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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

    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. 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

  4. Visualization of Intracellular Tyrosinase Activity in vitro

    PubMed Central

    Setty, Subba Rao Gangi

    2016-01-01

    Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes. PMID:27231711

  5. Intracellular angiotensin II activates rat myometrium.

    PubMed

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

    2011-09-01

    Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) 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 Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](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 Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) 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-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](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 AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.

  6. 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

  7. 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. PMID:2642767

  8. 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

  9. Intracellular Na+ and Ca2+ Modulation Increases the Tensile Properties of Developing Engineered Articular Cartilage

    PubMed Central

    Natoli, Roman M.; Skaalure, Stacey; Bijlani, Shweta; Chen, Ke X.; Hu, Jerry; Athanasiou, Kyriacos A.

    2010-01-01

    Objective Significant collagen content and tensile properties are difficult to achieve in articular cartilage tissue engineering. This study investigated whether treating developing tissue engineered cartilage constructs with modulators of intracellular Na+ or Ca2+ could increase collagen concentration and tensile properties. Methods Inhibitors of Na+ ion transporters and increasers of intracellular Ca2+ were investigated for their ability to affect articular cartilage development in a scaffold-less, 3D chondrocyte culture. Using a systematic approach, ouabain (Na+/K+ ATPase inhibitor), bumetanide (Na+/K+/2Cl− tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca2+ ionophore) were applied to tissue engineered constructs for 1 hr per day on days 10–14 of culture and examined at 2 or 4 wks. Gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed. Results Analysis showed that 20 µM ouabain, 0.3 µM ionomycin, or their combination increased the tensile modulus by 40–95% compared to untreated controls and resulted in increased collagen normalized to wet weight. In constructs exposed to ouabain, the increased collagen per wet weight was secondary to decreased GAG production on a per cell basis. Treatment with 20 µM ouabain also increased the neo-tissue’s ultimate tensile strength 56–86% at 4 wks. Other construct properties, such as construct growth and collagen type I production, were affected differently by Na+ modulation with ouabain versus Ca2+ modulation with ionomycin. Conclusions These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identify potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacological agents for increasing tensile integrity and directing

  10. Subcompartmentalized Nanoreactors as Artificial Organelle with Intracellular Activity.

    PubMed

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

    2016-04-01

    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.

  11. 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

  12. Engineered nonviral nanocarriers for intracellular gene delivery applications.

    PubMed

    Ojea-Jiménez, Isaac; Tort, Olivia; Lorenzo, Julia; Puntes, Victor F

    2012-10-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully.

  13. Engineered nonviral nanocarriers for intracellular gene delivery applications.

    PubMed

    Ojea-Jiménez, Isaac; Tort, Olivia; Lorenzo, Julia; Puntes, Victor F

    2012-10-01

    The efficient delivery of nucleic acids into mammalian cells is a central aspect of cell biology and of medical applications, including cancer therapy and tissue engineering. Non-viral chemical methods have been received with great interest for transfecting cells. However, further development of nanocarriers that are biocompatible, efficient and suitable for clinical applications is still required. In this paper, the different material platforms for gene delivery are comparatively addressed, and the mechanisms of interaction with biological systems are discussed carefully. PMID:22972254

  14. 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.

  15. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  16. Coupling of active motion and advection shapes intracellular cargo transport.

    PubMed

    Khuc Trong, Philipp; Guck, Jochen; Goldstein, Raymond E

    2012-07-13

    Intracellular cargo transport can arise from passive diffusion, active motor-driven transport along cytoskeletal filament networks, and passive advection by fluid flows entrained by such cargo-motor motion. Active and advective transport are thus intrinsically coupled as related, yet different representations of the same underlying network structure. A reaction-advection-diffusion system is used here to show that this coupling affects the transport and localization of a passive tracer in a confined geometry. For sufficiently low diffusion, cargo localization to a target zone is optimized either by low reaction kinetics and decoupling of bound and unbound states, or by a mostly disordered cytoskeletal network with only weak directional bias. These generic results may help to rationalize subtle features of cytoskeletal networks, for example as observed for microtubules in fly oocytes.

  17. A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.

    PubMed

    Pastuszka, Martha K; Janib, Siti M; Weitzhandler, Isaac; Okamoto, Curtis T; Hamm-Alvarez, Sarah; Mackay, J Andrew

    2012-11-12

    From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains. PMID:23088632

  18. 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.

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

    PubMed Central

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

    2015-01-01

    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 (А549) 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. PMID:25976444

  20. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  1. 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.

  2. 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

  3. Steroid signaling activation and intracellular localization of sex steroid receptors.

    PubMed

    Giraldi, Tiziana; Giovannelli, Pia; Di Donato, Marzia; Castoria, Gabriella; Migliaccio, Antimo; Auricchio, Ferdinando

    2010-12-01

    In addition to stimulating gene transcription, sex steroids trigger rapid, non-genomic responses in the extra-nuclear compartment of target cells. These events take place within seconds or minutes after hormone administration and do not require transcriptional activity of sex steroid receptors. Depending on cell systems, activation of extra-nuclear signaling pathways by sex steroids fosters cell cycle progression, prevents apoptosis, leads to epigenetic modifications and increases cell migration through cytoskeleton changes. These findings have raised the question of intracellular localization of sex steroid receptors mediating these responses. During the past years, increasing evidence has shown that classical sex steroid receptors localized in the extra-nuclear compartment or close to membranes of target cells induce these events. The emerging picture is that a process of bidirectional control between signaling activation and sex steroid receptor localization regulates the outcome of hormonal responses in target cells. This mechanism ensures cell cycle progression in estradiol-treated breast cancer cells, and its derangement might occur in progression of human proliferative diseases. These findings will be reviewed here together with unexpected examples of the relationship between sex steroid receptor localization, signaling activation and biological responses in target cells. We apologize to scientists whose reports are not mentioned or extensively discussed owing to space limitations.

  4. 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

  5. Engineering cells with intracellular agent–loaded microparticles to control cell phenotype

    PubMed Central

    Ankrum, James A; Miranda, Oscar R; Ng, Kelvin S; Sarkar, Debanjan; Xu, Chenjie; Karp, Jeffrey M

    2014-01-01

    Cell therapies enable unprecedented treatment options to replace tissues, destroy tumors and facilitate regeneration. The greatest challenge facing cell therapy is the inability to control the fate and function of cells after transplantation. We have developed an approach to control cell phenotype in vitro and after transplantation by engineering cells with intracellular depots that continuously release phenotype-altering agents for days to weeks. The platform enables control of cells’ secretome, viability, proliferation and differentiation, and the platform can be used to deliver drugs or other factors (e.g., dexamethasone, rhodamine and iron oxide) to the cell’s microenvironment. The preparation, efficient internalization and intracellular stabilization of ~1-μm drug-loaded microparticles are critical for establishing sustained control of cell phenotype. Herein we provide a protocol to generate and characterize micrometer-sized agent-doped poly(lactic-co-glycolic) acid (PLGA) particles by using a single-emulsion evaporation technique (7 h), to uniformly engineer cultured cells (15 h), to confirm particle internalization and to troubleshoot commonly experienced obstacles. PMID:24407352

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

    PubMed

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

    2016-01-01

    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

  7. 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.

  8. 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

  9. Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

    PubMed

    Miller, David J; Schwartz, Michael D; Dye, Billy T; Ahlquist, Paul

    2003-11-01

    Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

  10. 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.

  11. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide.

    PubMed

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A; Kopelman, Raoul

    2013-11-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.

  12. Aldosterone secretion, measurements of membrane potential and intracellular potassium activity in the isolated adrenal zone glomerulosa.

    PubMed

    Wiederholt, M; Hampel, J; Belkien, L; Oelkers, W

    1984-09-01

    Cell membrane potential and intracellular potassium activity (microelectrodes filled with ion-sensitive liquid ion exchanger) were measured in the zona glomerulosa of superfused hemi-adrenals of rats kept on different diets. Simultaneously, samples of the superfusate were collected and analyzed by radioimmunoassay for aldosterone content. Cell membrane potential and intracellular potassium activity were not influenced by high sodium, low sodium or high potassium diet. However, aldosterone secretion significantly changed. These results suggest that membrane potential and intracellular potassium activity per se may not be linked to changes in aldosterone secretion.

  13. The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

    PubMed

    Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

    2016-01-01

    To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

  14. Depollution potential of three macrophytes: exudated, wall-bound and intracellular peroxidase activities plus intracellular phenol concentrations.

    PubMed

    Larue, Camille; Korboulewsky, Nathalie; Wang, Runying; Mévy, Jean-Philippe

    2010-10-01

    The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 3.58 mg eq. [corrected] gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals.

  15. Intracellular disassembly and activity of pertussis toxin require interaction with ATP.

    PubMed

    Plaut, Roger D; Scanlon, Karen M; Taylor, Michael; Teter, Ken; Carbonetti, Nicholas H

    2016-08-01

    The active subunit (S1) of pertussis toxin (PT), a major virulence factor of Bordetella pertussis, ADP-ribosylates Gi proteins in the mammalian cell cytosol to inhibit GPCR signaling. The intracellular pathway of PT includes endocytosis and retrograde transport to the trans-Golgi network (TGN) and endoplasmic reticulum (ER). Subsequent translocation of S1 to the cytosol is presumably preceded by dissociation from the holotoxin. In vitro, such dissociation is stimulated by interaction of PT with ATP. To investigate the role of this interaction in cellular events, we engineered a form of PT (PTDM) with changes to two amino acids involved in the interaction with ATP. PTDM was reduced in (1) binding to ATP, (2) dissociability by interaction with ATP, (3) in vitro enzymatic activity and (4) cellular ADP-ribosylation activity. In cells treated with PTDM carrying target sequences for organelle-specific modifications, normal transport to the TGN and ER occurred, but N-glycosylation patterns of the S1 and S4 subunits were consistent with an inability of PTDM to dissociate in the ER. These results indicate a requirement for interaction with ATP for PT dissociation in the ER and cellular activity. They also indicate that the retrograde transport route is the cellular intoxication pathway for PT.

  16. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide

    NASA Astrophysics Data System (ADS)

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A.; Kopelman, Raoul

    2013-10-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers. Electronic supplementary information (ESI) available: Effect of Potassium depletion on F3 peptide subcellular localization, MTT

  17. Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation

    PubMed Central

    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

    2013-01-01

    Summary 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. PMID:24315997

  18. 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. PMID:24315997

  19. Intracellular Delivery and Antibacterial Activity of Gentamicin Encapsulated in pH-Sensitive Liposomes

    PubMed Central

    Lutwyche, Peter; Cordeiro, Carol; Wiseman, David J.; St-Louis, Maryse; Uh, Mitchell; Hope, Michael J.; Webb, Murray S.; Finlay, B. Brett

    1998-01-01

    Cell membranes are relatively impermeable to the antibiotic gentamicin, a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections. Liposomal encapsulation of this drug was used in order to achieve intracellular antibiotic delivery and therefore increase the drug’s therapeutic activity against intracellular pathogens. Gentamicin encapsulation in several dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoylphosphatidylethanolamine (DOPE)-based carrier systems was characterized. To systematically test the antibacterial efficacies of these formulations, a tissue culture assay system was developed wherein murine macrophage-like J774A.1 cells were infected with bacteria and were then treated with encapsulated drug. Of these formulations, DOPE–N-succinyl-DOPE and DOPE–N-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activities, killing greater than 75% of intracellular vacuole-resident wild-type Salmonella typhimurium compared to the level of killing of the control formulations. These formulations also efficiently eliminated intracellular infections caused by a recombinant hemolysin-expressing S. typhimurium strain and a Listeria monocytogenes strain, both of which escape the vacuole and reside in the cytoplasm. Control non-pH-sensitive liposomal formulations of gentamicin had poor antibacterial activities. A fluorescence resonance energy transfer assay indicated that the efficacious formulations undergo a pH-dependent lipid mixing and fusion event. Intracellular delivery of the fluorescent molecules encapsulated in these formulations was confirmed by confocal fluorescence microscopy and was shown to be dependent on endosomal acidification. This work shows that encapsulation of membrane-impermeative antibiotics in appropriately designed lipid-based delivery systems can enable their use in treating intracellular

  20. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  1. Ursolic Acid Activates Intracellular Killing Effect of Macrophages During Mycobacterium tuberculosis Infection.

    PubMed

    Podder, Biswajit; Jang, Woong Sik; Nam, Kung-Woo; Lee, Byung-Eui; Song, Ho-Yeon

    2015-05-01

    Tuberculosis is one of the most threatening infectious diseases to public health all over the world, for which Mycobacterium tuberculosis (MTB) is the etiological agent of pathogenesis. Ursolic acid (UA) has immunomodulatory function and exhibits antimycobacterial activity. However, the intracellular killing effect of UA has yet to be elucidated. The aim of this study was to evaluate the intracellular killing effect of UA during mycobacterial infection. The intracellular killing activity of UA was evaluated in the macrophage cell line THP-1 by the MGIT 960 system as well as by CFU count. The production of reactive oxygen species (ROS) and the level of nitric oxide (NO) were measured using DCF-DA and Griess reagent, respectively. Phagocytosis was observed by a fluorescence-based staining method, and the colony forming units were enumerated on 7H11 agar medium following infection. In addition, MRP8 mRNA expression was measured by qRT-PCR. UA significantly decreased the number of intracellular Mycobacterium through generation of ROS and NO. In addition, it profoundly activated the phagocytosis process of THP-1 cells during MTB-infection. Furthermore, our data demonstrated that UA activated the phagocytosis process in human monocyte cells through MRP8 induction. These data suggest that UA firmly contributes to the intracellular killing effect of macrophages during mycobacterial infection.

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

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-01

    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.

  3. 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

  4. Activation of the recombinant human alpha 7 nicotinic acetylcholine receptor significantly raises intracellular free calcium.

    PubMed

    Delbono, O; Gopalakrishnan, M; Renganathan, M; Monteggia, L M; Messi, M L; Sullivan, J P

    1997-01-01

    The alpha 7 nicotinic acetylcholine receptor (nAChR) subtype, unlike other neuronal nicotinic receptors, exhibits a relatively high permeability to Ca++ ions. Although Ca++ entry through this receptor subtype has been implicated in various Ca(++)-dependent processes in the central nervous system, little is known about how this receptor modulates mammalian intracellular Ca++ dynamics. Intracellular Ca++ responses evoked by activation of the human alpha 7 nAChRs stably expressed in HEK-293 (human embryonic kidney) cells were studied. Inward current and intracellular Ca++ transients were recorded simultaneously in response to a fast drug application system. Current recordings under whole-cell voltage-clamp and fast ratiometric intracellular Ca++ imaging acquisition were synchronized to drug pulses. The mean peak [Ca++]i observed with 100 microM (-)-nicotine was 356 +/- 48 nM (n = 8). The magnitude of the intracellular Ca++ elevation corresponds to a 20% fractional current carried by Ca++ ions. The EC50 of the intracellular Ca++ responses for (-)-nicotine, (+/-)-epibatidine, 1,1 dimethyl-4-phenyl-piperazinium and acetylcholine were 51, 3.5, 75 and 108 microM, respectively. These EC50 values strongly correlate with those recorded for the cationic inward current through alpha 7 nAChR. alpha-Bungarotoxin, methyllcaconitine or extracellular Ca++ chelation ablated (-)-nicotine-evoked increase in intracellular Ca++ concentration. This study provides evidence that cation influx through the human alpha 7 nAChR is sufficient to mediate a significant, transient, rise in intracellular Ca++ concentration.

  5. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  6. 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.

  7. Diverse intracellular pathogens activate Type III Interferon expression from peroxisomes

    PubMed Central

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

    2014-01-01

    Type I Interferon (IFN) 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 IFNs. The mechanisms controlling Type I IFN-independent responses are undefined. We have found that RIG-I like Receptors (RLRs) induce Type III IFN expression in a variety of human cell types, and identified factors that differentially regulate Type I and III IFN expression. We identified peroxisomes as a primary site that initiates Type III IFN expression, and revealed that the process of intestinal epithelial cell differentiation upregulates peroxisome biogenesis and promotes robust Type III IFN responses in human cells. These findings highlight the interconnections between innate immunity and cell biology. PMID:24952503

  8. 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...

  9. 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

  10. 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

  11. Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells.

    PubMed

    Ho, L H; Ratnaike, R N; Zalewski, P D

    2000-02-01

    Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.

  12. Tracking SERS-active nanoprobe intracellular uptake for chemical and biological sensing

    NASA Astrophysics Data System (ADS)

    Gregas, Molly K.; Yan, Fei; Scaffidi, Jonathan; Wang, Hsin-Neng; Khoury, Christopher; Zhang, Yan; Vo-Dinh, Tuan

    2007-09-01

    A critical aspect of the use of nanoprobes for intracellular studies in chemical and biological sensing involves a fundamental understanding of their uptake and trajectory in cells. In this study, we describe experiments using surface-enhanced Raman scattering (SERS) spectroscopy and mapping to track cellular uptake of plasmonics-active labeled nanoparticles. Three different Raman-active labels with positive, negative, and neutral charges were conjugated to silver colloidal nanoparticles with the aim of spatially and temporally profiling intracellular delivery and tracking of nanoprobes during uptake in single mammalian cells. 1-D Raman spectra and 2-D Raman mapping are used to identify and locate the probes via their SERS signal intensities. Because Raman spectroscopy is very specific for identification of chemical and molecular signatures, the development of functionalized plasmonics-active nanoprobes capable of exploring intracellular spaces and processes has the ability to provide specific information on the effects of biological and chemical pollutants in the intracellular environment. The results indicate that this technique will allow study of when, where, and how these substances affect cells and living organisms.

  13. Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators

    PubMed Central

    Akam, Eman A.; Chang, Tsuhen M.; Astashkin, Andrei V.

    2014-01-01

    Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species. PMID:25100578

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

    PubMed

    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

  15. LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages.

    PubMed

    Stephan, Alexander; Batinica, Marina; Steiger, Julia; Hartmann, Pia; Zaucke, Frank; Bloch, Wilhelm; Fabri, Mario

    2016-08-01

    As part of the innate host response neutrophils release neutrophil extracellular traps (NETs), protein:DNA complexes that contain a number of antimicrobial peptides (AMPs), such as cathelicidin. Human cathelicidin in its active form, LL37, has potent antimicrobial activity against bacteria. However, whether LL37 derived from NETs contributes to antimicrobial activity against intracellular pathogens remains unclear. Here, we report that NETs induced by mycobacteria contain cathelicidin. Human macrophages internalized NET-bound cathelicidin, which is transported to lysosomal compartments. Furthermore, using a model of in vitro-generated LL37:DNA complexes we found that LL37 derived from such complexes attacks mycobacteria in macrophage phagolysosomes resulting in antimicrobial activity. Taken together, our results suggest a mechanism by which LL37 in complex with DNA contributes to host defence against intracellular bacteria in human macrophages.

