-Associated Synthesis of SERS-Active Nanostructures and the Application for Monitoring of Intracellular Melanogenesis

    PubMed Central

    Dong, Haixin; Liu, Zhiming; Zhong, Huiqing; Yang, Hui; Zhou, Yan; Hou, Yuqing; Long, Jia; Lin, Jin; Guo, Zhouyi

    2017-01-01

    Melanin plays an indispensable role in the human body. It serves as a biological reducer for the green synthesis of precious metal nanoparticles. Melanin–Ag nanocomposites were successfully produced which exhibited very strong surface-enhanced Raman scattering (SERS) effect because of the reducibility property of melanin. A melanin–Ag composite structure was synthesized in situ in melanin cells, and SERS technique was performed for the rapid imaging and quantitative assay of intracellular melanin. This imaging technique was also used to successfully trace the formation and secretion of intracellular melanin after stimulation with melanin-stimulating hormones. Based on the self-reducing property of melanin, the proposed SERS imaging method can provide potentially powerful analytical detection tools to study the biological functions of melanin and to prevent and cure melanin-related diseases. PMID:28336903

  7. A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity

    PubMed Central

    Frost, William N.; Wang, Jean; Brandon, Christopher J.

    2007-01-01

    Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations. PMID:17306887

  8. Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA

    PubMed Central

    Werfel, Thomas A.; Swain, Corban; Nelson, Christopher E.; Kilchrist, Kameron V.; Evans, Brian C.; Miteva, Martina; Duvall, Craig L.

    2017-01-01

    Hydrolytically degrading nano-polyplexes (HDG-NPs) that reverse charge through conversion of tertiary amines to carboxylic acids were investigated to improve intracellular un-packaging of siRNA and target gene silencing compared to a non-degradable analog (non-HDG-NPs). Both NP types comprised reversible addition-fragmentation chaintransfer (RAFT) synthesized diblock copolymers of a poly(ethylene glycol) (PEG) corona-forming block and a cationic block for nucleic acid packaging that incorporated butyl methacrylate (BMA) and either dimethylaminoethyl methacrylate (DMAEMA, non-HDG-NPs) or dimethylaminoethyl acrylate (DMAEA, HDG-NPs). HDG-NPs decreased significantly in size and released significantly more siRNA (~40%) than non-HDG-NPs after 24 h in aqueous solution. While both HDG-NPs and non-HDG-NPs had comparable uptake and cytotoxicity up to 150 nM siRNA doses, HDG-NPs achieved significantly higher target gene silencing of the model gene luciferase in vitro. High resolution FRET confocal microscopy was used to monitor the intracellular un-packaging of siRNA. Non-HDG-NPs had significantly higher FRET efficiency than HDG-NPs, indicating that siRNA delivered from HDG-NPs was more fully un-packaged and therefore had improved intracellular bioavailability. PMID:26691570

  9. Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

    SciTech Connect

    Bond, P.L.; Keller, J.; Blackall, L.L.

    1999-06-05

    The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. from these observations, the authors postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimization of EBPR.

  10. Ziram and sodium N,N-dimethyldithiocarbamate inhibit ubiquitin activation through intracellular metal transport and increased oxidative stress in HEK293 cells.