  16. Dynamics of free intracellular Ca2+ during synaptic and spike activity of cricket tibial motoneurons.

    PubMed

    Baden, Tom; Hedwig, Berthold

    2009-04-01

    For all nervous systems, motoneurons are the main output pathway. They are involved in generating episodic motor activity as well as enduring motor rhythms. To determine whether changes in cytosolic Ca(2+) correlate with motor performance, we studied the spatiotemporal dynamics, mode of entry and role of free intracellular Ca(2+) in cricket (Gryllus bimaculatus) front leg tibial extensor and flexor motoneurons. Synaptic activation or intracellular depolarising current injection uniformly increased Ca(2+) with the same dynamics throughout the primary and secondary branches of the dendritic tree of all motoneurons. Ca(2+) rise times (mean tau(rise), 233-295 ms) were lower than decay times (mean tau(decay), 1927-1965 ms), and resulted in a Ca(2+) plateau during repetitive activation, such as during walking. The neurons therefore operate with a different Ca(2+) level during walking than during episodic leg movements. Ca(2+) enters the dendritic processes of motoneurons via a voltage-activated mechanism. Entry is driven by subthreshold excitation, and is largely independent of the neurons' spiking activity. To what extent ligand-activated mechanisms of Ca(2+) entry operate remains uncertain. We found no evidence for any prominent Ca(2+)-activated secondary currents in these motoneurons. Excitatory postsynaptic potentials evoked by extracellular stimulation of descending neurons were unaffected by the level of free intracellular Ca(2+). The activity of tibial motoneurons therefore appears to be only weakly dependent on the level of free intracellular Ca(2+) in dendrites. This is different to what has been found for many other neurons studied, and may represent an essential prerequisite for insect motoneurons to support a wide range of both episodic and rhythmic motor sequences underlying behaviour.

  17. Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

    PubMed

    Birkus, Gabriel; Bam, Rujuta A; Willkom, Madeleine; Frey, Christian R; Tsai, Luong; Stray, Kirsten M; Yant, Stephen R; Cihlar, Tomas

    2016-01-01

    Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

  18. 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.

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

    PubMed

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Martinez Molina, Daniel; 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

  20. Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs.

    PubMed

    Sasaki, Akira; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2015-09-24

    Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study.

  1. 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

  2. Raster image cross-correlation analysis for spatiotemporal visualization of intracellular degradation activities against exogenous DNAs.

    PubMed

    Sasaki, Akira; Yamamoto, Johtaro; Jin, Takashi; Kinjo, Masataka

    2015-01-01

    Reducing intracellular DNA degradation is critical to enhance the efficiency of gene therapy. Exogenous DNA incorporation into cells is strictly blocked by the defense machinery of intracellular nuclease activity. Raster image correlation spectroscopy (RICS) and raster image cross-correlation spectroscopy (cross-correlation RICS; ccRICS) are image-based correlation methods. These powerful tools allow the study of spatiotemporal molecular dynamics. Here we performed spatiotemporal ccRICS analyses of fluorescent DNA and directly monitored the process of exogenous DNA degradation in living cell cytoplasm. Such direct monitors of DNA degradation allow us to determine the fate of the exogenous DNA in living cells. On comparing the process in living cells, our study shows that cytoplasmic nuclease activity differs between cell lines; therefore, we propose that the difference of nuclease activity in cytoplasm dictates a different resistance to exogenous DNA incorporation. New insight on efficient gene delivery can be provided with our study. PMID:26400011

  3. Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study.

    PubMed

    Carnero, A; Lacal, J C

    1995-02-01

    Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II, a similar pattern to that induced by p21ras proteins. Thus, the comparison of activated enzymes after microinjection of p21ras or PLs indicated that only PLD microinjection mimetized p21ras signalling. Finally, inhibition of the endogenous PLD activity by neomycin substantially reduced the biological activity of p21ras. All these results suggest that PLD activation may constitute a relevant step in ras-induced germinal vesicle breakdown in Xenopus oocytes.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26462157

  5. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

    PubMed Central

    Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  6. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

    PubMed

    Tillotson, Benjamin J; Goulatis, Loukas I; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  7. Cellular Metabolic Activity and the Oxygen and Hydrogen Stable Isotope Composition of Intracellular Water and Metabolites

    NASA Astrophysics Data System (ADS)

    Kreuzer-Martin, H. W.; Hegg, E. L.

    2008-12-01

    Intracellular water is an important pool of oxygen and hydrogen atoms for biosynthesis. Intracellular water is usually assumed to be isotopically identical to extracellular water, but an unexpected experimental result caused us to question this assumption. Heme O isolated from Escherichia coli cells grown in 95% H218O contained only a fraction of the theoretical value of labeled oxygen at a position where the O atom was known to be derived from water. In fact, fewer than half of the oxygen atoms were labeled. In an effort to explain this surprising result, we developed a method to determine the isotope ratios of intracellular water in cultured cells. The results of our experiments showed that during active growth, up to 70% of the oxygen atoms and 50% of the hydrogen atoms in the intracellular water of E. coli are generated during metabolism and can be isotopically distinct from extracellular water. The fraction of isotopically distinct atoms was substantially less in stationary phase and chilled cells, consistent with our hypothesis that less metabolically-generated water would be present in cells with lower metabolic activity. Our results were consistent with and explained the result of the heme O labeling experiment. Only about 40% of the O atoms on the heme O molecule were labeled because, presumably, only about 40% of the water inside the cells was 18O water that had diffused in from the culture medium. The rest of the intracellular water contained 16O atoms derived from either nutrients or atmospheric oxygen. To test whether we could also detect metabolically-derived hydrogen atoms in cellular constituents, we isolated fatty acids from log-phase and stationary phase E. coli and determined the H isotope ratios of individual fatty acids. The results of these experiments showed that environmental water contributed more H atoms to fatty acids isolated in stationary phase than to the same fatty acids isolated from log-phase cells. Stable isotope analyses of

  8. Release of plasminogen activator inhibitor-1 from human astrocytes is regulated by intracellular ceramide.

    PubMed

    Kimura, M; Soeda, S; Oda, M; Ochiai, T; Kihara, T; Ono, N; Shimeno, H

    2000-12-15

    The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

    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.

  10. 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. PMID:27216383

  11. 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.

  12. Intracellular Signaling by Hydrolysis of Phospholipids and Activation of Protein Kinase C

    NASA Astrophysics Data System (ADS)

    Nishizuka, Yasutomi

    1992-10-01

    Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels. This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids, particularly choline phospholipids, by phospholipase D and phospholipase A_2 may also take part in cell signaling. The products of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.

  13. 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

  14. 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

  15. 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. PMID:26546608

  16. Simultaneous intracellular delivery of targeting antibodies and functional nanoparticles with engineered protein G system.

    PubMed

    Lim, Yong Taik; Cho, Mi Young; Lee, Jung Min; Chung, Sang Jeon; Chung, Bong Hyun

    2009-02-01

    Cellular internalization of functional nanoparticles that have optical and magnetic properties is very important in the cellular imaging and manipulation of specifically targeted biomolecules. In this study, a robust method to deliver functional nanoparticles and targeting antibodies into cells was suggested. The engineered protein G system, which contains an affinity tag and a cell penetration peptide in the N- and C-terminals, respectively, can capture surface-modified nanoparticles and antibodies without chemical reaction, and then non-invasively deliver them into the cells. Finally, gold-coated iron oxide nanoparticle/engineered protein G hybrid systems were successfully employed as multifunctional cargo systems for the targeting, imaging, and manipulation of mitochondria.

  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. An active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells.

    PubMed

    Bagh, Sangram; Mandal, Mahuya; Ang, Jordan; McMillen, David R

    2011-03-01

    Synthetic biology includes an effort to logically control cellular behavior. One long-term goal is to implement medical interventions inside living cells, creating intracellular "disease fighters"; one may imagine a system that detects viral infection and responds to halt the spread of the virus. Here, we explore a system designed to display some of the qualitative features that such disease prevention systems should have, while not claiming that the system itself has any medical application. An intracellular disease prevention mechanism should: lie dormant in the absence of the disease state; detect the onset of a lethal disease pathway; respond to halt or mitigate the disease's effects; and be subject to external deactivation when required. We have created a device that displays these properties, in the highly simplified case of a bacterial viral disease. Our system detects the onset of the lytic phase of bacteriophage lambda in Escherichia coli, responds by preventing this lethal pathway from being followed, and is deactivated by a temperature shift. We have formulated a mathematical model of the engineered system, using parameters obtained from the literature and by local experimental measurement, and shown that the model captures the essential experimental behavior of the system in most parameter regimes.

  19. 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.

  20. 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.

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

    PubMed

    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

  2. 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

  3. 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

  4. 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

  5. Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery.

    PubMed

    Taratula, Oleh; Garbuzenko, Olga B; Kirkpatrick, Paul; Pandya, Ipsit; Savla, Ronak; Pozharov, Vitaly P; He, Huixin; Minko, Tamara

    2009-12-16

    Low penetration ability of Small Interfering RNA (siRNA) through the cellular plasma membrane combined with its limited stability in blood, limits the effectiveness of the systemic delivery of siRNA. In order to overcome such difficulties, we constructed a nanocarrier-based delivery system by taking advantage of the lessons learned from the problems in the delivery of DNA. In the present study, siRNA nanoparticles were first formulated with Poly(Propyleneimine) (PPI) dendrimers. To provide lateral and steric stability to withstand the aggressive environment in the blood stream, the formed siRNA nanoparticles were caged with a dithiol containing cross-linker molecules followed by coating them with Poly(Ethylene Glycol) (PEG) polymer. A synthetic analog of Luteinizing Hormone-Releasing Hormone (LHRH) peptide was conjugated to the distal end of PEG polymer to direct the siRNA nanoparticles specifically to the cancer cells. Our results demonstrated that this layer-by-layer modification and targeting approach confers the siRNA nanoparticles stability in plasma and intracellular bioavailability, provides for their specific uptake by tumor cells, accumulation of siRNA in the cytoplasm of cancer cells, and efficient gene silencing. In addition, in vivo body distribution data confirmed high specificity of the proposed targeting delivery approach which created the basis for the prevention of adverse side effects of the treatment on healthy organs.

  6. Surface-Engineered Targeted PPI Dendrimer for Efficient Intracellular and Intratumoral siRNA Delivery

    PubMed Central

    Taratula, Oleh; Garbuzenko, Olga B.; Kirkpatrick, Paul; Pandya, Ipsit; Savla, Ronak; Pozharov, Vitaly P.; He, Huixin; Minko, Tamara

    2009-01-01

    Low penetration ability of Small Interfering RNA (siRNA) through the cellular plasma membrane combined with its limited stability in blood, limits the effectiveness of the systemic delivery of siRNA. In order to overcome such difficulties, we constructed a nanocarrier-based delivery system by taking advantage of the lessons learned from the problems in the delivery of DNA. In the present study, siRNA nanoparticles were first formulated with Poly(Propyleneimine) (PPI) dendrimers. To provide lateral and steric stability to withstand the aggressive environment in the blood stream, the formed siRNA nanoparticles were caged with a dithiol containing cross-linker molecules followed by coating them with Poly(Ethylene Glycol) (PEG) polymer. A synthetic analog of Luteinizing Hormone-Releasing Hormone (LHRH) peptide was conjugated to the distal end of PEG polymer to direct the siRNA nanoparticles specifically to the cancer cells. Our results demonstrated that this layer-by-layer modification and targeting approach confers the siRNA nanoparticles stability in plasma and intracellular bioavailability, provides for their specific uptake by tumor cells, accumulation of siRNA in the cytoplasm of cancer cells, and efficient gene silencing. In addition, in vivo body distribution data confirmed high specificity of the proposed targeting delivery approach which created the basis for the prevention of adverse side effects of the treatment on healthy organs. PMID:19567257

  7. AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides*

    PubMed Central

    Ishibashi, Yohei; Hirabayashi, Yoshio

    2015-01-01

    The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer. PMID:26048992

  8. The Peptide Microarray-Based Resonance Light Scattering Assay for Sensitively Detecting Intracellular Kinase Activity.

    PubMed

    Li, Tao; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-01-01

    The peptide microarray technology is a robust, reliable, and efficient technique for large-scale determination of enzyme activities, and high-throughput profiling of substrate/inhibitor specificities of enzymes. Here, the activities of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) in different cell lysates have been detected by a peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle (GNP) probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 (human glioma cell) cell lysate (corresponding to 200 cells) is achieved by a selected peptide substrate. The experimental results also demonstrate that the RLS assay can be employed to evaluate the chemical regulation of intracellular kinase activity. PMID:26490469

  9. 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

  10. 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

  11. Intracellular pathways linking hypoxia to activation of c-fos and AP-1.

    PubMed

    Premkumar, D R; Adhikary, G; Overholt, J L; Simonson, M S; Cherniack, N S; Prabhakar, N R

    2000-01-01

    Organisms respond to hypoxia through detection of blood oxygen levels by sensors at peripheral chemoreceptors and by receptors in certain key cells of the body. The pathways over which peripheral chemoreceptor signals are transmitted to respiratory muscles are well established. However, the intracellular pathways that transmit hypoxic stimulus to gene activation are just being identified. Using anti-sense c-fos strategy, we have shown that c-fos is essential for the activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of downstream genes such as tyrosine hydroxylase (TH; Mishra et al. 1998). The purpose of the present study was to identify intracellular pathways that link hypoxia to activation of c-fos. The results of the present study show that hypoxia causes Ca2+ influx through L-type voltage gated Ca2+ channels and that hypoxia-induced c-fos gene expression is Ca2+/calmodulin dependent. We also demonstrate that hypoxia activates the extracellular-regulated kinase (ERK) and p38, but not JNK. Further, phosphorylation of ERK is essential for c-fos activation via SRE cis-element. Further characterization of nuclear signalling pathways provides evidence for the involvement of Src, a non receptor protein tyrosine kinase, and Ras, a small G protein, in the hypoxia-induced c-fos gene expression. These results suggest a possible role for non-receptor protein tyrosine kinases in propagating signals from G-protein coupled receptors to the activation of immediate early genes such as c-fos during hypoxia.

  12. 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

  13. Cleaved CD44 intracellular domain supports activation of stemness factors and promotes tumorigenesis of breast cancer.

    PubMed

    Cho, Yunhee; Lee, Hyun-Woo; Kang, Hyeok-Gu; Kim, Hye-Young; Kim, Seok-Jun; Chun, Kyung-Hee

    2015-04-20

    CD44 plays a role in the progression of tumors and is expressed in cancer stem cells (CSCs). However, the mechanisms underlying the crosstalk of CD44 with stemness genes in CSC maintenance remains unclear. In this study, we demonstrated how the cleaved intracellular domain of CD44 (CD44ICD) activates stemness factors such as Nanog, Sox2 and Oct4, and contributes to the tumorigenesis of breast cancer. We have found that the overexpression of CD44ICD increased mammosphere formation in breast cancer cells. Treatment with a γ-secretase inhibitor (GSI), which blocks the cleavage of CD44ICD, interfered with mammosphere formation. Interestingly, CD44ICD decreased the expression levels and nuclear localization of stemness factors, but overexpression of CD44ICD reversed these effects. In addition, we showed that nuclear localization of CD44ICD is important for transcriptional activation of the stemness factors. Furthermore, CD44ICD-overexpressed cells exhibited strong tumorigenecity and greater metastatic potential than did the control cells or CD44-depleted cells in vivo in mice models. Taken together, it was supposed that CD44 promotes tumorigenesis through the interaction and nuclear-translocation of its intracellular domain and stemness factors. We suggest that the prevention of cleavage and nuclear-translocation of CD44ICD is a potential target in treating breast cancer. PMID:25909162

  14. 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

  15. 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.

  16. Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores*

    PubMed Central

    Patel, Vimal A.; Massenburg, Donald; Vujicic, Snezana; Feng, Lanfei; Tang, Meiyi; Litbarg, Natalia; Antoni, Angelika; Rauch, Joyce; Lieberthal, Wilfred; Levine, Jerrold S.

    2015-01-01

    Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. PMID:26183782

  17. 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.

  18. Inositol-Requiring Enzyme 1-Dependent Activation of AMPK Promotes Brucella abortus Intracellular Growth

    PubMed Central

    Liu, Ning; Li, Yingying; Dong, Chunyan; Xu, Xiaohan; Wei, Pan; Sun, Wanchun

    2016-01-01

    ABSTRACT AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is well conserved during evolution. AMPK activation inhibits production of reactive oxygen species (ROS) in cells via suppression of NADPH oxidase. However, the role of AMPK during the process of Brucella infection remains unknown. Our data demonstrate that B. abortus infection induces AMPK activation in HeLa cells in a time-dependent manner. The known AMPK kinases LKB1, CAMKKβ, and TAK1 are not required for the activation of AMPK by B. abortus infection. Instead, this activation is dependent on the RNase activity of inositol-requiring enzyme 1 (IRE1). Moreover, we also found that B. abortus infection-induced IRE1-dependent activation of AMPK promotes B. abortus intracellular growth with peritoneal macrophages via suppression of NADPH-derived ROS production. IMPORTANCE Previous studies showed that B. abortus infection does not promote any oxidative burst regulated by NADPH oxidase. However, the underlying mechanism remains elusive. We report for the first time that AMPK activation caused by B. abortus infection plays important role in NADPH oxidase-derived ROS production. PMID:26755628

  19. Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Croset, M; Crawford, N

    1986-02-01

    Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar

  20. 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.

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

    PubMed

    Heikal, Ahmed A

    2010-04-01

    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.

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

    PubMed

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

    2015-12-01

    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

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

    PubMed

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

    2015-12-01

    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.

  4. 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

  5. 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.

  6. Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

    PubMed

    Luster, Brennon R; Urbano, Francisco J; Garcia-Rill, Edgar

    2016-06-01

    The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types.

  7. Inhibition of intracellular growth of Histoplasma capsulatum yeast cells by cytokine-activated human monocytes and macrophages.

    PubMed Central

    Newman, S L; Gootee, L; Bucher, C; Bullock, W E

    1991-01-01

    Human monocytes/macrophages (M psi) were infected with Histoplasma capsulatum yeast cells, and intracellular growth was quantified after 24 h of incubation in medium alone or in medium containing cytokines. Yeast cells multiplied within freshly isolated monocytes, cultured M psi, and alveolar M psi with intracellular generation times of 14.2 +/- 1.4, 18.5 +/- 2.1, and 19.9 +/- 1.9 h (mean +/- standard error of the mean), respectively. Monocytes and M psi inhibited the intracellular growth of yeast cells in response to cytokine supernatant; maximum inhibition was obtained when cytokines were added to cell monolayers immediately after infection. Opsonization of yeast cells in normal serum or in H. capsulatum-immune serum did not affect the intracellular generation time of yeast cells in either control M psi or cytokine-activated M psi. PMID:1898916

  8. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

    PubMed Central

    Wu, T C; Guarnieri, F G; Staveley-O'Carroll, K F; Viscidi, R P; Levitsky, H I; Hedrick, L; Cho, K R; August, J T; Pardoll, D M

    1995-01-01

    The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency. Images Fig. 2 Fig. 3 PMID:8524826

  9. 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

    2015-09-01

    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.

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

    PubMed Central

    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

    2015-01-01

    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 crosslink 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 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 crosslinking to produce synergistic benefits. PMID:26317395

  11. 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

  12. Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

    PubMed Central

    Reiland, J; Ott, V L; Lebakken, C S; Yeaman, C; McCarthy, J; Rapraeger, A C

    1996-01-01

    Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane

  13. Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus.

    PubMed

    Kim, Kihoon; Umamoto, Sean; Trang, Phong; Hai, Rong; Liu, Fenyong

    2004-03-01

    A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.

  14. G protein coupled receptor signaled apoptosis is associated with activation of a cation insensitive acidic endonuclease and intracellular acidification.

    PubMed

    Sharma, K; Srikant, C B

    1998-01-01

    Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.

  15. 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.

  16. 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

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

    PubMed

    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

  19. 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

  20. 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.

  1. 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

  2. Intracellular mechanisms modulating gamma band activity in the pedunculopontine nucleus (PPN).

    PubMed

    Luster, Brennon R; Urbano, Francisco J; Garcia-Rill, Edgar

    2016-06-01

    The pedunculopontine nucleus is a part of the reticular activating system, and is active during waking and REM sleep. Previous results showed that all PPN cells tested fired maximally at gamma frequencies when depolarized. This intrinsic membrane property was shown to be mediated by high-threshold N- and P/Q-type Ca(2+) channels. Recent studies show that the PPN contains three independent populations of neurons which can generate gamma band oscillations through only N-type channels, only P/Q-type channels, or both N- and P/Q-type channels. This study investigated the intracellular mechanisms modulating gamma band activity in each population of neurons. We performed in vitro patch-clamp recordings of PPN neurons from Sprague-Dawley rat pups, and applied 1-sec ramps to induce intrinsic membrane oscillations. Our results show that there are two pathways modulating gamma band activity in PPN neurons. We describe populations of neurons mediating gamma band activity through only N-type channels and the cAMP/PKA pathway (presumed "REM-on" neurons), through only P/Q-type channels and the CaMKII pathway (presumed "Wake-on" neurons), and a third population which can mediate gamma activity through both N-type channels and cAMP/PK and P/Q-type channels and CaMKII (presumed "Wake/REM-on" neurons). These novel results suggest that PPN gamma oscillations are modulated by two independent pathways related to different Ca(2+) channel types. PMID:27354537

  3. Inhibition of spontaneous network activity in neonatal hippocampal slices by energy substrates is not correlated with intracellular acidification.

    PubMed

    Mukhtarov, Marat; Ivanov, Anton; Zilberter, Yuri; Bregestovski, Piotr

    2011-01-01

    Several energy substrates complementary to glucose, including lactate, pyruvate and β-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO(3) -free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and β-hydroxybutyrate on network excitability in neonatal brain slices.

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

    PubMed

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

    2014-10-15

    We recently proposed a new bioinformatic algorithm called OncoFinder for quantifying the activation of intracellular signaling pathways. It was proved advantageous for minimizing errors of high-throughput gene expression analyses and showed strong potential for identifying new biomarkers. Here, for the first time, we applied OncoFinder for normal and cancerous tissues of the human bladder to identify biomarkers of bladder cancer. Using Illumina HT12v4 microarrays, we profiled gene expression in 17 cancer and seven non-cancerous bladder tissue samples. These experiments were done in two independent laboratories located in Russia and Canada. We calculated pathway activation strength values for the investigated transcriptomes and identified signaling pathways that were regulated differently in bladder cancer (BC) tissues compared with normal controls. We found, for both experimental datasets, 44 signaling pathways that serve as excellent new biomarkers of BC, supported by high area under the curve (AUC) values. We conclude that the OncoFinder approach is highly efficient in finding new biomarkers for cancer. These markers are mathematical functions involving multiple gene products, which distinguishes them from "traditional" expression biomarkers that only assess concentrations of single genes. PMID:25296972

  5. Antagonism of the STING Pathway via Activation of the AIM2 Inflammasome by Intracellular DNA.

    PubMed

    Corrales, Leticia; Woo, Seng-Ryong; Williams, Jason B; McWhirter, Sarah M; Dubensky, Thomas W; Gajewski, Thomas F

    2016-04-01

    Recent evidence has indicated that innate immune sensing of cytosolic DNA in dendritic cells via the host STING pathway is a major mechanism leading to spontaneous T cell responses against tumors. However, the impact of the other major pathway triggered by intracellular DNA, the absent in melanoma 2 (AIM2) inflammasome, on the functional output from the stimulator of IFN genes (STING) pathway is poorly understood. We found that dendritic cells and macrophages deficient in AIM2, apoptosis-associated specklike protein, or caspase-1 produced markedly higher IFN-β in response to DNA. Biochemical analyses showed enhanced generation of cyclic GMP-AMP, STING aggregation, and TANK-binding kinase 1 and IFN regulatory factor 3 phosphorylation in inflammasome-deficient cells. Induction of pyroptosis by the AIM2 inflammasome was a major component of this effect, and inhibition of caspase-1 reduced cell death, augmenting phosphorylation of TANK-binding kinase 1/IFN regulatory factor 3 and production of IFN-β. Our data suggest that in vitro activation of the AIM2 inflammasome in murine macrophages and dendritic cells leads to reduced activation of the STING pathway, in part through promoting caspase-1-dependent cell death.

  6. Delineation of Platelet Activation Pathway of Scutellarein Revealed Its Intracellular Target as Protein Kinase C.

    PubMed

    Tian, Xiaoxuan; Chang, Lianying; Ma, Guangyin; Wang, Taiyi; Lv, Ming; Wang, Zhilong; Chen, Liping; Wang, Yuefei; Gao, Xiumei; Zhu, Yan

    2016-01-01

    Erigeron breviscapus has been widely used in traditional Chinese medicine (TCM) and its total flavonoid component is commonly used to treat ischemic stroke, coronary heart disease, diabetes and hypertension. Scutellarin is the major ingredient of E. breviscapus and scutellarein is one of the main bioactive metabolites of scutellarin in vivo, but the latter's pharmacological activities have not been fully characterized. Provided evidence that could inhibit platelet aggregation, the effect of scutellarein on rat washed platelets and its underlying mechanisms were evaluated in our research. Scutellarein inhibited platelet adhesion and aggregation induced by multiple G protein coupled receptor agonists such as thrombin, U46619 and ADP, in a concentration-dependent manner. Furthermore, the mild effect of scutellarein on intracellular Ca(2+) mobilization and cyclic AMP (cAMP) level was observed. On the other hand, the role of scutellarein as potential protein kinase C (PKC) inhibitor was confirmed by PKC activity analysis and molecular docking. The phorbol myristate acetate-induced platelets aggregation assay with or without ADP implied that the scutellarein takes PKC(s) as its primary target(s), and acts on it in a reversible way. Finally, scutellarein as a promising agent exhibited a high inhibition effect on ADP-induced platelet aggregation among its analogues. This study clarifies the PKC-related signaling pathway involved in antiplatelet action of scutellarein, and may be beneficial for the treatment of cardiovascular diseases. PMID:26581323

  7. α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

    PubMed

    Cabrera, Ingrid; Abasolo, Ibane; Corchero, José L; Elizondo, Elisa; Gil, Pilar Rivera; Moreno, Evelyn; Faraudo, Jordi; Sala, Santi; Bueno, Dolores; González-Mira, Elisabet; Rivas, Merche; Melgarejo, Marta; Pulido, Daniel; Albericio, Fernando; Royo, Miriam; Villaverde, Antonio; García-Parajo, Maria F; Schwartz, Simó; Ventosa, Nora; Veciana, Jaume

    2016-04-01

    Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest. PMID:26890358

  8. α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

    PubMed

    Cabrera, Ingrid; Abasolo, Ibane; Corchero, José L; Elizondo, Elisa; Gil, Pilar Rivera; Moreno, Evelyn; Faraudo, Jordi; Sala, Santi; Bueno, Dolores; González-Mira, Elisabet; Rivas, Merche; Melgarejo, Marta; Pulido, Daniel; Albericio, Fernando; Royo, Miriam; Villaverde, Antonio; García-Parajo, Maria F; Schwartz, Simó; Ventosa, Nora; Veciana, Jaume

    2016-04-01

    Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

  9. Carbon dioxide, membrane potential and intracellular potassium activity in frog skeletal muscle.

    PubMed Central

    Huguenin, F; Reber, W; Zeuthen, T

    1980-01-01

    1. The membrane potential of isolated frog muscle fibres has been measured in absence and in presence of CO2, at constant external pH. 2. At a normal external Cl concentration, CO2 (PCO2 = 97 mmHg; pH = 7.0) applied for 10 min caused a highly variable depolarization, the average potential change being 8 mV after 5 min. The effect was reversible 3. In Cl-free solutions, CO2 (PCO2 = 97 mmHg; pH = 7.0) caused a biphasic depolarization of 20 mV after 5 min. The effect was fully reversible on CO2 removal. 4. The same effect appeared at a lower partial pressure (PCO2 = 38 mmHg; pH 7.3) in the presence of tetrodotoxin (10(-7) M). 5. In order to investigate the cause of the CO2-induced depolarization, membrane potential and intracellular K activity (ai/K,)( of surface muscle fibres were measured with voltage and ion-sensitive micro-electrodes. 6. At a normal external Cl concentration, CO2(PCO2 approximately equal to 97 mmHg; pH = 7.0) decreased ai/K by 5 mM after 5 min. 7. The same effect appeared at low external Cl concentration (11 mM). 8. At high partial pressure (PCO2 approximately equal to 588 mmHg; pH = 6.8), CO2 reduced ai/K by 19 mM in 10 min. 9. In long-term experiments performed over 4 h with a normal external Cl concentration, CO2 (PCO2 approximately equal to 97 mmHg; pH 5.8 or 7) changed practically neither membrane potential, nor ai/K. 10. It is concluded that increasing the PCO2 when keeping the external pH constant causes an early depolarization of muscle. This effect is particularly marked in the absence of chloride. It can be explained partly, in surface muscle fibres, by a decrease of the intracellular K activity. PMID:6776259

  10. 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

  11. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  12. 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

  13. 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.

  14. Cationic triblock copolymer micelles enhance antioxidant activity, intracellular uptake and cytotoxicity of curcumin.

    PubMed

    Yoncheva, Krassimira; Kamenova, Katya; Perperieva, Teodora; Hadjimitova, Vera; Donchev, Petar; Kaloyanov, Kaloyan; Konstantinov, Spiro; Kondeva-Burdina, Magdalena; Tzankova, Virginia; Petrov, Petar

    2015-07-25

    The aim of the present study was to develop curcumin loaded cationic polymeric micelles and to evaluate their loading, preservation of curcumin antioxidant activity and intracellular uptake ability. The micelles were prepared from a triblock copolymer consisting of poly(ϵ-caprolactone) and very short poly(2-(dimethylamino) ethyl methacrylate) segments (PDMAEMA9-PCL70-PDMAEMA9). The micelles showed monomodal size distribution, mean diameter of 145 nm, positive charge (+72 mV), critical micellar concentration around 0.05 g/l and encapsulation efficiency of 87%. The ability of the micellar curcumin to scavenge the ABTS radical and hypochlorite ions was higher than that of the free curcumin. Confocal microscopy revealed that the uptake of curcumin by chronic myeloid leukemia derived K-562 cells and human multiple myeloma cells U-266 was more intensive when curcumin was loaded into the micelles. These results correlated with the higher cytotoxicity of the micellar curcumin compared to free curcumin. Intraperitoneal treatment of Wistar rats indicated that PDMAEMA-PCL-PDMAEMA copolymer, comprising very short cationic chains, did not change the levels of malondialdehyde and glutathione in livers indicating an absence of oxidative stress. Thus, PDMAEMA-PCL-PDMAEMA triblock micelles could be considered efficient and safe platform for curcumin delivery. PMID:26026253

  15. Electrogenic glutamate uptake in glial cells is activated by intracellular potassium

    NASA Astrophysics Data System (ADS)

    Barbour, Boris; Brew, Helen; Attwell, David

    1988-09-01

    Uptake of glutamate into glial cells in the CNS maintains the extracellular glutamate concentration below neurotoxic levels and helps terminate its action as a neurotransmitter 1. The co-transport of two sodium ions on the glutamate carrier is thought to provide the energy needed to transport glutamate into cells2,3. We have shown recently that glutamate uptake can be detected electrically because the excess of Na+ ions transported with each glutamate anion results in a net current flow into the cell4. We took advantage of the control of the environment, both inside and outside the cell, provided by whole-cell patch-clamping and now report that glutamate uptake is activated by intracellular potassium and inhibited by extracellular potassium. Our results indicate that one K+ ion is transported out of the cell each time a glutamate anion and three Na+ ions are transported in. A carrier with this stoichiometry can accumulate glutamate against a much greater concentration gradient than a carrier co-transporting one glutamate anion and two Na+ ions. Pathological rises in extracellular potassium concentration will inhibit glutamate uptake by depolarizing glial cells and by preventing the loss of K+ from the glutamate carrier. This will facilitate a rise in the extracellular glutamate concentration to neurotoxic levels and contribute to the neuronal death occurring in brain anoxia and ischaemia.

  16. Anti-angiogenic activity and intracellular distribution of epigallocatechin-3-gallate analogs.

    PubMed

    Piyaviriyakul, Suratsawadee; Shimizu, Kosuke; Asakawa, Tomohiro; Kan, Toshiyuki; Siripong, Pongpun; Oku, Naoto

    2011-01-01

    Angiogenesis, a process of construction of new blood capillaries, is crucial for tumor progression and metastasis. Our previous studies demonstrated that a component of green tea, epigallocatechin-3-gallate (EGCG), suppressed angiogenesis and subsequent tumor growth. In this study, to elucidate the detailed mechanism of the anti-angiogenic effect of EGCG and to enhance the antiangiogenic activity of EGCG, we designed and synthesized EGCG derivatives and examined their biological effect and intracellular localization in human umbilical vein endothelial cells (HUVECs). EGCG derivatives aminopentyl dideoxyEGCG and aminopentyl dideoxygallocatechin-3-gallate (cis-APDOEGCG and trans-APDOEGCG) had an enhanced inhibitory effect on the proliferation when used at more than 30 µM. To elucidate antiangiogenic effect of EGCG, we used a 1 µM concentration for subsequent experiments where no effect on proliferation was observed. These EGCG derivatives showed a stronger inhibitory effect on migration, invasion, and tube formation by HUVECs than the non-derivatized EGCG. Furthermore, the derivatives induced a change in the distribution of F-actin and subsequent morphology of the HUVECs. Next, we synthesized fluorescent TokyoGreen-conjugated EGCG derivative (EGCG-TG) and observed the distribution in HUVECs under a confocal laser scanning microscope. Abundant fluorescence was observed in the cells after a 3-h incubation, and was localized in mitochondria as well as in cytoplasm. These results suggest that EGCG was incorporated into the HUVECs, that a portion of it entered into their mitochondria. PMID:21372391

  17. A Novel Intracellular Isoform of Matrix Metalloproteinase-2 Induced by Oxidative Stress Activates Innate Immunity

    PubMed Central

    Lovett, David H.; Mahimkar, Rajeev; Raffai, Robert L.; Cape, Leslie; Maklashina, Elena; Cecchini, Gary; Karliner, Joel S.

    2012-01-01

    Background Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress. Methodology and Principal Findings Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M77, thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes. Conclusion A novel N-terminal truncated intracellular isoform of MMP-2 is

  18. 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

  19. An arsenate-activated glutaredoxin from the arsenic hyperaccumulator fern Pteris vittata L. regulates intracellular arsenite.

    PubMed

    Sundaram, Sabarinath; Rathinasabapathi, Bala; Ma, Lena Q; Rosen, Barry P

    2008-03-01

    To elucidate the mechanisms of arsenic resistance in the arsenic hyperaccumulator fern Pteris vittata L., a cDNA for a glutaredoxin (Grx) Pv5-6 was isolated from a frond expression cDNA library based on the ability of the cDNA to increase arsenic resistance in Escherichia coli. The deduced amino acid sequence of Pv5-6 showed high homology with an Arabidopsis chloroplastic Grx and contained two CXXS putative catalytic motifs. Purified recombinant Pv5-6 exhibited glutaredoxin activity that was increased 1.6-fold by 10 mm arsenate. Site-specific mutation of Cys(67) to Ala(67) resulted in the loss of both GRX activity and arsenic resistance. PvGrx5 was expressed in E. coli mutants in which the arsenic resistance genes of the ars operon were deleted (strain AW3110), a deletion of the gene for the ArsC arsenate reductase (strain WC3110), and a strain in which the ars operon was deleted and the gene for the GlpF aquaglyceroporin was disrupted (strain OSBR1). Expression of PvGrx5 increased arsenic tolerance in strains AW3110 and WC3110, but not in OSBR1, suggesting that PvGrx5 had a role in cellular arsenic resistance independent of the ars operon genes but dependent on GlpF. AW3110 cells expressing PvGrx5 had significantly lower levels of arsenite when compared with vector controls when cultured in medium containing 2.5 mm arsenate. Our results are consistent with PvGrx5 having a role in regulating intracellular arsenite levels, by either directly or indirectly modulating the aquaglyceroporin. To our knowledge, PvGrx5 is the first plant Grx implicated in arsenic metabolism.

  20. Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation.

    PubMed

    Paciello, Ida; Silipo, Alba; Lembo-Fazio, Luigi; Curcurù, Laura; Zumsteg, Anna; Noël, Gaëlle; Ciancarella, Valeria; Sturiale, Luisa; Molinaro, Antonio; Bernardini, Maria Lina

    2013-11-12

    LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.

  1. Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

    PubMed Central

    Paciello, Ida; Silipo, Alba; Lembo-Fazio, Luigi; Curcurù, Laura; Zumsteg, Anna; Noël, Gaëlle; Ciancarella, Valeria; Sturiale, Luisa; Molinaro, Antonio; Bernardini, Maria Lina

    2013-01-01

    LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection. PMID:24167293

  2. Lack of regulation by intracellular Ca2+ of the hyperpolarization-activated cation current in rat thalamic neurones.

    PubMed Central

    Budde, T; Biella, G; Munsch, T; Pape, H C

    1997-01-01

    1. The regulation of the hyperpolarization-activated cation current, Ih, in thalamocortical neurones by intracellular calcium ions has been implemented in a number of mathematical models on the waxing and waning behaviour of synchronized rhythmic activity in thalamocortical circuits. In the present study, the Ca2+ dependence of Ih in thalamocortical neurones was experimentally investigated by combining Ca2+ imaging and patch-clamp techniques in the ventrobasal thalamic complex (VB) in vitro. 2. Properties of Ih were analysed before and during rhythmic stimulation of Ca2+ entry by trains of depolarizing voltage pulses. Despite a significant increase in intracellular Ca2+ concentration ([Ca2+]i) from resting levels of 74 +/- 23 nM to 251 +/- 78 nM upon rhythmic stimulation, significant differences in the voltage dependence of Ih activation did not occur (half-maximal activation at -86.4 +/- 1.3 mV vs. -85.2 +/- 2.9 mV; slope of the activation curve, 11.2 +/- 2.4 mV vs. 12.5 +/- 2.5 mV). Recording of Ih with predefined values of [Ca2+]i (13.2 nM or 10.01 microM in the patch pipette) revealed no significant differences in the activation curve or the fully activated I-V relationship of Ih. 3. In comparison, stimulation of the intracellular cyclic adenosine monophosphate (cAMP) pathway induced a significantly positive shift in Ih voltage dependence of +5.1 +/- 1.9 mV, with no alteration in the fully activated I-V relationship. 4. These data argue against a direct regulation of Ih by intracellular Ca2+, and particularly do not support a primary role of Ca(2+)-dependent modulation of the Ih channels in the waxing and waning of sleep spindle oscillations in thalamocortical neurones. Images Figure 1 PMID:9288676

  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

    2016-01-01

    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

  4. 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

  5. [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.