    PubMed

    Dennis, Kathleen E; Valentine, William M

    2015-04-20

    Ubiquitin activating enzyme E1 plays a pivotal role in ubiquitin based protein signaling through regulating the initiating step of the cascade. Previous studies demonstrated that E1 is inhibited by covalent modification of reactive cysteines contained within the ubiquitin-binding groove and by conditions that increase oxidative stress and deplete cellular antioxidants. In this study, we determined the relative contribution of covalent adduction and oxidative stress to E1 inhibition produced by ziram and sodium N,N-dimethyldithiocarbamate (DMDC) in HEK293 cells. Although no dithiocarbamate-derived E1 adducts were identified on E1 using shotgun LC/MS/MS for either ziram or DMDC, both dithiocarbamates significantly decreased E1 activity, with ziram demonstrating greater potency. Ziram increased intracellular levels of zinc and copper, DMDC increased intracellular levels of only copper, and both dithiocarbamates enhanced oxidative injury evidenced by elevated levels of protein carbonyls and expression of heme oxygenase-1. To assess the contribution of intracellular copper transport to E1 inhibition, coincubations were performed with the copper chelator triethylenetetramine hydrochloride (TET). TET significantly protected E1 activity for both of the dithiocarbamates and decreased the associated oxidative injury in HEK293 cells as well as prevented dithiocarbamate-mediated lipid peroxidation assayed using an ethyl aracidonate micelle system. Because TET did not completely ameliorate intracellular transport of copper or zinc for ziram, TET apparently maintained E1 activity through its ability to diminish dithiocarbamate-mediated oxidative stress. Experiments to determine the relative contribution of elevated intracellular zinc and copper were performed using a metal free incubation system and showed that increases in either metal were sufficient to inhibit E1. To evaluate the utility of the HEK293 in vitro system for screening environmental agents, a series of additional

  11. Recent geothermal reservoir engineering activities at Lawrence Berkeley Laboratory

    SciTech Connect

    Lippmann, M.J.; Bodvarsson, G.S.; Benson, S.M.; Pruess, K.

    1987-09-01

    This paper briefly describes the most recent activities in reservoir engineering for the geothermal group of Lawrence Berkeley Laboratory (LBL). The primary emphasis of the geothermal program of LBL is dedicated to reservoir engineering including theoretical investigations, the development and application of mathematical models, and field studies. The objectives of these activities are to develop and validate methods and instruments which will be utilized in the determination of the parameters of geothermal systems, and the identification and evaluation of the importance of the distinct processes which occur in reservoirs. The ultimate goal of the program is the development of state of the art technologies which characterize geothermal reservoirs and evaluate their productive capacity and longevity.

  12. Intracellular amyloid beta interacts with SOD1 and impairs the enzymatic activity of SOD1: implications for the pathogenesis of amyotrophic lateral sclerosis.

    PubMed

    Yoon, Eun Jin; Park, Hyo Jin; Kim, Goo Young; Cho, Hyung Min; Choi, Jung Ha; Park, Hye Yoon; Jang, Ja Young; Rhim, Hyang Shuk; Kang, Seong Man

    2009-09-30

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1), including G93A, were reportedly linked to familial ALS. SOD1 is a key antioxidant enzyme, and is also one of the major targets for oxidative damage in the brains of patients suffering from Alzheimers disease (AD). Several lines of evidence suggest that intracellular amyloid beta (Abeta) is associated with the pathogenesis of AD. In this report we demonstrate that intracellular Abeta directly interacts with SOD1, and that this interaction decreases the enzymatic activity of the enzyme. We observed Abeta-SOD1 aggregates in the perinuclear region of H4 cells, and mapped the SOD1 binding region to Abeta amino acids 26-42. Interestingly, intracellular Ab binds to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. This resulted in considerably less mutant enzymatic activity. Our study implicates a potential role for Abeta in the development of ALS by interacting with the SOD1 G93A mutant.

  13. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells

    PubMed Central

    Ji, Yun; Dai, Zhaolai; Wu, Guoyao; Wu, Zhenlong

    2016-01-01

    Excessive reactive oxygen species (ROS) induces oxidative damage to cellular constituents, ultimately leading to induction of apoptotic cell death and the pathogenesis of various diseases. The molecular mechanisms for the action of ROS in intestinal diseases remain poorly defined. Here, we reported that 4-hydroxy-2-nonenal (4-HNE) treatment led to capses-3-dependent apoptosis accompanied by increased intracellular ROS level and reduced glutathione concentration in intestinal epithelial cells. These effects of 4-HNE were markedly abolished by the antioxidant L-cysteine derivative N-acetylcysteine (NAC). Further studies demonstrated that the protective effect of NAC was associated with restoration of intracellular redox state by Nrf2-related regulation of expression of genes involved in intracellular glutathione (GSH) biosynthesis and inactivation of 4-HNE-induced phosphorylation of extracellular signal-regulated protein kinases (ERK1/2). The 4-HNE-induced ERK1/2 activation was mediated by repressing mitogen-activated protein kinase phosphatase-1 (MKP-1), a negative regulator of ERK1/2, through a proteasome-dependent degradation mechanism. Importantly, either overexpression of MKP-1 or NAC treatment blocked 4-HNE-induced MKP-1 degradation, thereby protecting cell from apoptosis. These novel findings provide new insights into a functional role of MKP-1 in oxidative stress-induced cell death by regulating ERK1/2 MAP kinase in intestinal epithelial cells. PMID:27620528

  14. Dense Clouds near the Central Engine of Active Galactic Nuclei

    NASA Technical Reports Server (NTRS)

    Sivron, R.; Tsuruta, S

    1993-01-01

    A model is presented which assumes the existence of cold dense clouds near the central engine of Active Galactic Nuclei (AGNs). The effects of such clouds on the observed spectrum are explored. It is shown that this model is consistent with the complicated observed spectra and variability behavior of most extensively studied Seyfert nuclei. The results are compared with other proposed models. The existing observational evidence appears to support the "cloud-model."

  15. A Single Amino Acid Substitution in the v-Eyk Intracellular Domain Results in Activation of Stat3 and Enhances Cellular Transformation

    PubMed Central

    Besser, Daniel; Bromberg, Jacqueline F.; Darnell, James E.; Hanafusa, Hidesaburo

    1999-01-01

    The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3 subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher numbers of transformants in soft agar and stronger activation of Stat3; levels of Stat1 activation by the two Eyk molecules were similar. A mutation in the sequence Y933VPL, present in c-Eyk, to the v-Eyk sequence Y933VPQ led to increased activation of Stat3 and increased transformation efficiency. However, altering another sequence, Y862VNT, present in both Eyk molecules to F862VNT markedly decreased transformation without impairing Stat3 activation. These results indicate that activation of Stat3 enhances transformation efficiency and cooperates with another pathway to induce transformation. PMID:9891073

  16. Elementary teachers committed to actively teaching science and engineering

    NASA Astrophysics Data System (ADS)

    Opperman, Julianne Radkowski

    Committed elementary teachers of science and engineering, members of a professional learning community called Collaborative Conversations in STEM, were studied to elicit their perceptions of experiences that influenced their commitment to, and their pedagogical content knowledge of, STEM teaching and learning. The hermeneutic phenomenological interviews enabled the teachers to express their beliefs in their own words. Data analysis employed a theoretical framework that investigated teacher epistemology and knowledge in light of their experiences. Findings revealed a web of lifelong experiences unique to each individual, and evidential of the committed elementary scientist-teachers' present day values, teaching epistemology, lifelong learning, and emotional and intellectual engagement. Scientist-teachers are individuals whose teaching and learning characteristics reflect those of scientists and engineers. Evidence indicated that no single transformative learning experience resulted in those elementary teachers' commitment to STEM teaching and learning, but recent professional development activities were influential. Formal K-16 STEM learning was not uniformly or positively influential to the teachers' commitment to, or knowledge of, STEM. Findings suggest that ongoing professional development for STEM teaching and learning can influence elementary teachers to become committed to actively teaching STEM. The Collaborative Conversations in STEM provided intellectual and emotional engagement that empowered the teachers to provide STEM teaching and learning for their students and their colleagues overcoming impediments encountered in a literacy-focused curriculum. Elementary teachers actively committed to teaching science and engineering can undergo further transformation and emerge as leaders.