  6. Intracellular staining and detection of cytokines by fluorescence-activated flow cytometry.

    PubMed

    Freer, Giulia

    2014-01-01

    The detection of cytokines inside cells producing them has made a tremendous impact on the way immune reactivity is measured. Intracellular cytokine staining is the only immunological technique allowing determination of antigen-specific T cell function and phenotype at the same time; for this reason, it is one of the most popular methods to measure antigenicity in the evaluation of vaccine efficacy and in the study of infectious diseases. It is a flow cytometric technique based on staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies after short term culture of stimulated immune cells in the presence of a protein secretion inhibitor, followed by fixation and permeabilization. Most experiments involve detection of five to ten different colors but many more can be detected by modern flow cytometers. Here, we discuss our experience using a standard protocol for intracellular cytokine staining. PMID:24908309

  7. Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

    PubMed Central

    Faletto, M B; Miller, W H; Garvey, E P; St Clair, M H; Daluge, S M; Good, S S

    1997-01-01

    The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity. PMID:9145876

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

    PubMed

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

    2015-12-01

    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 Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-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 Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Sandia Cognitive Runtime Engine with Active Memory

    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 datamore » 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.« less

  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. 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

  16. Simultaneous intracellular β-D-glucosidase and phosphodiesterase I activities measurements based on a triple-signaling fluorescent probe.

    PubMed

    Li, Yinhui; Wang, Hao; Li, Jishan; Zheng, Jing; Xu, Xinhua; Yang, Ronghua

    2011-02-15

    Despite that considerable efforts have been devoted to the design of various fluorogenic enzyme substrates, they are single-enzyme assay approaches that cannot afford detection of multienzyme activity. In this study, we set out our first attempt to design a new probe that could measure intracellular β-D-glucosidase and phosphodiesterase I activities. Unlike the commonly used fluorogenic enzyme substrates that contain one recognition site and signaling reporter, the new probe molecule possesses two cleavage sites, specificly corresponding to β-D-glucosidase and phosphodiesterase I, and three fluorescent reporters, 7-β-D-glucopyranosyloxycoumarin, 7-hydroxycoumarin, and meso-tetraphenylporphyrin. On the basis of intramolecular photoinduced electron transfer and fluorescence resonance energy transfer mechanisms, interaction of the probe with the two enzymes, whether only one or both, produces different signal readouts with high sensitivity. Remarkably, the probe is chemically stable in complex biological fluids. Fluorescence outputs are not significantly affected by biologically related metal ions, anions, amino acids, and proteins. Furthermore, fluorescence microscopy confirmed that the probe is an excellent candidate for intracellular delivery and can be accumulated intensively in cells. We demonstrated the applicability for the simultaneous images of intracellular β-D-glucosidase and phosphodiesterase I activities using the different optical imaging modes.

  17. Comparative activities of new fluoroquinolones, alone or in combination with amoxicillin, trimethoprim-sulfamethoxazole, or rifampin, against intracellular Listeria monocytogenes.

    PubMed Central

    Michelet, C; Avril, J L; Arvieux, C; Jacquelinet, C; Vu, N; Cartier, F

    1997-01-01

    We studied the activities of the new fluoroquinolones clinafloxacin, levofloxacin, ofloxacin, and sparfloxacin alone or in combination on the intracellular growth of Listeria monocytogenes. Against intracellular growth of the four strains tested, a similar reduction of the bacterial count was obtained with clinafloxacin at the dose of 10 x MIC (delta log10 CFU/ml = -2.19 +/- 0.24), with levofloxacin at 8 x MIC (delta log10 CFU/ml = -2.28 +/- 0.25), and with sparfloxacin at 4 x MIC (delta log10 CFU/ml = -2.16 +/- 0.21) after 24 h of incubation. The combination of the quinolones with trimethoprim-sulfamethoxazole or amoxicillin did not show a substantial increase in activity compared to the fluoroquinolone alone. Antagonism with rifampin was strongly suggested. No modification of the MIC was observed after 20 successive infections of HeLa cells and contact with subinhibitory concentrations of clinafloxacin, levofloxacin, and sparfloxacin for 24 h. We conclude that clinafloxacin, levofloxacin, or sparfloxacin could represent a therapeutic alternative to amoxicillin for the treatment of Listeria infections in adults, especially clinafloxacin, whose MIC is low (0.06 to 0.12 micrograms/ml), and whose best activity against intracellular L. monocytogenes was obtained at a concentration of 1.2 micrograms/ml, which is similar to clinically achievable levels. The results must be confirmed in an experimental model. PMID:8980755

  18. Intracellular Ca(2+) mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.

    PubMed

    Werthén, M; Lundgren, T

    2001-03-01

    Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones (AHLs) and a transcription factor, activated by the AHLs. In this study, a possible involvement of intracellular Ca(2+) as second messenger and/or protein kinase activity during signal transduction is analyzed. When N-hexanoyl-l-homoserine lactone was added to a suspension of Fura-2-loaded Serratia liquefaciens, there was a decline in [Ca(2+)](i), measured as a decrease in the Fura-2 fluorescence ratio. As controls, the addition of the signal molecule N-3-oxohexanoyl-l-homoserine lactone, which is not produced by S. liquefaciens, did not induce changes in [Ca(2+)](i). Using a protein kinase activity assay on AHL-stimulated cells, an increase in kinase activity after N-butanoyl-l-homoserine lactone stimulation of S. liquefaciens cells was detected, whereas the kinase activity induced by N-3-oxohexanoyl-l-homoserine lactone was not statistically significant. The conclusion from this study is that changes in [Ca(2+)](i) are involved in quorum sensing signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in S. liquefaciens upon AHL stimulation. We suggest that the transient intracellular [Ca(2+)] changes and kinase activity, activated by the AHL signal, are critical for the quorum-sensing signal transduction.

  19. 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

    2016-01-01

    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 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

  1. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    PubMed

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

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas 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. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA 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.

  2. 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.

  3. 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

  4. Proteomes of host cell membranes modified by intracellular activities of Salmonella enterica.

    PubMed

    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.

  5. Effects of chronic sleep deprivation on autonomic activity by examining heart rate variability, plasma catecholamine, and intracellular magnesium levels.

    PubMed

    Takase, Bonpei; Akima, Takashi; Satomura, Kimio; Ohsuzu, Fumitaka; Mastui, Takemi; Ishihara, Masayuki; Kurita, Akira

    2004-10-01

    Chronic sleep deprivation is associated with cardiovascular events. In addition, autonomic activity determined from the levels of the heart rate variability (HRV), plasma catecholamine, and intracellular magnesium (Mg) are important in the pathophysiology of cardiovascular events. This study therefore aimed to determine the effects of chronic sleep deprivation on autonomic activity by examining the HRV, plasma catecholamine, and intracellular magnesium levels. Thirty (30) healthy male college students ranging in age from 20 to 24 years of age (average 22 +/- 1 years; mean +/- SD) with no coronary risk factors such as hypertension, diabetes mellitus, hyperlipidemia or a family history of premature coronary artery disease (CAD) were included in the study. Over a 4-week period, the volunteers' plasma levels of epinephrine, norepinephrine, and erythrocyte-Mg were measured. The study was made during the 4 weeks before and immediately after college finals exams. HRV, obtained from 24-hour ambulatory ECG monitoring, included time and frequency domain indices. The HRV indices and erythrocyte-Mg decreased while norepinephrine increased during chronic sleep deprivation. It is concluded that chronic sleep deprivation causes an autonomic imbalance and decreases intracellular Mg, which could be associated with chronic sleep deprivation-induced cardiovascular events. PMID:15754837

  6. 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

  7. 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

  8. Mechanisms of intracellular defense and activity of free radical oxidation in rat myocardium in the dynamics of chronic fluorine intoxication.

    PubMed

    Zhukova, A G; Alekhina, D A; Sazontova, T G; Prokop'ev, Yu A; Gorokhova, L G; Stryapko, N V; Mikhailova, N N

    2013-12-01

    The mechanisms of intracellular defense and activity of free radical oxidation in the myocardium were studied in the dynamics of chronic fluorine intoxication. At the early stages of fluorine intoxication (day 3-week 3), the concentrations of defense proteins HIF-1α, HSC73, and HOx-2 and activity of the main metabolic enzymes increased, which promoted maintenance of cardiomyocyte structure and function at the normal physiological level. At late stages of fluorine intoxication (weeks 6 and 9), metabolic changes in the myocardium attest to high strain of the adaptive mechanisms.

  9. 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.

  10. Electrochemical Activation of Engineered Protein Switches

    PubMed Central

    Choi, Jay H.; Zayats, Maya; Searson, Peter C.; Ostermeier, Marc

    2016-01-01

    Engineered protein switches have a large dynamic range, high specificity for the activating ligand, and a modular architecture, and have been explored for a wide range of applications including biosensors and therapeutics. The ability to externally control switch function is important in extending applications for protein switches. We recently demonstrated that the on/off state could be controlled by the redox state of disulfide bonds introduced into the switches at select locations. Here, we demonstrate that an electrochemical signal can be used as an exogenous input to control switch function via reduction of the engineered disulfide bonds. This study suggests that disulfide-containing protein switch is a potentially useful platform for bioelectronic sensors with remote control of the sensing ability. PMID:26241391

  11. 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

  12. 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

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

    PubMed

    Reniere, Michelle L; Whiteley, Aaron T; Portnoy, Daniel A

    2016-07-01

    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. 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

  15. Cell uptake, intracellular distribution, fate and reactive oxygen species generation of polymer brush engineered CeO(2-x) NPs.

    PubMed

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

    2015-04-21

    Cerium Oxide nanoparticles (CeO(2-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). CeO(2-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 CeO(2-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 CeO(2-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 CeO(2-x) NPs. The brush coating does not prevent CeO(2-x) NPs from displaying antioxidant properties.

  16. 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

  17. Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization.

    PubMed

    Carter, Russell E; Seidel, Jessica L; Lindquist, Britta E; Sheline, Christian T; Shuttleworth, C William

    2013-06-01

    Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn(2+) release and intracellular accumulation occurs with SD, and elevated intracellular Zn(2+) ([Zn(2+) ]i ) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn(2+) ]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn(2+) ]i loading prior to SD, by transient ZnCl2 application with the Zn(2+) ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn(2+) ]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn(2+) release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn(2+) ]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn(2+) ]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn(2+) ]i accumulation in a range of brain injury models. PMID:23495967

  18. Intracellular mechanisms coupled to NPY Y2 and Y5 receptor activation and lipid accumulation in murine adipocytes.

    PubMed

    Rosmaninho-Salgado, Joana; Cortez, Vera; Estrada, Marta; Santana, Magda M; Gonçalves, Alexandra; Marques, Ana Patrícia; Cavadas, Cláudia

    2012-12-01

    The formation of adipose tissue is a process that includes the pre-adipocyte proliferation and differentiation to adipocytes that are cells specialized in lipid accumulation. The adipocyte differentiation is a process driven by the coordinated expression of various transcription factors, such as peroxisome proliferator-activated receptor (PPAR-γ). Neuropeptide Y (NPY) induces adipocyte proliferation and differentiation but the NPY receptors and the intracellular pathways involved in these processes are still not clear. In the present work we studied the role of NPY receptors and the intracellular pathways involved in the stimulatory effect of NPY on lipid accumulation. The murine pre-adipocyte cell line, 3T3-L1, was used as a cell model. Adipogenesis was evaluated by quantifying lipid accumulation by Oil red-O assay and by analyzing PPAR-γ expression using the Western blotting assay. Adipocytes were incubated with NPY (100nM) and a decrease on lipid accumulation and PPAR-γ expression was observed in the presence of NPY Y(2) receptor antagonist (BIIE0246, 1μM) or NPY Y(5) antagonist. Furthermore, NPY Y(2) (NPY(3-36), 100nM) or NPY Y(5) (NPY(19-23)(GLY(1), Ser(3), Gln(4), Thr(6), Ala(31), Aib(32), Gln(34)) PP, 100nM) receptor agonists increased lipid accumulation and PPAR-γ expression. We further investigate the intracellular pathways associated with NPY Y(2) and NPY Y(5) receptor activation. Our results show NPY induces PPAR-γ expression and lipid accumulation through NPY Y(2) and NPY Y(5) receptors activation. PKC and PLC inhibitors inhibit lipid accumulation induced by NPY Y(5) receptor agonist. Moreover, our results suggest that lipid accumulation induced by NPY Y(2) receptor activation occurs through PKA, MAPK and PI3K pathways. In conclusion, this study contributes to a step forward on the knowledge of intracellular mechanisms associated with NPY receptors activation on adipocytes and contributes to a better understanding and the development of new

  19. 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

  20. 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. PMID:27303304

  1. Engineering Clostridia Neurotoxins with elevated catalytic activity.

    PubMed

    Guo, Jiubiao; Pan, Xuehua; Zhao, Yanxiang; Chen, Sheng

    2013-11-01

    BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, yet these two toxins showed different efficiency on substrate hydrolysis and had different requirements for the recognition of P2' site of VAMP2, E(78). These differences may be due to their different composition of their substrate recognition pockets in the active site. Swapping of LC/T S1' pocket residue, L(230), with the corresponding isoleucine in LC/B increased LC/T activity by ∼25 fold, while swapping of LC/B S1' pocket residue, S(201), with the corresponding proline in LC/T increased LC/B activity by ∼10 fold. Optimization of both S1 and S1' pocket residues of LC/T, LC/T (K(168)E, L(230)I) elevated LC/T activity by more than 100-fold. The highly active LC/T derivative engineered in this study has the potential to be used as a more effective tool to study mechanisms of exocytosis in central neuron. The LC/B derivative with elevated activity has the potential to be developed into novel therapy to minimize the impact of immunoresistance during BoNT/B therapy.

  2. Rationally Engineering Phototherapy Modules of Eosin-Conjugated Responsive Polymeric Nanocarriers via Intracellular Endocytic pH Gradients.

    PubMed

    Liu, Guhuan; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2015-07-15

    Spatiotemporal switching of respective phototherapy modes at the cellular level with minimum side effects and high therapeutic efficacy is a major challenge for cancer phototherapy. Herein we demonstrate how to address this issue by employing photosensitizer-conjugated pH-responsive block copolymers in combination with intracellular endocytic pH gradients. At neutral pH corresponding to extracellular and cytosol milieu, the copolymers self-assemble into micelles with prominently quenched fluorescence emission and low (1)O2 generation capability, favoring a highly efficient photothermal module. Under mildly acidic pH associated with endolysosomes, protonation-triggered micelle-to-unimer transition results in recovered emission and enhanced photodynamic (1)O2 efficiency, which synergistically actuates release of encapsulated drugs, endosomal escape, and photochemical internalization processes.

  3. Entry of Sanfetrinem into Human Polymorphonuclear Granulocytes and Its Cell-Associated Activity against Intracellular, Penicillin-Resistant Streptococcus pneumoniae

    PubMed Central

    Cuffini, Anna Maria; Tullio, Vivian; Bonino, Alessandro; Allocco, Alessandra; Palarchio, Angela Ianni; Carlone, Nicola A.

    1998-01-01

    The entry of antibiotics into phagocytes is necessary for activity against intracellular pathogens. The ability of sanfetrinem, the first member of a new class of antibiotics, to penetrate human polymorphonuclear granulocytes and its consequences upon subsequent phagocytosis and killing of ingested penicillin-resistant Streptococcus pneumoniae have been evaluated. Sanfetrinem penetrated into human polymorphonuclear leukocytes (PMNs) at all concentrations tested, with cellular concentration/extracellular concentration ratios of 6.6 to 5.03 and 4.21 when sanfetrinem was used at 0.25 to 0.5 and 1 μg/ml, respectively, within 30 min of incubation. The uptake was complete within 5 min and was not energy dependent, since it was not affected by cell viability, environmental temperature, or the addition of a metabolic inhibitor. At a concentration of one-half the MIC, sanfetrinem significantly enhanced human PMN phagocytosis and increased intracellular bactericidal activity against penicillin-resistant S. pneumoniae. Following preexposure of PMNs to a concentration of one-half the MIC of sanfetrinem, there was a significant increase in both phagocytosis and killing compared with that for the controls, indicating the ability of sanfetrinem to interact with biological membranes and remain active within PMNs. Preexposure of streptococci to sanfetrinem made penicillin-resistant S. pneumoniae more susceptible to the bactericidal mechanisms of human PMNs than untreated organisms. PMID:9661015

  4. 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

  5. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron.

    PubMed Central

    Byrd, T F; Horwitz, M A

    1989-01-01

    We have investigated the role of iron in the intracellular biology of Legionella pneumophila in human monocytes and in the effector arm of cell-mediated immune defense against this intracellular bacterial pathogen. To determine if L. pneumophila intracellular multiplication is iron dependent, we studied the effect of the iron chelator deferoxamine on L. pneumophila infection of monocytes. Deferoxamine at 15 microM completely inhibited L. pneumophila intracellular multiplication. The inhibitory effect of deferoxamine was reversed with equimolar iron-saturated transferrin but not apotransferrin. To examine the potential role of iron in monocyte activation, we investigated the influence of iron-saturated transferrin on L. pneumophila multiplication in IFN gamma-activated monocytes. Iron transferrin, but not apotransferrin, neutralized the capacity of activated monocytes to inhibit L. pneumophila multiplication. To explore a potential mechanism by which activated monocytes might limit the availability of intracellular iron, we examined transferrin receptor expression on nonactivated and activated monocytes cultured in vitro for 5 d. By fluorescence-activated flow cytometry, activated monocytes exhibited markedly fewer transferrin receptors than nonactivated monocytes. By Scatchard analysis of 125I-transferrin binding to monocytes, nonactivated monocytes had 38,300 +/- 12,700 (mean +/- SE) transferrin binding sites, whereas activated monocytes had 10,300 +/- 1,600, a reduction of 73%. Activated and nonactivated monocytes had a similar mean Kd (1.8 +/- 0.2 nM). This study demonstrates that (a) L. pneumophila intracellular multiplication is iron dependent; (b) activated monocytes inhibit L. pneumophila multiplication by limiting the availability of intracellular iron; and (c) transferrin receptors are downregulated on IFN gamma-activated monocytes. Images PMID:2496141

  6. Single-cell codetection of metabolic activity, intracellular functional proteins, and genetic mutations from rare circulating tumor cells.