  17. High-Salt Intake Augments the Activity of the RhoA/ROCK Pathway and Reduces Intracellular Calcium in Arteries From Rats.

    PubMed

    Crestani, Sandra; Webb, Robert Clinton; da Silva-Santos, José Eduardo

    2017-04-01

    We investigated the influence of salt overconsumption on the functionality of the RhoA/Rho-associated kinase (ROCK) pathway and calcium regulation in arteries. The aorta and small mesenteric arteries from rats fed a chow containing 2%, 4%, or 8% NaCl were evaluated in organ baths for the activity of the RhoA/ROCK pathway and intracellular calcium mobilization. Components of these pathways and intracellular calcium levels were also assessed in samples from 4% NaCl group. In arteries from animals fed regular chow, the ROCK inhibitor Y-27632 reduced the responses to phenylephrine, even when the smallest concentrations (1 and 3 μM) were tested. However, only higher concentrations of Y-27632 (10 and 50 μM) reduced phenylephrine-induced contraction in vessels from high-salt groups. Immunoblotting revealed augmented phosphorylation of the myosin phosphatase targeting subunit 1 and increased amounts of RhoA in the membrane fraction of aorta homogenates from the 4% NaCl group. Under calcium-free solution, vessels from NaCl groups presented reduced contractile responses to phenylephrine and caffeine, compared with the regular chow group. Moreover, decreased intracellular calcium at rest and after stimulation with ATP were found in aortic smooth muscle cells from 4% NaCl-fed rats, which also showed diminished levels of SERCA2 and SERCA3, but not of IP3 and ryanodine receptors, or STIM1 and Orai1 proteins. Arteries from rats subjected to high-salt intake are unable to properly regulate intracellular calcium levels and present augmented activity of the calcium sensitization pathway RhoA/ROCK. These changes may precede the development of vascular diseases induced by high-salt intake.

  18. Mechanism of the Intracellular Killing and Modulation of Antibiotic Susceptibility of Listeria monocytogenes in THP-1 Macrophages Activated by Gamma Interferon

    PubMed Central

    Ouadrhiri, Youssef; Scorneaux, Bernard; Sibille, Yves; Tulkens, Paul M.

    1999-01-01

    Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of l-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225–1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by l-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of l-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection. PMID:10223943

  19. Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death.

    PubMed

    Martin Muñoz, Patricia; Ortega Ferrusola, Cristina; Vizuete, Guillermo; Plaza Dávila, Maria; Rodriguez Martinez, Heriberto; Peña, Fernando J

    2015-12-01

    Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard technique. The parameters of sperm quality (motility, velocity, and membrane integrity) and markers of sperm senescence (caspase 3, 4-hydroxynonenal, and mitochondrial membrane potential) were assessed before and after cryopreservation. Changes in the intracellular thiol content were also monitored. Cryopreservation caused significant increases in senescence markers as well as dramatic depletion of intracellular thiols to less than half of the initial values (P < 0.001) postthaw. Interestingly, very high and positive correlations were observed among thiol levels with sperm functionality postthaw: total motility (r = 0.931, P < 0.001), progressive motility (r = 0.904, P < 0.001), and percentage of live spermatozoa without active caspase 3 (r = 0.996, P < 0.001). In contrast, negative correlations were detected between active caspase 3 and thiol content both in living (r = -0.896) and dead (r = -0.940) spermatozoa; additionally, 4-hydroxynonenal levels were negatively correlated with thiol levels (r = -0.856). In conclusion, sperm functionality postthaw correlates with the maintenance of adequate levels of intracellular thiols. The accelerated senescence of thawed spermatozoa is related to oxidative and electrophilic stress induced by increased production of 4-hydroxynoneal in thawed samples once intracellular thiols are depleted.

  20. Active control of fan noise from a turbofan engine

    NASA Technical Reports Server (NTRS)

    Thomas, Russell H.; Burdisso, Ricardo A.; Fuller, Christopher R.; O'Brien, Walter F.

    1993-01-01

    A three channel active control system is applied to an operational turbofan engine in order to reduce tonal noise produced by both the fan and high pressure compressor. The control approach is the feedforward filtered-x least-mean-square algorithm implemented on a digital signal processing board. Reference transducers mounted on the engine case provides blade passing and harmonics frequency information to the controller. Error information is provided by large area microphones placed in the acoustic far field. In order to minimize the error signal, the controller actuates loudspeakers mounted on the inlet to produce destructive interference. The sound pressure level of the fundamental tone of the fan was reduced using the three channel controller by up to 16 dB over a 60 deg angle about the engine axis. A single channel controller could produce reduction over a 30 deg angle. The experimental results show the control to be robust. Simultaneous control of two tones is done with parallel controllers. The fundamental and the first harmonic tones of the fan were controlled simultaneously with reductions of 12 dBA and 5 dBA, respectively, measured on the engine axis. Simultaneous control was also demonstrated for the fan fundamental and the high pressure compressor fundamental tones.

  1. Active control of fan noise from a turbofan engine

    NASA Technical Reports Server (NTRS)

    Thomas, Russell H.; Burdisso, Ricardo A.; Fuller, Christopher R.; O'Brien, Walter F.

    1994-01-01

    A three-channel active control system is applied to an operational turbofan engine to reduce tonal noise produced by both the fan and the high-pressure compressor. The control approach is the feedforward filtered-x least-mean-square algorithm implemented on a digital signal processing board. Reference transducers mounted on the engine case provide blade passing and harmonics frequency information to the controller. Error information is provided by large area microphones placed in the acoustic far field. To minimize the error signal, the controller actuates loudspeakers mounted on the inlet to produce destructive interference. The sound pressure level of the fundamental tone of the fan was reduced using the three-channel controller by up to 16 dB over a +/- 30-deg angle about the engine axis. A single-channel controller could produce reduction over a +/- 15-deg angle. The experimental results show the control to be robust. Outside of the areas contolled, the levels of the tone actually increased due to the generation of radial modes by the control sources. Simultaneous control of two tones is achieved with parallel controllers. The fundamental and the first harmonic tones of the fan were controlled simultaneously with reductions of 12 and 5 dBA, respectively, measured on the engine axis. Simultaneous control was also demonstrated for the fan fundamental and the high-pressure compressor fundamental tones.

  2. Active control of fan noise from a turbofan engine

    NASA Technical Reports Server (NTRS)

    Thomas, Russell H.; Burdisso, Ricardo A.; Fuller, Christopher R.; O'Brien, Walter F.

    1993-01-01

    A three channel active control system is applied to an operational turbofan engine in order to reduce tonal noise produced by both the fan and high pressure compressor. The control approach is the feedforward filtered-x least-mean-square algorithm implemented on a digital signal processing board. Reference transducers mounted on the engine case provides blade passing and harmonics frequency information to the controller. Error information is provided by large area microphones placed in the acoustic far field. In order to minimize the error signal, the controller actuates loudspeakers mounted on the inlet to produce destructive interference. The sound pressure level of the fundamental tone of the fan was reduced using the three channel controller by up to 16 dB over a 60 deg angle about the engine axis. A single channel controller could produce reduction over a 30 deg angle. The experimental results show the control to be robust. Simultaneous control of two tones is done with parallel controllers. The fundamental and the first harmonic tones of the fan were controlled simultaneously with reductions of 12 dBA and 5 dBA, respectively, measured on the engine axis. Simultaneous control was also demonstrated for the fan fundamental and the high pressure compressor fundamental tones.