    PubMed

    Zhang, Yu; Tang, Yin; Sun, Shuai; Wang, Zhihua; Wu, Wenjun; Zhao, Xiaodong; Czajkowsky, Daniel M; Li, Yan; Tian, Jianhui; Xu, Ling; Wei, Wei; Deng, Yuliang; Shi, Qihui

    2015-10-01

    The high glucose uptake and activation of oncogenic signaling pathways in cancer cells has long made these features, together with the mutational spectrum, prime diagnostic targets of circulating tumor cells (CTCs). Further, an ability to characterize these properties at a single cell resolution is widely believed to be essential, as the known extensive heterogeneity in CTCs can obscure important correlations in data obtained from cell population-based methods. However, to date, it has not been possible to quantitatively measure metabolic, proteomic, and genetic data from a single CTC. Here we report a microchip-based approach that allows for the codetection of glucose uptake, intracellular functional proteins, and genetic mutations at the single-cell level from rare tumor cells. The microchip contains thousands of nanoliter grooves (nanowells) that isolate individual CTCs and allow for the assessment of their glucose uptake via imaging of a fluorescent glucose analog, quantification of a panel of intracellular signaling proteins using a miniaturized antibody barcode microarray, and retrieval of the individual cell nuclei for subsequent off-chip genome amplification and sequencing. This approach integrates molecular-scale information on the metabolic, proteomic, and genetic status of single cells and permits the inference of associations between genetic signatures, energy consumption, and phosphoproteins oncogenic signaling activities in CTCs isolated from blood samples of patients. Importantly, this microchip chip-based approach achieves this multidimensional molecular analysis with minimal cell loss (<20%), which is the bottleneck of the rare cell analysis. PMID:26378744

  7. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling.

    PubMed

    Seki, Shinsuke; Mazur, Peter

    2008-06-01

    Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below -40 degrees C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158-166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to -25 degrees C, holding them 10 min, rapidly cooling them to -70 degrees C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at -56 degrees C during warming and was complete by -46 degrees C. The present study differs in that the oocytes were warmed rapidly from -70 degrees C to temperatures between -65 and -50 degrees C and held for 3-60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E(a)) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 degrees C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 degrees C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 degrees C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

  8. Extraction and in vitro antioxidant activity of intracellular polysaccharide by Pholiota adiposa SX-02.

    PubMed

    Deng, Peng; Zhang, Guquan; Zhou, Bo; Lin, Rongshan; Jia, Le; Fan, Keming; Liu, Xiaonan; Wang, Guoyi; Wang, Li; Zhang, Jianjun

    2011-01-01

    Response surface methodology (RSM) was used to optimize the extraction parameters for Pholiota adiposa SX-02 intracellular polysaccharide (IPS) produced during submerged culture. The optimum conditions of IPS extraction were predicted to be, ultrasonic power at 564.93 W, precipitation time 30.34 h and pH 8.28, and IPS yield was estimated at 19.75%. The actual value of IPS under these conditions was 20.51%. The in vitro antioxidant results showed that the inhibition effects of IPS at a dosage of 250 mg/l on superoxide anion, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were 74.66 ± 5.31%, 69.20 ± 5.13%, and 75.20 ± 6.73%, respectively, which were 27.51 ± 2.23%, 16.58 ± 1.33%, and 9.46 ± 0.72% higher than that of butylated hydroxytoluene (BHT), respectively. The reducing power of IPS was 0.32 ± 0.02 (absorbance at 700 nm), 39.13 ± 3.47% higher than that of BHT. The results provide a reference for large-scale extraction of IPS by P. adiposa SX-02 in industrial fermentation and the IPS can be used as a potential antioxidant which enhances adaptive immune responses.

  9. 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

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

    PubMed

    Passalacqua, Karla D; Charbonneau, Marie-Eve; Donato, Nicholas J; Showalter, Hollis D; Sun, Duxin; Wen, Bo; He, Miao; Sun, Hanshi; O'Riordan, Mary X D; Wobus, Christiane E

    2016-07-01

    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

  11. 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.

  12. 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

  13. Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells.

    PubMed Central

    Iredale, P. A.; Hill, S. J.

    1993-01-01

    1. Increases in intracellular calcium ([Ca2+]i) were measured in chinese hamster cultured ovary cells (clone, CHO-K1), by use of the fluorescent, calcium-sensitive dye, fura-2. 2. Addition of both ATP and UTP elicited rapid increases in [Ca2+]i due to mobilization from intracellular stores and calcium entry across the plasma membrane. 3. Omission of calcium from the extracellular medium and pre-incubation with the inorganic calcium channel blocker, nickel (Ni2+) prevented the calcium entry components of the responses. 4. Investigation of the concentration-response relationships of various analogues of ATP suggests the presence of a purinoceptor which cannot be characterized as P2X or P2Y. In addition, there appears to be a sub-population of P2Y-purinoceptors which do not cross-react with the 'nucleotide' receptor population. 5. Cross-desensitization and additivity experiments suggest that both ATP and UTP activate the same receptor. 6. Pre-incubation with the tumour-promoting agent, beta-phorbol-12,13 dibutyrate (PDBu), caused a reduction in the increases in [Ca2+]i, suggesting a role for protein kinase C in feedback inhibition of purinoceptor responses in this cell line. 7. In summary, we present evidence for the existence of an endogenous P2U-purinoceptor (or 'nucleotide receptor') which is linked to increases in [Ca2+]i in CHO-K1 cells. PMID:8306069

  14. Monitoring intracellular calcium in response to GPCR activation using thin-film silicon photodiodes with integrated fluorescence filters.

    PubMed

    Martins, S A M; Moulas, G; Trabuco, J R C; Monteiro, G A; Chu, V; Conde, J P; Prazeres, D M F

    2014-02-15

    G-protein coupled receptor (GPCRs) drug discovery is a thriving strategy in the pharmaceutical industry. The standard approach uses living cells to test millions of compounds in a high-throughput format. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluorescence or luminescence read-out systems, which require external equipment for signal acquisition. In this work, thin-film amorphous silicon photodiodes with an integrated fluorescence filter were developed to capture the intracellular calcium dynamics in response to the activation of the endogenous muscarinic M1 GPCR of HEK 293T cells. Using the new device it was possible to characterize the potency of carbachol (EC50=10.5 µM) and pirenzepine (IC50=4.2 μM), with the same accuracy as standard microscopy optical systems. The smaller foot-print provided by the detection system makes it an ideal candidate for the future integration in microfluidic devices for drug discovery. PMID:24055937

  15. Relationship of free intracellular calcium to the cytolytic activity of Entamoeba histolytica.

    PubMed

    Ravdin, J I; Moreau, F; Sullivan, J A; Petri, W A; Mandell, G L

    1988-06-01

    Entamoeba histolytica adherence and destruction of host cells is required for in vivo pathogenicity; amebic in vitro adherence is mediated by a galactose- or N-acetyl-D-galactosamine-inhibitable surface lectin (Gal/GalNAc adherence lectin). Free intracellular Ca2+ concentration [( Ca2+]i) was measured in living amebae and target cells during amebic cytolysis of Chinese hamster ovary (CHO) cells and human polymorphonuclear neutrophils by utilizing the Ca2+ probe Fura-2 and computer-enhanced digitized microscopy. Motile E. histolytica trophozoites had oscillatory increases in [Ca2+]i in head or tail regions; however, there was no increase in regional or total amebic [Ca2+]i upon contact with a target CHO cell. Target CHO cells and polymorphonuclear neutrophils demonstrated marked irreversible increases in [Ca2+]i within 30 to 300 s following contact by an ameba (P less than 0.01); increased [Ca2+]i preceded the occurrence of nonspecific surface membrane permeability and death of the target cell. Target CHO cells contiguous on a monolayer to a cell contacted by an ameba experienced a rapid but reversible rise in [Ca2+]i (P less than 0.01) and were not killed. Galactose (40 mg/ml) totally abrogated the rise in target CHO cell [Ca2+]i that followed contact by amebae (P less than 0.01); immunoaffinity-purified amebic Gal/GalNAc adherence lectin (0.25 micrograms/ml) induced a rapid and reversible rise in CHO cell [Ca2+]i (P less than 0.01) which was inhibited by galactose. Amebic [Ca2+]i was not elevated following parasite adherence to target cells; a rapid and substantial rise in target cell [Ca2+]i occurred which was mediated, at least in part, by the Gal/GalNAc adherence lectin of the parasite and led to the death of target cells. PMID:2897335

  16. 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

  17. 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

  18. Platelet Activating Factor Enhances Synaptic Vesicle Exocytosis Via PKC, Elevated Intracellular Calcium, and Modulation of Synapsin 1 Dynamics and Phosphorylation

    PubMed Central

    Hammond, Jennetta W.; Lu, Shao-Ming; Gelbard, Harris A.

    2016-01-01

    Platelet activating factor (PAF) is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analog of PAF (cPAF) enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity. PMID:26778968

  19. Smart vesicle kit for in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe.

    PubMed

    Qian, Ruocan; Ding, Lin; Yan, Liwen; Lin, Manfei; Ju, Huangxian

    2014-09-01

    A smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3' end, which was just complementary with the loop of MB assembled on probe surface. Thus, the MB was opened upon hybridization to switch the fluorescent state from "off" to "on". The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of the cytoplasmic telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2 × 10(-11), 2.4 × 10(-11), and 8.6 × 10(-13) IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells. The proposed method could be employed for dynamic monitoring of the cytoplasmic telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs.

  20. Intracellular and Extracellular Carbonic Anhydrases Cooperate Non-enzymatically to Enhance Activity of Monocarboxylate Transporters*

    PubMed Central

    Klier, Michael; Andes, Fabian T.; Deitmer, Joachim W.; Becker, Holger M.

    2014-01-01

    Proton-coupled monocarboxylate transporters (MCTs) are carriers of high-energy metabolites such as lactate, pyruvate, and ketone bodies and are expressed in most tissues. It has previously been shown that transport activity of MCT1 and MCT4 is enhanced by the cytosolic carbonic anhydrase II (CAII) independent of its catalytic activity. We have now studied the influence of the extracellular, membrane-bound CAIV on transport activity of MCT1/4, heterologously expressed in Xenopus oocytes. Coexpression of CAIV with MCT1 and MCT4 resulted in a significant increase in MCT transport activity, even in the nominal absence of CO2/HCO3−. CAIV-mediated augmentation of MCT activity was independent of the CAIV catalytic function, since application of the CA-inhibitor ethoxyzolamide or coexpression of the catalytically inactive mutant CAIV-V165Y did not suppress CAIV-mediated augmentation of MCT transport activity. The interaction required CAIV at the extracellular surface, since injection of CAIV protein into the oocyte cytosol did not augment MCT transport function. The effects of cytosolic CAII (injected as protein) and extracellular CAIV (expressed) on MCT transport activity, were additive. Our results suggest that intra- and extracellular carbonic anhydrases can work in concert to ensure rapid shuttling of metabolites across the cell membrane. PMID:24338019

  1. Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

    PubMed

    Ypey, D L; Weidema, A F; Höld, K M; Van der Laarse, A; Ravesloot, J H; Van Der Plas, A; Nijweide, P J

    1992-12-01

    Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone

  2. 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

  3. Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity.

    PubMed

    Kloster, Antonina; Olsen, Lars Folke

    2012-05-01

    We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations.

  4. The role of reduced intracellular concentrations of active drugs in the lack of response to anticancer chemotherapy

    PubMed Central

    Marin, Jose JG; Monte, Maria J; Blazquez, Alba G; Macias, Rocio IR; Serrano, Maria A; Briz, Oscar

    2014-01-01

    A major difficulty in the treatment of cancers is the poor response of many tumors to pharmacological regimens. This situation can be accounted for by the existence of a variety of complex mechanisms of chemoresistance (MOCs), leading to reduced intracellular concentrations of active agents, changes in the molecular targets of the drugs, enhanced repair of drug-induced modifications in macromolecules, stimulation of anti-apoptotic mechanisms, and inhibition of pro-apoptotic mechanisms. The present review focuses on alterations in the expression and appearance of the genetic variants that affect the genes involved in reducing the amount of active agents inside tumor cells. These alterations can occur through two mechanisms: either by lowering uptake or enhancing efflux (so-called MOC-1a and MOC-1b, respectively), or by decreasing the activation of prodrugs or enhancing inactivation of active agents through their biotransformation (MOC-2). The development of chemosensitizers that are useful in implementing the pharmacological manipulation of these processes constitutes a challenge to modern pharmacology. Nevertheless, the important physiological roles of the most relevant genes involved in MOC-1a, MOC-1b, and MOC-2 make it difficult to prevent the side effects of chemosensitizers. A more attainable goal in this area of pharmacological enquiry is the identification of proteomic profiles that will permit oncologists to accurately predict a lack of response to a given regimen, which would be useful for adapting treatment to the personal situation of each patient. PMID:24317012

  5. Trypanocidal activity and selectivity in vitro of aromatic amidine compounds upon bloodstream and intracellular forms of Trypanosoma cruzi.

    PubMed

    De Souza, E M; da Silva, P B; Nefertiti, A S G; Ismail, M A; Arafa, R K; Tao, B; Nixon-Smith, C K; Boykin, D W; Soeiro, M N C

    2011-02-01

    Trypanosoma cruzi is the etiological agent of Chagas disease, an important neglected illness affecting about 12-14 million people in endemic areas of Latin America. The chemotherapy of Chagas disease is quite unsatisfactory mainly due to its poor efficacy especially during the later chronic phase and the considerable well-known side effects. These facts emphasize the need to search for find new drugs. Diamidines and related compounds are minor groove binders of DNA at AT-rich sites and present excellent anti-trypanosomal activity. In the present study, six novel aromatic amidine compounds (arylimidamides and diamidines) were tested in vitro to determine activity against the infective and intracellular stages of T. cruzi, which are responsible for sustaining the infection in the mammalian hosts. In addition, their selectivity and toxicity towards primary cultures of cardiomyocyte were evaluated since these cells represent important targets of infection and inflammation in vivo. The aromatic amidines were active against T. cruzi in vitro, the arylimidamide DB1470 was the most effective compound presenting a submicromolar LD(50) values, good selectivity index, and good activity at 4 °C in the presence of blood constituents. Our results further justify trypanocidal screening assays with these classes of compounds both in vitro and in vivo in experimental models of T. cruzi infection.

  6. 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

    2015-10-01

    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.

  7. 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

  8. Ethnic differences in pre-adipocyte intracellular lipid accumulation and alkaline phosphatase activity.

    PubMed

    Ali, Aus T; Chirambo, George; Penny, Clement; Paiker, Janice E; Ikram, Faisel; Psaras, George; Crowther, Nigel J

    2015-01-01

    Alkaline phosphatase (ALP) increases lipid accumulation in human pre-adipocytes. This study was performed to assess whether ethnic differences in the prevalence of obesity in African and European females are related to differences in pre-adipocyte lipid accretion and ALP activity. Pre-adipocytes were isolated from 13 black and 14 white females. Adipogenesis was quantified using the lipid dye, Oil red O, whilst ALP activity was assayed in cell extracts on day zero and 12days after initiating adipogenesis. Lipid levels (OD units/mg protein) were lower in pre-adipocytes from white than black females on day 0 (0.36±0.05 versus 0.44±0.03, respectively; p<0.0005) and day 12 (1.18±0.14 versus 1.80±0.22, respectively; p<0.0005), as was ALP activity (mU/mg protein) on day zero (36.5±5.8 versus 136.4±10.9, respectively; p<0.0005) and day 12 (127±16 versus 278±27, respectively; p<0.0005). Treatment of pre-adipocytes with histidine, an ALP inhibitor, blocked lipid accumulation. Thus, lipid uptake is higher in pre-adipocytes isolated from black compared to white females which parallels the obesity prevalence rates in these population groups. The reason for higher fat accumulation in pre-adipocytes isolated from black females may be related to higher ALP activity.

  9. Activated protein kinase C binds to intracellular receptors in rat hepatocytes.

    PubMed Central

    Robles-Flores, M; García-Sáinz, J A

    1993-01-01

    The aim of this study was to identify in rat hepatocytes cellular polypeptides that bind protein kinase C (PKC) and may influence its activity and its compartmentation. At least seven proteins, with apparent M(r) values between 12,000 and 36,000, that behave like Receptors for Activated C-Kinase (RACKs) were found in the Triton-X-100-insoluble fraction of these cells; i.e. PKC bound to these polypeptides when it was in its active form. RACKS seem to be PKC substrates. Studies using isotype-specific PKC antibodies suggested some selectivity of RACKs, i.e. RACKs in the M(r) approximately 28,000-36,000 region bound PKC-alpha and PKC-beta in the presence of phosphatidylserine, diolein and Ca2+, whereas those of M(r) approximately 12,000-14,000 bound all isoforms studied, and, in contrast with the other RACKs, they did this even in the absence of Ca2+. Peptide I (KGDYEKILVALCGGN), which has a sequence suggested to be involved in the PKC-RACKs interaction [Mochly-Rosen, Khaner, Lopez and Smith (1991) J. Biol. Chem. 266, 14866-14868], inhibited PKC activity. Preincubation of RACKs with antisera directed against peptide I prevented PKC binding to them. The data suggest that peptide I blocks PKC binding to RACKs by two mechanisms: inhibition of PKC activity and competition with a putative binding site. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8257439

  10. 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…

  11. 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

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

    2016-09-30

    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.

  12. 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

  13. Mathematical modeling of the intracellular protein dynamics: the importance of active transport along microtubules.

    PubMed

    Szymańska, Zuzanna; Parisot, Martin; Lachowicz, Mirosław

    2014-12-21

    In this paper we propose a mathematical model of protein and mRNA transport inside a cell. The spatio-temporal model takes into account the active transport along microtubules in the cytoplasm as well as diffusion and is able to reproduce the oscillatory changes in protein concentration observed in many experimental data. In the model the protein and the mRNA interact with each other that allows us to classify the model as a simple gene regulatory network. The proposed model is generic and may be adapted to specific signaling pathways. On the basis of numerical simulations, we formulate a new hypothesis that the oscillatory dynamics is allowed by the mRNA active transport along microtubules from the nucleus to distant locations.

  14. Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase**

    PubMed Central

    Pizzirani, Daniela; Bach, Anders; Realini, Natalia; Armirotti, Andrea; Mengatto, Luisa; Bauer, Inga; Girotto, Stefania; Pagliuca, Chiara; De Vivo, Marco; Summa, Maria; Ribeiro, Alison; Piomelli, Daniele

    2015-01-01

    The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents. PMID:25395373

  15. Sustained Axenic Metabolic Activity by the Obligate Intracellular Bacterium Coxiella burnetii▿ †

    PubMed Central

    Omsland, Anders; Cockrell, Diane C.; Fischer, Elizabeth R.; Heinzen, Robert A.

    2008-01-01

    Growth of Coxiella burnetii, the agent of Q fever, is strictly limited to colonization of a viable eukaryotic host cell. Following infection, the pathogen replicates exclusively in an acidified (pH 4.5 to 5) phagolysosome-like parasitophorous vacuole. Axenic (host cell free) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short-lived, with bacterial protein synthesis halting after a few hours. Here, we describe a complex axenic medium that supports sustained (>24 h) C. burnetii metabolic activity. As an initial step in medium development, several biological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness. Based on [35S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citrate buffer. To compensate for C. burnetii auxotrophies and other potential metabolic deficiencies, we developed a citrate buffer-based medium termed complex Coxiella medium (CCM) that contains a mixture of three complex nutrient sources (neopeptone, fetal bovine serum, and RPMI cell culture medium). Optimal C. burnetii metabolism occurred in CCM with a high chloride concentration (140 mM) while the concentrations of sodium and potassium had little effect on metabolism. CCM supported prolonged de novo protein and ATP synthesis by C. burnetii (>24 h). Moreover, C. burnetii morphological differentiation was induced in CCM as determined by the transition from small-cell variant to large-cell variant. The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investigate the physiology of this organism including developmental transitions and responses to antimicrobial factors associated with the host cell. PMID:18310349

  16. 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.