  3. Ultra Fine Particles from Diesel Engines Induce Vascular Oxidative Stress via JNK Activation

    PubMed Central

    Li, Rongsong; Ning, Zhi; Cui, Jeffery; Khalsa, Bhavraj; Ai, Lisong; Takabe, Wakako; Beebe, Tyler; Majumdar, Rohit; Sioutas, Constantinos; Hsiai, Tzung

    2011-01-01

    Exposure of particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultra fine particles (UFP) from diesel vehicle engines have been shown to be pro-atherogenic in apoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induced vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intra-cellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O2·-) production in human aortic endothelial cells (HAEC). Flow cytometry (FACS) showed that UFP increased MitoSOX Red intensity specific for mitochondrial superoxide. Protein carbonyl content is increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated hemeoxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pre-treatment with antioxidant, N-acetyl cysteine (NAC), significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP stimulated O2·- production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation play an important role in UFP-induced oxidative stress and stress response gene expression. PMID:19154785

  4. Ultrafine particles from diesel engines induce vascular oxidative stress via JNK activation.

    PubMed

    Li, Rongsong; Ning, Zhi; Cui, Jeffery; Khalsa, Bhavraj; Ai, Lisong; Takabe, Wakako; Beebe, Tyler; Majumdar, Rohit; Sioutas, Constantinos; Hsiai, Tzung

    2009-03-15

    Exposure to particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultrafine particles (UFP) from diesel vehicle engines have been shown to be proatherogenic in ApoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induce vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intracellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O(2)(-)) production in human aortic endothelial cells (HAEC). Flow cytometry showed that UFP increased MitoSOX red intensity specific for mitochondrial superoxide. Protein carbonyl content was increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated heme oxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pretreatment with the antioxidant N-acetylcysteine significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with the JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP-stimulated O(2)(-) production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation plays an important role in UFP-induced oxidative stress and stress response gene expression.

  5. Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments.

    PubMed

    Zhang, Xiaoxu; Gao, Yanyun; Yin, Ying; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2017-02-07

    Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.

  6. Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells.

    PubMed

    Imai, Seiji; Okayama, Naotsuka; Shimizu, Manabu; Itoh, Makoto

    2003-04-04

    SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.

  7. Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells

    PubMed Central

    Kim, Donghee; Kang1,2, Dawon; Martin, Elizabeth A.; Kim, Insook; Carroll, John L.

    2014-01-01

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K+ channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1 mM AICAR and 0.1–0.5 mM A769662) did not inhibit TASK activity or cause depolarization during acute (10 min) or prolonged (2–3 hr) exposure. Hypoxia inhibited TASK activity by ~70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15–40 min) failed to increase [Ca2+]i in glomus cells. Compound C (40 µM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca2+] in isolated rat carotid body glomus cells. PMID:24530802

  8. Receptor-activated Ca2+ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2+ signalling requirements.

    PubMed Central

    Barritt, G J

    1999-01-01

    Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage-operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Na+, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals. PMID:9882611

  9. Engineering Ascorbate Peroxidase Activity Into Cytochrome C Peroxidase

    SciTech Connect

    Meharenna, Y.T.; Oertel, P.; Bhaskar, B.; Poulos, T.L.

    2009-05-26

    Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each others activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303--307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of {approx}12 min{sup -1}, indicating that the engineered ascorbate-binding loop can bind ascorbate.

  10. Scope for active noise abatement in vehicle diesel engines

    NASA Astrophysics Data System (ADS)

    Summerauer, I.; Boesch, N.

    1984-04-01

    Noise reduction measures must be directed to the engine, the exhaust system, and the cooling system (fan) all of which contribute approximately 90% of the sound energy emitted from commercial diesel trucks. The noise generation processes were visualized and limiting conditions fixed by law were considered in establishing criteria for active solar noise abatement measures. A more effective silencer and better vibration damping on the surface of the silencer and exhaust pipes can reduce noise from the exhaust system. Acoustic emission generated by the fan and air flow can be reduced by decreasing flow velocity or by turning on the fan only when a full cooling output is required (10% of the time). Active measures are needed on the engine itself either at the point of the solid-borne sound transmission or at the point of the solid-borne vibrations. The predominant effect is on the engine casing; oil sump; air suction pipe or air charge line; the flywheel casing; and the clutch housing.