  17. Nicotine enhances murine airway contractile responses to kinin receptor agonists via activation of JNK- and PDE4-related intracellular pathways

    PubMed Central

    2010-01-01

    Background Nicotine plays an important role in cigarette-smoke-associated airway disease. The present study was designed to examine if nicotine could induce airway hyperresponsiveness through kinin receptors, and if so, explore the underlying mechanisms involved. Methods Murine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in presence of nicotine (1 and 10 μM) or vehicle (DMSO). Contractile responses induced by kinin B1 receptor agonist, des-Arg9-bradykinin, and B2 receptor agonist, bradykinin, were monitored with myographs. The B1 and B2 receptor mRNA expressions were semi-quantified using real-time PCR and their corresponding protein expressions assessed with confocal-microscopy-based immunohistochemistry. Various pharmacological inhibitors were used for studying intracellular signaling pathways. Results Four days of organ culture with nicotine concentration-dependently increased kinin B1 and B2 receptor-mediated airway contractions, without altering the kinin receptor-mediated relaxations. No such increase was seen at day 1 or day 2. The airway contractile responses to 5-HT, acetylcholine and endothelin receptor agonists remained unaffected by nicotine. Two different neuronal nicotinic receptor antagonists MG624 and hexamethonium blocked the nicotine-induced effects. The enhanced contractile responses were accompanied by increased mRNA and protein expression for both kinin receptors, suggesting the involvement of transcriptional mechanisms. Confocal-microscopy-based immunohistochemistry showed that 4 days of nicotine treatment induced activation (phosphorylation) of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38. Inhibition of JNK with its specific inhibitor SP600125 abolished the nicotine-induced effects on kinin receptor-mediated contractions and reverted the enhanced receptor mRNA expression. Administration of phosphodiesterase inhibitors (YM976 and theophylline

  18. 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

  19. Modeling and analysis of early events in T-lymphocyte antigen-activated intracellular-signaling pathways

    NASA Astrophysics Data System (ADS)

    Zheng, Yanan; Balakrishnan, Venkataramanan; Buzzard, Greg; Geahlen, Robert; Harrison, Marietta; Rundell, Ann

    2005-12-01

    The T-cell antigen-activated signaling pathway is a highly regulated intracellular biochemical system that is crucial for initiating an appropriate adaptive immune response. To improve the understanding of the complex regulatory mechanisms controlling the early events in T-cell signaling, a detailed mathematical model was developed that utilizes ordinary differential equations to describe chemical reactions of the signaling pathway. The model parameter values were constrained by experimental data on the activation of a specific signaling intermediate and indicated an initial rapid cascade of phosphorylation events followed by a comparatively slow signal downregulation. Nonlinear analysis of the model suggested that thresholding and bistability occur as a result of the embedded positive and negative feedback loops within the model. These nonlinear system properties may enhance the T-cell receptor specificity and provide sub-threshold noise filtering with switch-like behavior to ensure proper cell response. Additional analysis using a reduced second-order model led to further understanding of the observed system behavior. Moreover, the interactions between the positive and negative feedback loops enabled the model to exhibit, among a variety of other feasible dynamics, a sustained oscillation that corresponds to a stable limit cycle in the two-dimensional phase plane. Quantitative analysis in this paper has helped identify potential regulatory mechanisms in the early T-cell signaling events. This integrated approach provides a framework to quantify and discover the ensemble of interconnected T-cell antigen-activated signaling pathways from limited experimental data.

  20. Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth.

    PubMed

    Giannoni, Elisa; Buricchi, Francesca; Raugei, Giovanni; Ramponi, Giampietro; Chiarugi, Paola

    2005-08-01

    Src tyrosine kinases are central components of adhesive responses and are required for cell spreading onto the extracellular matrix. Among other intracellular messengers elicited by integrin ligation are reactive oxygen species, which act as synergistic mediators of cytoskeleton rearrangement and cell spreading. We report that after integrin ligation, the tyrosine kinase Src is oxidized and activated. Src displays an early activation phase, concurrent with focal adhesion formation and driven mainly by Tyr527 dephosphorylation, and a late phase, concomitant with reactive oxygen species production, cell spreading, and integrin-elicited kinase oxidation. In addition, our results suggest that reactive oxygen species are key mediators of in vitro and in vivo v-Src tumorigenic properties, as both antioxidant treatments and the oxidant-insensitive C245A and C487A Src mutants greatly decrease invasivity, serum-independent and anchorage-independent growth, and tumor onset. Therefore we propose that, in addition to the known phosphorylation/dephosphorylation circuitry, redox regulation of Src activity is required during both cell attachment to the extracellular matrix and tumorigenesis.

  1. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    PubMed

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  2. 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.

  3. A computational model of adipose tissue metabolism: Evidence for intracellular compartmentation and differential activation of lipases

    PubMed Central

    Kim, Jaeyeon; Saidel, Gerald M.; Kalhan, Satish C.

    2008-01-01

    Regulation of lipolysis in adipose tissue is critical to whole body fuel homeostasis and to the development of insulin resistance. Due to the challenging nature of laboratory investigations of regulatory mechanisms in adipose tissue, mathematical models could provide a valuable adjunct to such experimental work. We have developed a computational model to analyze key components of adipose tissue metabolism in vivo in human in the fasting state. The various key components included triglyceride-fatty acid cycling, regulation of lipolytic reactions, and glyceroneogenesis. The model, consisting of spatially lumped blood and cellular compartments, included essential transport processes and biochemical reactions. Concentration dynamics for major substrates were described by mass balance equations. Model equations were solved numerically to simulate dynamic responses to intravenous epinephrine infusion. Model simulations were compared with the corresponding experimental measurements of the arteriovenous difference across the abdominal subcutaneous fat bed in humans. The model can simulate physiological responses arising from the different expression levels of lipases. Key findings of this study are as follows: (1) Distinguishing the active metabolic subdomain (~3% of total tissue volume) is critical for simulating data. (2) During epinephrine infusion, lipases are differentially activated such that diglyceride breakdown is ~4 times faster than triglyceride breakdown. (3) Glyceroneogenesis contributes more to glycerol-3-phosphate synthesis during epinephrine infusion when pyruvate oxidation is inhibited by a high acetyl-CoA/free-CoA ratio. PMID:18234232

  4. Cationic Polyrotaxanes as a Feasible Framework for the Intracellular Delivery and Sustainable Activity of Anionic Enzymes: A Comparison Study with Methacrylate-Based Polycations.

    PubMed

    Tamura, Atsushi; Ikeda, Go; Nishida, Kei; Yui, Nobuhiko

    2015-08-01

    We have developed cationic polyrotaxanes composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (α-CDs) threaded along a poly(ethylene glycol) (PEG) chain capped with a terminal bulky stopper (DMAE-PRX) for the intracellular delivery of proteins through the polyelectrolyte complexation. Herein, to ascertain the effect of supramolecular backbone structure of cationic polyrotaxanes, the physicochemical properties and biological activity of polyelectrolyte complex with anionic β-galactosidase (β-gal) were investigated in comparison to a cationic linear polymer, poly[2-(N,N-dimethylaminoethyl) methacrylate] (PDMAEMA). In the cellular experiments, the DMAE-PRX/β-gal complexes exhibited higher intracellular uptake of β-gal and sustainable enzymatic activity of delivered β-gal than the PDMAEMA/β-gal complexes. It is considered that the cationic polyrotaxanes are promising supramolecular backbone structure for the intracellular protein delivery.

  5. Effect of dialyser membranes on extracellular and intracellular granulocyte and monocyte activation in ex vivo pyrogen-free conditions.

    PubMed

    Mahiout, A; Courtney, J M

    1994-10-01

    This study examined effects of blood-contacting materials on the monocyte reaction following the first contact of human blood with hollow fibre dialyser membranes under pyrogen-free conditions. Membrane materials were the unchanged regenerated cellulose, the synthetic polysulphone (PS), a positively charged diethylaminoethyl cellulose (DEAE-C), the negatively charged carboxymethyl cellulose (CMC) and acrylonitrile copolymer (AN). The experimental system involved perfusion with human fresh venous blood through different modules containing the materials in the form of hollow fibre membranes. Extracellular and intracellular aspects of blood reactions after the first contact with the materials were investigated in Ficoll-separated granulocytes and peripheral blood mononuclear cells. Investigations were done by release reactions of platelet activating factor (PAF), oxygen radical (O2-), leukotriene B4, prostaglandin E2 (PGE2) and cytokines (IL-1 beta, TNF-alpha, IL-6). The intracellular activation of peripheral blood mononuclear cells was done by mRNA transcription of IL-1 beta, TNF-alpha, IL-6, IL-8 and beta 2-microglobulin (beta 2-MG). From the set of parameters, release reactions were only measurable for PAF, PGE2 and O2- if a second stimulus (phorbol myristate acetate, lipopolysaccharide, zymosan and calcium ionophore) was used after blood-membrane interaction. Although the extent of the release reaction was weak, negatively charged membranes were, in general, more active. All dialysers exhibited the same increase in beta 2-MG mRNA transcription, suggesting that all blood-contacting membranes initiate the gene expression of beta 2-MG at the same level. TNF-alpha, IL-6, IL-1 beta and IL-8 mRNAs were demonstrated in the AN and CMC membranes rather than the other materials, which exhibit a lower transcription than the tubing set. As has been found, an enhanced generation of PGE2 for both CMC and AN membranes supports, therefore, the concept of an effect of the negative

  6. The antihyperlipidemic activities of enzymatic and acidic intracellular polysaccharides by Termitomyces albuminosus.

    PubMed

    Zhao, Huajie; Li, Shangshang; Zhang, Jianjun; Che, Gen; Zhou, Meng; Liu, Min; Zhang, Chen; Xu, Nuo; Lin, Lin; Liu, Yu; Jia, Le

    2016-10-20

    Two polysaccharides, EIPS and AIPS were obtained by the hydrolysis of IPS from Termitomyces albuminosus, and their pharmacological effects on blood lipid profiles metabolism and oxidative stress were investigated. The results demonstrated that EIPS was superior to IPS and AIPS on reducing hepatic lipid levels and preventing oxidative stress by improving serum enzyme activities (ALT, AST, and ALP), serum lipid levels (TC, TG, HDL-C, LDL-C and VLDL-C), hepatic lipid levels (TC and TG), and antioxidant status (SOD, GSH-Px, CAT, T-AOC, MDA, and LPO). These conclusions indicated that EIPS, AIPS and IPS might be suitable for functional foods and natural drugs on preventing the high-fat emulsion-induced hyperlipidemia. In addition, the monosaccharide compositions of IPS and its hydrolyzate were also processed. PMID:27474674

  7. Intracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

    PubMed Central

    Decleva, Eva; Menegazzi, Renzo; Fasolo, Alba; Defendi, Federica; Sebastianutto, Michele; Dri, Pietro

    2013-01-01

    Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn2+, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2− and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn2+ results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2− generated within gp91phox in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux. PMID:23578765

  8. 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. PMID:18708250

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Light- and pH-activated intracellular drug release from polymeric mesoporous silica nanoparticles.

    PubMed

    Tian, Ye; Kong, Yi; Li, Xiaojian; Wu, Jun; Ko, Alex C-T; Xing, Malcolm

    2015-10-01

    Surface modified mesoporous silica nanoparticles (MSNs) with reduced toxicity were prepared for light and pH dual triggerable drug delivery system. Both 413 nm light and acidic environment can activate the drug release process, improving the pharmacological action. By applying rhodamine B (RhB) as a model drug, the accumulative RhB release is as high as 95% in pH 5.0 and in irradiation of 413 nm light, compared to only 55% in pH 7.4 and in dark. The anti-cancer drug camptothecin (CPT) loaded nanoparticles can kill cancer cells with IC₅₀ value of 0.02 μg mL(-1) in exposure of 413 nm light, which is much lower than free CPT (about 0.1 μg mL(-1)). Multimodal nonlinear optical imaging microscopy (NLOM) was employed to acquire in vitro coherent anti-Stokes Raman (CARS) and two-photon excited fluorescence (TPEF) images of live MCF-7 cells and showed that the nanoparticles can be taken up by breast tumor cell MCF-7 with high efficiency, indicating its great potential for anti-cancer drug delivery system. PMID:26188470

  14. Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

    PubMed

    Rolón-Reyes, Kimberleve; Kucheryavykh, Yuriy V; Cubano, Luis A; Inyushin, Mikhail; Skatchkov, Serguei N; Eaton, Misty J; Harrison, Jeffrey K; Kucheryavykh, Lilia Y

    2015-01-01

    Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells. PMID:26098895

  15. Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway

    PubMed Central

    Rolón-Reyes, Kimberleve; Kucheryavykh, Yuriy V.; Cubano, Luis A.; Inyushin, Mikhail; Skatchkov, Serguei N.; Eaton, Misty J.; Harrison, Jeffrey K.; Kucheryavykh, Lilia Y.

    2015-01-01

    Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells. PMID:26098895

  16. 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. PMID:23650602

  17. Intracellular proton-mediated activation of TRPV3 channels accounts for the exfoliation effect of α-hydroxyl acids on keratinocytes.

    PubMed

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, Kewei

    2012-07-27

    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.

  18. Activation of α7 nicotinic acetylcholine receptors increases intracellular cAMP levels via activation of AC1 in hippocampal neurons

    PubMed Central

    Cheng, Qing; Yakel, Jerrel L.

    2015-01-01

    The activation of α7 nAChRs has been shown to improve hippocampal-dependent learning and memory. However, the molecular mechanism of α7 nAChRs’ action remains elusive. We previously reported that activation of α7 nAChRs induced a prolonged enhancement of glutamatergic synaptic transmission in a PKA-dependent manner. Here, we investigated any connection between the activation of the α7 nAChR and cAMP signaling in hippocampal neurons. To address this question, we employed a FRET-based biosensor to measure the intracellular cAMP levels directly via live cell imaging. We found that application of the α7 nAChR-selective agonist choline, in the presence of the α7 nAChR positive allosteric modulator PNU-120596, induced a significant change in emission ratio of F535/F470, which indicated an increase in intracellular cAMP levels. This choline-induced increase was abolished by the α7 nAChR antagonist MLA and the calcium chelator BAPTA, suggesting that the cAMP increase depends on the α7 nAChR activation and subsequent intracellular calcium rise. The selective AC1 inhibitor CB-6673567 and siRNA-mediated deletion of AC1 both blocked the choline-induced cAMP increase, suggesting that calcium-dependent AC1 is required for choline’s action. Furthermore, α7 nAChR activation stimulated the phosphorylation of synapsin, which serves as a downstream effector to regulate neurotransmitter release. Our findings provide the first direct evidence to link activation of α7 nAChRs to a cAMP rise via AC1, which defines a new signaling pathway employed by α7 nAChRs. Our study sheds light into potential molecular mechanisms of the positive cognitive actions of α7 nAChR agonists and development of therapeutic treatments for cognitive impairments. PMID:25937212

  19. 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.

  20. 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.

  1. 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.

  2. 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. PMID:27480055

  3. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  4. Sensitization of H2O2-induced TRPM2 activation and subsequent interleukin-8 (CXCL8) production by intracellular Fe(2+) in human monocytic U937 cells.

    PubMed

    Shimizu, Shunichi; Yonezawa, Ryo; Negoro, Takaharu; Yamamoto, Shinichiro; Numata, Tomohiro; Ishii, Masakazu; Mori, Yasuo; Toda, Takahiro

    2015-11-01

    Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca(2+)-permeable channel. In monocytes/macrophages, H2O2-induced TRPM2 activation causes cell death and/or production of chemokines that aggravate inflammatory diseases. However, relatively high concentrations of H2O2 are required for activation of TRPM2 channels in vitro. Thus, in the present study, factors that sensitize TRPM2 channels to H2O2 were identified and subsequent physiological responses were examined in U937 human monocytes. Temperature increase from 30°C to 37°C enhanced H2O2-induced TRPM2-mediated increase in intracellular free Ca(2+) ([Ca(2+)]i) in TRPM2-expressing HEK 293 cells (TRPM2/HEK cells). The H2O2-induced TRPM2 activation enhanced by the higher temperature was dramatically sensitized by intracellular Fe(2+)-accumulation following pretreatment with FeSO4. Thus intracellular Fe(2+)-accumulation sensitizes H2O2-induced TRPM2 activation at around body temperature. Moreover, intracellular Fe(2+)-accumulation increased poly(ADP-ribose) levels in nuclei by H2O2 treatment, and the sensitization of H2O2-induced TRPM2 activation were almost completely blocked by poly(ADP-ribose) polymerase inhibitors, suggesting that intracellular Fe(2+)-accumulation enhances H2O2-induced TRPM2 activation by increase of ADP-ribose production through poly(ADP-ribose) polymerase pathway. Similarly, pretreatment with FeSO4 stimulated H2O2-induced TRPM2 activation at 37°C in U937 cells and enhanced H2O2-induced ERK phosphorylation and interleukin-8 (CXCL8) production. Although the addition of H2O2 to cells under conditions of intracellular Fe(2+)-accumulation caused cell death, concentration of H2O2 required for CXCL8 production was lower than that resulting in cell death. These results indicate that intracellular Fe(2+)-accumulation sensitizes TRPM2 channels to H2O2 and subsequently produces CXCL8 at around body temperature. It is possible that sensitization of H2O2-induced TRPM2

  5. 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. PMID:25757566

  6. Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation.

    PubMed

    Matias, Andreza C; Manieri, Tânia M; Cipriano, Samantha S; Carioni, Vivian M O; Nomura, Cassiana S; Machado, Camila M L; Cerchiaro, Giselle

    2013-02-01

    Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions. PMID:22951949

  7. 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.

  8. Isolation of Microarray-Quality RNA from Primary Human Cells after Intracellular Immunostaining and Fluorescence-Activated Cell Sorting

    PubMed Central

    Iglesias-Ussel, Maria; Marchionni, Luigi; Romerio, Fabio

    2013-01-01

    Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that – although the RNA isolated from these samples presented some degradation – it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis. PMID:23434645

  9. 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.

  10. Nesfatin-1 increases intracellular calcium concentration by protein kinase C activation in cultured rat dorsal root ganglion neurons.

    PubMed

    Ozcan, Mete; Gok, Zeynep Betul; Kacar, Emine; Serhatlioglu, Ihsan; Kelestimur, Haluk

    2016-04-21

    Nesfatin-1 is a recently identified anorexigenic hypothalamic polypeptide derived from the posttranslational processing of nucleobindin 2 (NUCB2). Several studies have indicated that this neuropeptide may be participated in somatosensory and visceral transmission including pain signals in addition to energy metabolism. The aim of this study was to explore the possible role of nesfatin-1 in the transmission of peripheral neural signals by investigating the effects of nesfatin-1 on intracellular free calcium levels ([Ca(2+)]i) in cultured neonatal rat dorsal root ganglion (DRG) neurons. The effects of nesfatin-1 on [Ca(2+)]i in DRG neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1-or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of nesfatin-1 on [Ca(2+)]i and role of the protein kinase C (PKC)-mediated pathway in nesfatin-1 effect were assessed. Nesfatin-1 elevated [Ca(2+)]i in cultured DRG neurons. The response was prevented by pretreating the cells with pertussis toxin. The protein kinase C inhibitor chelerythrine chloride suppressed nesfatin-1-induced rise in [Ca(2+)]i. The result shows that nesfatin-1 interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)]i, which is linked to protein kinase C activation in cultured rat DRG neurons.

  11. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

    PubMed

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael

    2004-07-01

    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

  12. Glucose enhances insulin promoter activity in MIN6 beta-cells independently of changes in intracellular Ca2+ concentration and insulin secretion.

    PubMed Central

    Kennedy, H J; Rafiq, I; Pouli, A E; Rutter, G A

    1999-01-01

    Recent studies have suggested that glucose may activate insulin gene transcription through increases in intracellular Ca(2+) concentration, possibly acting via the release of stored insulin. We have investigated this question by dynamic photon-counting imaging of insulin- and c-fos-promoter-firefly luciferase reporter construct activity. Normalized to constitutive viral promoter activity, insulin promoter activity in MIN6 beta-cells was increased 1.6-fold after incubation at 30 mM compared with 3 mM glucose, but was unaltered at either glucose concentration by the presence of insulin (100 nM) or the Ca(2+) channel inhibitor, verapamil (100 microM). Increases in intracellular [Ca(2+)] achieved by plasma membrane depolarization with KCl failed to enhance either insulin or c-fos promoter activity in MIN6 cells, but increased c-fos promoter activity 5-fold in AtT20 cells. Together, these results demonstrate that glucose can exert a direct effect on insulin promoter activity in islet beta-cells, via a signalling pathway which does not require increases in intracellular [Ca(2+)] nor insulin release and insulin receptor activation. PMID:10455011

  13. 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.

  14. 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.

  15. 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.

  16. Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1

    PubMed Central

    Al-Samir, Samer; Papadopoulos, Symeon; Scheibe, Renate J; Meißner, Joachim D; Cartron, Jean-Pierre; Sly, William S; Alper, Seth L; Gros, Gerolf; Endeward, Volker

    2013-01-01

    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 () 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 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

  17. Comparison of glutathione reductase activity and the intracellular glutathione reducing effects of 13 derivatives of 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells.

    PubMed

    Xu, Shenghui; Kojima-Yuasa, Akiko; Azuma, Hideki; Kennedy, David Opare; Konishi, Yotaro; Matsui-Yuasa, Isao

    2010-05-14

    In a previous study, we showed that (1'S)-acetoxychavicol acetate ((S)-ACA) caused a rapid decrease in glutathione (GSH) levels less than 15 min after exposure. (S)-ACA-induced cell death was reversed by the addition of N-acetylcysteine. In the current study, we investigated the inhibitory activities of 13 derivatives of (S)-ACA on tumor cell viability, intracellular GSH level and GR activity. Correlations were found among a decrease in cell viability, intracellular GSH levels and the activity of GR in Ehrlich ascites tumor cells treated with the various ACA analogues. A test of the 13 derivatives revealed that the structural factors regulating activity were as follows: (1) the para or 1'-position of acetoxyl group (or other acyl group) was essential, (2) the presence of a C2'-C3' double or triple bond was essential, and (3) the S configuration of the 1'-acetoxyl group was preferable.

  18. Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

    PubMed Central

    De Kimpe, S. J.; Thiemermann, C.; Vane, J. R.

    1995-01-01

    1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h

  19. Intracellular labile iron determines H2O2-induced apoptotic signaling via sustained activation of ASK1/JNK-p38 axis.

    PubMed

    Mantzaris, M D; Bellou, S; Skiada, V; Kitsati, N; Fotsis, T; Galaris, D

    2016-08-01

    Hydrogen peroxide (H2O2) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular "labile iron" were able to modulate signal transduction in H2O2-induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H2O2-induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H2O2-mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H2O2, influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H2O2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions.

  20. 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

  1. ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

    PubMed

    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 Ca(2+) and Mg(2+) and the role of these changes in SR Ca(2+) 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 [Ca(2+)] <1 µM, ß-adrenergic stimulation increased luminal Ca(2+) activation of single RyR channels, decreased luminal Mg(2+) inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg(2+). At cytoplasmic [Ca(2+)] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+) and Ca(2+) inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+) 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 Ca(2+) binding to the luminal Ca(2+) site and decreasing its affinity for luminal Mg(2+) and 2) decreasing affinity of the low-affinity Ca(2+)/Mg(2+) cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

  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. 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

  4. Intracellular chromium reduction.

    PubMed

    Arslan, P; Beltrame, M; Tomasi, A

    1987-10-22

    Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid. PMID:2820507

  5. 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.

  6. Tumoricidal effector mechanisms of murine Bacillus Calmette-Guérin-activated macrophages: mediation of cytolysis, mitochondrial respiration inhibition, and release of intracellular iron by distinct mechanisms.

    PubMed

    Klostergaard, J; Leroux, M E; Ezell, S M; Kull, F C

    1987-04-15

    Murine Bacillus Calmette-Guérin-activated macrophages mediate discrete cytotoxic effects in cocultured tumor target cells in vitro. These effects include: the loss of intracellular iron, in part associated with reversible inhibition of the Kreb's cycle enzyme, aconitase; cytostasis, associated with reversible lesions inflicted in the electron transport chain (ETC) of the mitochondria resulting in reversible loss of proliferative capacity; and cytolysis, manifested by eventual gross perturbation of the integrity of the plasma membrane. We demonstrate that these manifestations of cytotoxicity are the result of three independent mechanisms employing apparently distinct macromolecules for their commission. Analysis of target cells that are highly susceptible (L-929), highly resistant (L-1210), or have incomplete resistance (EMT-6) to the cytolytic effects of cocultured activated macrophages indicates that there is no consistent relationship between the release of intracellular 59Fe and 51Cr. Thus, perturbation of intracellular iron pools did not appear to be an obligatory step on the pathway to cytolysis. Further evidence for this dissociation was obtained by employing a specific heteroantiserum reactive with cytolytic molecule(s). This antiserum could block the cytolytic response (51Cr release of cocultured L-929 and EMT-6 targets) but had no effect on the extent of iron release from viable EMT-6 or L-1210 targets. Furthermore, the cytolytic factor itself was incapable of mediating effects on the ETC or in causing release of intracellular iron. Two lines of evidence suggested that effects on the ETC are not linked with loss of intracellular iron. First, the monokine respiration inhibitory factor was incapable of causing release of intracellular iron from target cells in which the mitochondria were strongly suppressed. Second, the kinetics of release of respiration inhibitory factor from endotoxin-triggered Bacillus Calmette-Guérin-activated macrophages indicate a

  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. Impact of the oxaliplatin-5 fluorouracil-folinic acid combination on respective intracellular determinants of drug activity

    PubMed Central

    Fischel, J L; Formento, P; Ciccolini, J; Rostagno, P; Etienne, M C; Catalin, J; Milano, G

    2002-01-01

    The combination of 5-fluorouracil-folinic acid and oxaliplatin has led to a significant improvement of chemotherapy efficacy in advanced pretreated colorectal cancer. The objective of the present study was, considering the oxaplatin-5-fluorouracil-folinic acid combination, to examine the impact of one given drug on the cellular determinants of cytotoxic activity of the other drug. These cellular factors were analysed on the human colon cancer cell line WiDr in clinically relevant conditions of drug exposure (‘De Gramont’ schedule) with oxaliplatin-folinic acid during 2 h followed by 5-fluorouracil 48 h. The DNA binding of oxaliplatin was significantly reduced by the presence of 5-fluorouracil but this effect was time-dependent and after 50 h the platinum incorporated into DNA was identical in controls and in the drug combination. In the presence of oxaliplatin, there was less formation of FUH2 which is the first catabolite produced in the cascade of 5-fluorouracil metabolic degradation. The effects of drugs on cell cycle were quite different from one drug to the other with oxaliplatin inducing a shift towards G2 accumulation and 5-fluorouracil-folinic acid to a greater proportion of cells in G1–S. When oxaliplatin and 5-fluorouracil-folinic acid were combined the cell cycle effects were very similar to that of the 5-fluorouracil-folinic acid sequence alone. Oxaliplatin was able to reduce thymidylate synthase activity with a marked impact 28 h after the beginning of cell exposure to the drug. The 5-fluorouracil-folinic acid drug sequence led to a profound reduction in thymidylate synthase activity and this decrease was not markedly enhanced by the presence of oxaliplatin. Regarding apoptosis, changes in mitochondrial membrane permeability were observed in the presence of the tested drugs and the impact of 5-fluorouracil-folinic acid was greater than that of oxaliplatin. The addition of oxaliplatin did not amplify the action of 5-fluorouracil

  9. Engineering design activities and conceptual change in middle school science

    NASA Astrophysics Data System (ADS)

    Schnittka, Christine G.

    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 groups. One eighth-grade physical science teacher and her students participated in a unit on heat transfer and thermal energy. One class served as the control while two others received variations of an engineering design treatment. Data were gathered from teacher and student entrance and exit interviews, audio recordings of student dialog during group work, video recordings and observations of all classes, pre- and posttests on science content and engineering attitudes, and artifacts and all assignments completed by students. Qualitative and quantitative data were collected concurrently, but analysis took place in two phases. Qualitative data were analyzed in an ongoing manner so that the researcher could explore emerging theories and trends as the study progressed. These results were compared to and combined with the results of the quantitative data analysis. Analysis of the data was carried out in the interpretive framework of analytic induction. Findings indicated that students overwhelmingly possessed alternative conceptions about heat transfer, thermal energy, and engineering prior to the interventions. While all three classes made statistically significant gains in their knowledge about heat and energy, students in the engineering design class with the targeted demonstrations made the most significant gains over the other two other classes. Engineering attitudes changed significantly in the two classes that received the engineering design intervention. Implications from this study can inform teachers' use of engineering design activities in science classrooms. These implications are: (1) Alternative conceptions will

  10. 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

  11. Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge, a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions.

    PubMed

    Nguyen, Hien Thi Thu; Kristiansen, Rikke; Vestergaard, Mette; Wimmer, Reinhard; Nielsen, Per Halkjær

    2015-07-01

    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. (13)C-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 (3)H-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

  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

    DOE PAGES

    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 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

  14. 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

  15. PlyC, a bacteriophage endolysin that is internalized by epithelial cells and retains bacteriolytic activity against intracellular streptococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. 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.

  17. 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…

  18. In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide.

    PubMed

    Ji, Yanbin; Majumder, Subhabrata; Millard, Melissa; Borra, Radhika; Bi, Tao; Elnagar, Ahmed Y; Neamati, Nouri; Shekhtman, Alexander; Camarero, Julio A

    2013-08-01

    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.

  19. 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

  20. 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. PMID:25687775

  1. 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.

  2. Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells.

    PubMed

    Paola, D; Domenicotti, C; Nitti, M; Vitali, A; Borghi, R; Cottalasso, D; Zaccheo, D; Odetti, P; Strocchi, P; Marinari, U M; Tabaton, M; Pronzato, M A

    2000-02-16

    Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop. PMID:10679257

  3. Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine.

    PubMed

    de Muys, J M; Gourdeau, H; Nguyen-Ba, N; Taylor, D L; Ahmed, P S; Mansour, T; Locas, C; Richard, N; Wainberg, M A; Rando, R F

    1999-08-01

    The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a

  4. Intracellular survival of Staphylococcus aureus due to alteration of cellular activity in arsenic and lead intoxicated mature Swiss albino mice.

    PubMed

    Bishayi, Biswadev; Sengupta, Mahuya

    2003-02-14

    The role of heavy metals like arsenic (As) and lead (Pb) as environmental toxicants is established. However, the exact mechanism of their effect on immunocompetent cell activity is not well known. Staphylococcus aureus is a virulent pathogen that has the ability to cause a variety of potentially life-threatening infections. The objective of our study was to demonstrate in an experimental mouse model of bacteremic S. aureus infection, bacterial clearance from blood and spleen in arsenic, lead treated and control group of mice. Bacterial density was measured in blood and spleen after 0, 24, 48 and 72 h post-infection. Our findings show a significant increase in bacterial load in blood (P<0.025 for arsenic and P<0.01 for lead) and delayed bacterial clearance by spleen in both arsenic (P<0.05) and lead (P<0.025) treated groups as compared to control, thus highlighting an immuno-compromised state following heavy metal exposure. To further elucidate immunomodulatory effects of both arsenic and lead, cell function studies were performed on splenic macrophages (M(phi)) isolated from lead and arsenic treated as well as control group of mice. Our findings show a decrease in cell adhesion property (P<0.005) of splenic M(phi)s from 2.9925+/-0.053 in control to 1.395+/-0.106 in arsenic and 0.8835+/-0.0106 in lead treated mice at 60 min. Morphologic alteration of the splenic M(phi)s showed an increase (As: P<0.05, Pb: P<0.0005) in both arsenic (6.876+/-0.3287%) and lead (16.55+/-1.051%) treated mice to control (2.649+/-1.238%) which may be responsible for the formers' reduced functional status. The chemotactic index, a measure of chemotactic migration of the macrophages toward immune serum, was 16.43+/-1.007 in control cell and was reduced (P<0.0005) to 4.19+/-0.393 in arsenic and 2.92+/-0.649 in lead treated mice at 60 min. These altered cell functions could probably explain the intracellular survival of S. aureus but such a causal relationship awaits further detailed

  5. 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.

  6. A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity.

    PubMed

    Frost, William N; Wang, Jean; Brandon, Christopher J

    2007-05-15

    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.

  7. Ziram and Sodium N,N-Dimethyldithiocarbamate Inhibit Ubiquitin Activation through Intracellular Metal Transport and Increased Oxidative Stress in HEK293 Cells

    PubMed Central

    2015-01-01

    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

  8. 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

  9. 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

  10. [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.

  11. Hydrolytic charge-reversal of PEGylated polyplexes enhances intracellular un-packaging and activity of siRNA.

    PubMed

    Werfel, Thomas A; Swain, Corban; Nelson, Christopher E; Kilchrist, Kameron V; Evans, Brian C; Miteva, Martina; Duvall, Craig L

    2016-04-01

    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 chain-transfer (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.

  12. 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.

  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. 4-Hydroxy-2-nonenal induces apoptosis by activating ERK1/2 signaling and depleting intracellular glutathione in intestinal epithelial cells.

    PubMed

    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

  15. 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.

  16. 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.

  17. Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering.

    PubMed

    Bednarek, Radoslaw; Boncela, Joanna; Smolarczyk, Katarzyna; Cierniewska-Cieslak, Aleksandra; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2008-01-18

    Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.

  18. Facilitation by intracellular carbonic anhydrase of Na+–HCO3− co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte

    PubMed Central

    Villafuerte, Francisco C; Swietach, Pawel; Youm, Jae-Boum; Ford, Kerrie; Cardenas, Rosa; Supuran, Claudiu T; Cobden, Philip M; Rohling, Mala; Vaughan-Jones, Richard D

    2014-01-01

    Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3− ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3− or H+ transport via the native acid-extruding proteins, Na+–HCO3− cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and –impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3− ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3− buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3− transport, and hence pHi regulation in the heart. PMID:24297849

  19. Facilitation by intracellular carbonic anhydrase of Na+ -HCO3- co-transport but not Na+ / H+ exchange activity in the mammalian ventricular myocyte.

    PubMed

    Villafuerte, Francisco C; Swietach, Pawel; Youm, Jae-Boum; Ford, Kerrie; Cardenas, Rosa; Supuran, Claudiu T; Cobden, Philip M; Rohling, Mala; Vaughan-Jones, Richard D

    2014-03-01

    Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3- ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein-protein binding (a 'transport metabolon') or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3- or H+ transport via the native acid-extruding proteins, Na+ -HCO3- cotransport (NBC) and Na+ / H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and -impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3- ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3- buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3- transport, and hence pHi regulation in the heart.

  20. 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.

  1. 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.

  2. Active Learning in a Finnish Engineering University Course

    ERIC Educational Resources Information Center

    Larson, Debra; Ahonen, Anna-Maija

    2004-01-01

    This paper is a case study on the use of active learning techniques in an upper-level engineering course at the Helsinki University of Technology. The paper describes how these techniques were introduced and successfully used within the Finnish university classroom. The cultural subtext is explored and attention is given to teaching techniques…

  3. 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.

  4. The respiratory burst is not required for killing of intracellular and extracellular parasites by a lymphokine-activated macrophage cell line.

    PubMed

    Scott, P; James, S; Sher, A

    1985-06-01

    The macrophage cell line, IC-21, was found to be incapable of producing the oxygen products associated with the respiratory burst. However, IC-21 cells were activated by lymphokine (LK) to kill intracellular (Leishmania donovani amastigotes) and extracellular (Schistosoma mansoni larvae) parasites, as well as tumor cells. In each case, the cytotoxicity exhibited by activated IC-21 cells and activated peritoneal macrophages was indistinguishable. However, nonactivated IC-21 cells were unable to kill L. donovani log-growth phase promastigotes, while nonactivated peritoneal macrophages destroyed greater than 90% of the initial infection. These results indicate that amastigotes and schistosome larvae are susceptible to killing by nonoxidative cytotoxic mechanism induced by lymphokine activation but, on the other hand, support the concept that the killing of log-growth phase promastigotes by nonactivated cells is dependent upon the respiratory burst. We propose that the IC-21 cell line may be a useful model for studying nonoxidative killing functions of activated macrophages. PMID:2988973

  5. 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.

  6. The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

    NASA Astrophysics Data System (ADS)

    Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

    2013-09-01

    We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

  7. Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.

    PubMed

    Seo, Hye Sook; Leclercq, Guy

    2002-01-01

    The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the

  8. Influence of changes in external potassium and chloride ions on membrane potential and intracellular potassium ion activity in rabbit ventricular muscle.

    PubMed

    Fozzard, H A; Lee, C O

    1976-04-01

    1. The membrane responses of rabbit papillary muscles to rapid changes in [K](o) and [Cl](o) were measured with open-tipped micropipettes and with closed micropipettes made from K-selective glass.2. The muscle cells behaved primarily as a K electrode, and responses to changes in [K](o) with constant [Cl](o) or with constant [K](o) x [Cl](o) were substantially the same.3. When [Cl](o) was changed at a constant [K](o) the membrane potentials changed rapidly and symmetrically by a small value and remained constant for 30 min.4. Measurement of potential with K(+)-selective micro-electrodes in these experiments showed no change in intracellular K activity. In addition to permitting calculation of K permeability, these measurements reassured us that the K(+)-selective electrodes were well insulated and not influenced by electrical shunts at the impalement site.5. Although the membrane response to changes in [Cl](o) was small, it was possible to calculate that the permeability ratio (P(Cl)/P(K)), was 0.11. The Cl and K conductances were about 0.015 mmho/cm(2) and 0.09 mmho/cm(2) respectively, resulting in a conductance ratio (g(Cl)/g(K)) of about 0.17.6. The time course of depolarization by increase in [K](o) was rapid (half-time 5 sec), but repolarization on return to lower [K](o) was much slower (half-time 50 sec). The depolarization time course was easily fitted by the potential change calculated by assuming the need for K diffusion into the extracellular spaces and taking account of the logarithmic relation between membrane potential and [K](o). These calculations did not fit the time course of repolarization, which was slowed in the fashion expected from an inward-rectifying membrane.7. The influence of [K](i) on membrane potential was investigated by changes in tonicity of the external solution. Hypotonic solution produced a change in intracellular K activity close to that produced by ideal water movement. However, in hypertonic solution, intracellular K activity

  9. Mitogenic hormone-induced intracellular message: assay and partial characterization of an activator of DNA replication induced by epidermal growth factor.