  11. Factors affecting the activation and inhibition of intracellular enzymes for degradation of 1,2 diamino benzene: kinetics and thermodynamic studies.

    PubMed

    P, Saranya; G, Sekaran

    2015-11-01

    Citrobacter freundii, the bacterium isolated from marine sediments was capable of degrading 1,2 diamino benzene (DAB), an endocrine disruptor. The mixed intracellular enzymes from C. freundii were extracted and purified. The mixed intracellular enzymes were used for the degradation of DAB and degree of degradation was evaluated in terms of pyruvic acid, the end product, formed. The variables such as effect of pH, temperature and metal ions on the degradation of DAB using mixed intracellular enzymes (MICE) were investigated. The maximum amount of pyruvic acid formed was found to be 569 ± 5 µg with 96% degradation efficiency at pH 7; temperature 25 °C; zinc nitrate 0.1 mM; and copper sulphate ions 0.15 mM. The stability of MICE at different temperatures and the interaction of MICE with metal ions were confirmed using FT-IR spectroscopy. The formation of pyruvic acid from degradation of DAB followed pseudo-second-order rate kinetics and it was a spontaneous, exothermic process. The activation energy of degradation of DAB by MICE was found to be 82.55 kJ/mol.

  12. Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

    PubMed Central

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-01-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases. PMID:16199568

  13. Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16.

    PubMed

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-10-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

  14. Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation.

    PubMed

    Rivers, David B; Crawley, Timothy; Bauser, Holly

    2005-02-01

    Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.

  15. Engineering principles and concepts for active solar systems

    NASA Astrophysics Data System (ADS)

    Hunn, B. D.; Carlisle, N.; Franta, G.; Kolar, W.

    1987-07-01

    This publication is a much refined and updated version of a solar design handbook originally prepared in 1978 to accompany a series of week-long courses conducted in support of the Solar Federal Buildings Program. The 1978 material was published in 1981 as the Solar Design Workbook (SERI/SP-62-308). This current document represents the culmination of an eight-year effort to compile a comprehensive state-of-the-art reference and instructional tool for practicing design professionals, architects, and engineers. It is intended to cover all phases of the design and installation of active solar energy systems for buildings. Although it contains many design guidelines, the emphasis is on providing sufficient knowledge of how these systems work to allow an engineer or architect to make well-informed decisions. It is aimed primarily at commercial building applications, but most of the material is also applicable to residential buildings.

  16. Cellular accumulation of fluoroquinolones is not predictive of their intracellular activity: studies with gemifloxacin, moxifloxacin and ciprofloxacin in a pharmacokinetic/pharmacodynamic model of uninfected and infected macrophages.

    PubMed

    Vallet, Coralie M; Marquez, Béatrice; Ngabirano, Eva; Lemaire, Sandrine; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M; Van Bambeke, Françoise

    2011-09-01

    Fluoroquinolones enter eukaryotic cells but the correlation between cellular accumulation and activity remains poorly established. Gemifloxacin is known to accumulate to a larger extent than most other fluoroquinolones in tissues. Using murine J774 macrophages and human THP-1 monocytes, we show that gemifloxacin accumulates more than ciprofloxacin and even moxifloxacin. Whilst showing indistinguishable kinetics of accumulation in J774 macrophages, gemifloxacin was released at an approximately two-fold slower rate than ciprofloxacin and its release was only partial. Gemifloxacin was also a weaker substrate than ciprofloxacin for the efflux transporter Mrp4 active in J774 macrophages. In cells infected with Listeria monocytogenes or Staphylococcus aureus (typical cytoplasmic and phagolysosomal organisms, respectively), gemifloxacin was equipotent to moxifloxacin and ciprofloxacin in concentration-dependent experiments if data are normalised based on the minimum inhibitory concentration (MIC) in broth. T