    PubMed Central

    Das, M

    1980-01-01

    This paper explores the pathway from nuclear quiescence to mitogenesis. It describes an in vitro assay for an activator of DNA replication induced by epidermal growth factor (EGF) in responsive cells. Cytoplasmic extracts from EGF-treated 3T3 cells were found to contain substances that can stimulate DNA synthesis in isolated nuclei from spleen cells of adult frogs. Extracts from untreated resting 3T3 cells lack this activity, and EGF itself is incapable of stimulating DNA synthesis in these cell-free systems. The extract-induced stimulation of incorporation of [3H]dTTP into nuclear DNA is ATP dependent and requires the presence of the four deoxyribonucleoside triphosphates, suggesting the occurrence of replication rather than repair synthesis. This cell-free assay has been used to obtain some initial insights into the mechanism of induction and biochemical characterization of the intermediate in EGF action. Half-maximal induction of the active intracellular substance is achieved at about 0.08 nM EGF, a concentration that correlates well with the concentration required for half-maximal mitogenesis. Studies on the biochemical characteristics of this active substance strongly suggest that the activity is associated with a protein. The activity is nondialyzable and sensitive to trypsin and heat. Sucrose gradient centrifugation of the extract revealed three peaks of activity with molecular weights of 46,000, 110,000, and 270,000 (sedimentation coefficients: 3.7 S, 6.6 S, and 12 S, respectively). These results indicate that receptor-EGF interaction at the cell surface leads to the intracellular generation of protein that are capable of stimulating quiescent nuclei into activity. PMID:6965791

  10. Spatio-temporal modelling of the NF-κB intracellular signalling pathway: the roles of diffusion, active transport, and cell geometry.

    PubMed

    Terry, Alan J; Chaplain, Mark A J

    2011-12-01

    The nuclear factor kappa B (NF-κB) intracellular signalling pathway is central to many stressful, inflammatory, and innate immune responses. NF-κB proteins themselves are transcription factors for hundreds of genes. Experiments have shown that the NF-κB pathway can exhibit oscillatory dynamics-a negative feedback loop causes oscillatory nuclear-cytoplasmic translocation of NF-κB. Given that cell size and shape are known to influence intracellular signal transduction, we consider a spatio-temporal model of partial differential equations for the NF-κB pathway, where we model molecular movement by diffusion and, for several key species including NF-κB, by active transport as well. Through numerical simulations we find values for model parameters such that sustained oscillatory dynamics occur. Our spatial profiles and animations bear a striking resemblance to experimental images and movie clips employing fluorescent fusion proteins. We discover that oscillations in nuclear NF-κB may occur when active transport is across the nuclear membrane only, or when no species are subject to active transport. However, when active transport is across the nuclear membrane and NF-κB is additionally actively transported through the cytoplasm, oscillations are lost. Hence transport mechanisms in a cell will influence its response to activation of its NF-κB pathway. We also demonstrate that sustained oscillations in nuclear NF-κB are somewhat robust to changes in the shape of the cell, or the shape, location, and size of its nucleus, or the location of ribosomes. Yet if the cell is particularly flat or the nucleus sufficiently small, then oscillations are lost. Thus the geometry of a cell may partly determine its response to NF-κB activation. The NF-κB pathway is known to be constitutively active in several human cancers. Our spatially explicit modelling approach will allow us, in future work, to investigate targeted drug therapy of tumours.

  11. The Drosophila FTZ-F1 Nuclear Receptor Mediates Juvenile Hormone Activation of E75A Gene Expression through an Intracellular Pathway*

    PubMed Central

    Dubrovsky, Edward B.; Dubrovskaya, Veronica A.; Bernardo, Travis; Otte, Valerie; DiFilippo, Robert; Bryan, Heather

    2011-01-01

    Juvenile hormone (JH) regulates a wide variety of biological activities in holometabolous insects, ranging from vitellogenesis and caste determination in adults to the timing of metamorphosis in larvae. The mechanism of JH signaling in such a diverse array of processes remains either unknown or contentious. We previously found that the nuclear receptor gene E75A is activated in S2 cells as a primary response to JH. Here, by expressing an intracellular form of JH esterase, we demonstrate that JH must enter the cell in order to activate E75A. To find intracellular receptors involved in the JH response, we performed an RNAi screen against nuclear receptor genes expressed in this cell line and identified the orphan receptor FTZ-F1. Removal of FTZ-F1 prevents JH activation of E75A, whereas overexpression enhances activation, implicating FTZ-F1 as a critical component of the JH response. FTZ-F1 is bound in vivo to multiple enhancers upstream of E75A, suggesting that it participates in direct JH-mediated gene activation. To better define the role of FTZ-F1 in JH signaling, we investigated interactions with candidate JH receptors and found that the bHLH-PAS proteins MET and GCE both interact with FTZ-F1 and can activate transcription through the FTZ-F1 response element. Removal of endogenous GCE, but not MET, prevents JH activation of E75A. We propose that FTZ-F1 functions as a competence factor by loading JH signaling components to the promoter, thus facilitating the direct regulation of E75A gene expression by JH. PMID:21832074

  12. Spatio-temporal modelling of the NF-κB intracellular signalling pathway: the roles of diffusion, active transport, and cell geometry.

    PubMed

    Terry, Alan J; Chaplain, Mark A J

    2011-12-01

    The nuclear factor kappa B (NF-κB) intracellular signalling pathway is central to many stressful, inflammatory, and innate immune responses. NF-κB proteins themselves are transcription factors for hundreds of genes. Experiments have shown that the NF-κB pathway can exhibit oscillatory dynamics-a negative feedback loop causes oscillatory nuclear-cytoplasmic translocation of NF-κB. Given that cell size and shape are known to influence intracellular signal transduction, we consider a spatio-temporal model of partial differential equations for the NF-κB pathway, where we model molecular movement by diffusion and, for several key species including NF-κB, by active transport as well. Through numerical simulations we find values for model parameters such that sustained oscillatory dynamics occur. Our spatial profiles and animations bear a striking resemblance to experimental images and movie clips employing fluorescent fusion proteins. We discover that oscillations in nuclear NF-κB may occur when active transport is across the nuclear membrane only, or when no species are subject to active transport. However, when active transport is across the nuclear membrane and NF-κB is additionally actively transported through the cytoplasm, oscillations are lost. Hence transport mechanisms in a cell will influence its response to activation of its NF-κB pathway. We also demonstrate that sustained oscillations in nuclear NF-κB are somewhat robust to changes in the shape of the cell, or the shape, location, and size of its nucleus, or the location of ribosomes. Yet if the cell is particularly flat or the nucleus sufficiently small, then oscillations are lost. Thus the geometry of a cell may partly determine its response to NF-κB activation. The NF-κB pathway is known to be constitutively active in several human cancers. Our spatially explicit modelling approach will allow us, in future work, to investigate targeted drug therapy of tumours. PMID:21907212

  13. In vitro extracellular and intracellular activities of clavulanic acid and those of piperacillin and ceftriaxone alone and in combination with tazobactam against clinical isolates of Legionella species.

    PubMed Central

    Edelstein, P H; Edelstein, M A

    1994-01-01

    The activities of ceftriaxone, piperacillin, tazobactam, clavulanic acid, and combinations of ceftriaxone or piperacillin with tazobactam against 22 clinical Legionella isolates were measured by broth microdilution and macrodilution methods and in macrophages. The broth microdilution MICs that inhibited 90% of strains tested were 2 and 1 microgram/ml for ceftriaxone and tazobactam, respectively. Broth macrodilution MICs were 8 and 1 microgram/ml, respectively, for the two Legionella pneumophila strains tested with piperacillin and were 0.25 and 0.5 microgram/ml, respectively, for clavulanate. No significant intracellular anti-L. pneumophila activity was observed for ceftriaxone (32 micrograms/ml), piperacillin (32 micrograms/ml), tazobactam alone (16 micrograms/ml), clavulanate alone (2 micrograms/ml), or tazobactam in combination with ceftriaxone (ceftriaxone/tazobactam at 32/4 and 16/16 micrograms/ml) or piperacillin (32/4 micrograms/ml). Erythromycin (1 microgram/ml) was active against intracellular L. pneumophila in the same macrophage model of infection. It is very unlikely that tazobactam or clavulanate, alone or in combination with beta-lactam antimicrobial agents, will be effective for the treatment of Legionnaires' disease in humans. PMID:8192443

  14. Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes

    PubMed Central

    Mukherjee, Debarati; Sen, Arpita; Boettner, Douglas R.; Fairn, Gregory D.; Schlam, Daniel; Bonilla Valentin, Fernando J.; Michael McCaffery, J.; Hazbun, Tony; Staiger, Chris J.; Grinstein, Sergio; Lemmon, Sandra K.; Claudio Aguilar, R.

    2013-01-01

    Summary Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking. PMID:23943876

  15. Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells.

    PubMed

    Deng, Zhengyu; Qian, Yinfeng; Yu, Yongqiang; Liu, Guhuan; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2016-08-24

    Reactive oxygen species (ROS) and oxidative stress are implicated in various physiological and pathological processes, and this feature provides a vital biochemical basis for designing novel therapeutic and diagnostic nanomedicines. Among them, oxidation-responsive micelles and vesicles (polymersomes) of amphiphilic block copolymers have been extensively explored; however, in previous works, oxidation by ROS including H2O2 exclusively leads to microstructural destruction of polymeric assemblies. For oxidation-responsive polymersomes, fast release of encapsulated hydrophilic drugs and bioactive macromolecules will occur upon microstructural disintegration. Under certain application circumstances, this does not meet design requirements for sustained-release drug nanocarriers and long-acting in vivo nanoreactors. Also note that conventional polymersomes possess thick hydrophobic bilayers and compromised membrane permeability, rendering them as ineffective nanocarriers and nanoreactors. We herein report the fabrication of oxidation-responsive multifunctional polymersomes exhibiting intracellular milieu-triggered vesicle bilayer cross-linking, permeability switching, and enhanced imaging/drug release features. Mitochondria-targeted H2O2 reactive polymersomes were obtained through the self-assembly of amphiphilic block copolymers containing arylboronate ester-capped self-immolative side linkages in the hydrophobic block, followed by surface functionalization with targeting peptides. Upon cellular uptake, intracellular H2O2 triggers cascade decaging reactions and generates primary amine moieties; prominent amidation reaction then occurs within hydrophobic bilayer membranes, resulting in concurrent cross-linking and hydrophobic-to-hydrophilic transition of polymersome bilayers inside live cells. This process was further utilized to achieve integrated functions such as sustained drug release, (combination) chemotherapy monitored by fluorescence and magnetic resonance (MR

  16. The role of intracellular high-mobility group box 1 in the early activation of Kupffer cells and the development of Con A-induced acute liver failure.

    PubMed

    Yang, Qiao; Liu, Yanning; Shi, Yu; Zheng, Min; He, Jiliang; Chen, Zhi

    2013-10-01

    Acute liver failure (ALF) is a highly complex syndrome characterized by devastating activation of early activation of Kupffer cells (KCs) has been implicated in the pathogenesis of ALF. However, the factors regulating KC early activation are virtually unexplored. The aim of present study was to determine the role of the intracellular high-mobility group box 1 (HMGB1) in modulating the early activation of KCs during ALF. The intravenous injection of Concanavalin A (Con A) was used to establish a mouse model of ALF. The dynamic pro-inflammatory properties and MHC II expression of KCs were measured by qRT-PCR and flow cytometry. HMGB1 expression in KCs was measured by qRT-PCR and Western blotting. The immunofluorescence was implemented to determine the relocation of HMGB1 in KCs, and the siRNA against HMGB1 was utilized to assess the impact of HMGB1 on KC pro-inflammatory properties. The peak of pro-inflammatory cytokines production and MHC II expression in KCs appeared at the early stage of ALF. The up-regulation of HMGB1 expression and the translocation of HMGB1 in KCs were in parallel with the early activation of KCs. The blockade of intracellular HMGB1 expression caused by siRNA significantly inhibited the production of KC-derived pro-inflammatory cytokines, and led to a down-regulation of MAP kinase activation in KCs. The self-derived HMGB1 is an "early alarmin" of KC activation during Con A-induced ALF. HMGB1 might be a potential target for cell-specific strategy in ALF.

  17. 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."

  18. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  19. An inside job: hacking into Janus kinase/signal transducer and activator of transcription signaling cascades by the intracellular protozoan Toxoplasma gondii.

    PubMed

    Denkers, Eric Y; Bzik, David J; Fox, Barbara A; Butcher, Barbara A

    2012-02-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype.

  20. An Inside Job: Hacking into Janus Kinase/Signal Transducer and Activator of Transcription Signaling Cascades by the Intracellular Protozoan Toxoplasma gondii

    PubMed Central

    Bzik, David J.; Fox, Barbara A.; Butcher, Barbara A.

    2012-01-01

    The intracellular protozoan Toxoplasma gondii is well known for its skill at invading and living within host cells. New discoveries are now also revealing the astounding ability of the parasite to inject effector proteins into the cytoplasm to seize control of the host cell. This review summarizes recent advances in our understanding of one such secretory protein called ROP16. This molecule is released from rhoptries into the host cell during invasion. The ROP16 molecule acts as a kinase, directly activating both signal transducer and activator of transcription 3 (STAT3) and STAT6 signaling pathways. In macrophages, an important and preferential target cell of parasite infection, the injection of ROP16 has multiple consequences, including downregulation of proinflammatory cytokine signaling and macrophage deviation to an alternatively activated phenotype. PMID:22104110

  1. 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.

  2. A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi.

    PubMed

    Miranda, Cristian Gabriel; Solana, Maria Elisa; Curto, Maria de Los Angeles; Lammel, Estela Maria; Schijman, Alejandro Gabriel; Alba Soto, Catalina Dirney

    2015-12-01

    Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication.

  3. A flow cytometer-based method to simultaneously assess activity and selectivity of compounds against the intracellular forms of Trypanosoma cruzi.

    PubMed

    Miranda, Cristian Gabriel; Solana, Maria Elisa; Curto, Maria de Los Angeles; Lammel, Estela Maria; Schijman, Alejandro Gabriel; Alba Soto, Catalina Dirney

    2015-12-01

    Chagas disease is a major unsolved health issue in Latin America and an emerging threat worldwide. New drugs are urgently needed for chemotherapy as those available (benznidazole and nifurtimox) have variable efficacy and elevated toxicity. Efforts are actually oriented to improve tools and technologies (e.g. transgenic parasites, flow cytometry or image-based systems) for the screening of large numbers of candidate compounds for their activity against Trypanosoma cruzi (T. cruzi). Methods that test drug efficacy and selectivity in the same assay are suitable to accelerate the process of drug discovery. Here, we developed a GFP expressing T. cruzi from a moderate virulence stock and confirmed that the transgenic parasite retained the biological characteristics of the parental strain. With this tool, we established a flow cytometer-based method to simultaneously test drug activity against intracellular amastigotes and toxicity to the host cell. This one-step procedure allows determining the selectivity index of the tested compound in a sensitive and accurate manner even with low infection rates. This method can provide additional information on the interactions between drug, parasites and host cell and could be adapted to other trypanosomatids and protozoa with intracellular multiplication. PMID:26272680

  4. 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.

  5. 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.

  6. Engineering Translational Activators with CRISPR-Cas System.

    PubMed

    Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

    2016-01-15

    RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility. PMID:26414660

  7. Inhibitory Activity of the Isoflavone Biochanin A on Intracellular Bacteria of Genus Chlamydia and Initial Development of a Buccal Formulation

    PubMed Central

    Hanski, Leena; Genina, Natalja; Uvell, Hanna; Malinovskaja, Kristina; Gylfe, Åsa; Laaksonen, Timo; Kolakovic, Ruzica; Mäkilä, Ermei; Salonen, Jarno; Hirvonen, Jouni; Elofsson, Mikael; Sandler, Niklas; Vuorela, Pia M.

    2014-01-01

    Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC50 = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications. PMID:25514140

  8. 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.

  9. 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.

  10. Educational Activities At The Nuclear Engineering Teaching Laboratory

    NASA Astrophysics Data System (ADS)

    Tipping, Tracy N.

    2011-06-01

    The Nuclear Engineering Teaching Laboratory (NETL) at the University of Texas at Austin performs a wide variety of educational activities for students at various levels. Regular on-site courses in the areas of health physics, radiochemistry, and reactor operations are offered for university credit. Along with on-site courses, access to the reactor facility via a remote console connection allows students in an off-site classroom to conduct experiments via a "virtual" control console. In addition to the regularly scheduled courses, other programs, such as the Nuclear Regulatory Commission Summer Nuclear Engineering Institute and Office of Naval Research partnerships with Historically Black Colleges and Universities, provide access to the facility for students from other universities both domestic and foreign. And NETL hosts professional development programs such as training programs for Nuclear Regulatory Commission personnel and International Atomic Energy Agency fellowships.

  11. C-terminal clipping of chemokine CCL1/I-309 enhances CCR8-mediated intracellular calcium release and anti-apoptotic activity.

    PubMed

    Denis, Catherine; Deiteren, Kathleen; Mortier, Anneleen; Tounsi, Amel; Fransen, Erik; Proost, Paul; Renauld, Jean-Christophe; Lambeir, Anne-Marie

    2012-01-01

    Carboxypeptidase M (CPM) targets the basic amino acids arginine and lysine present at the C-terminus of peptides or proteins. CPM is thought to be involved in inflammatory processes. This is corroborated by CPM-mediated trimming and modulation of inflammatory factors, and expression of the protease in inflammatory environments. Since the function of CPM in and beyond inflammation remains mainly undefined, the identification of natural substrates can aid in discovering the (patho)physiological role of CPM. CCL1/I-309, with its three C-terminal basic amino acids, forms a potential natural substrate for CPM. CCL1 plays a role not only in inflammation but also in apoptosis, angiogenesis and tumor biology. Enzymatic processing differently impacts the biological activity of chemokines thereby contributing to the complex regulation of the chemokine system. The aim of the present study was to investigate whether (i) CCL1/I-309 is prone to trimming by CPM, and (ii) the biological activity of CCL1 is altered after C-terminal proteolytic processing. CCL1 was identified as a novel substrate for CPM in vitro using mass spectrometry. C-terminal clipping of CCL1 augmented intracellular calcium release mediated by CCR8 but reduced the binding of CCL1 to CCR8. In line with the higher intracellular calcium release, a pronounced increase of the anti-apoptotic activity of CCL1 was observed in the BW5147 cellular model. CCR8 signaling, CCR8 binding and anti-apoptotic activity were unaffected when CPM was exposed to the carboxypeptidase inhibitor DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid. The results of this study suggest that CPM is a likely candidate for the regulation of biological processes relying on the CCL1-CCR8 system. PMID:22479563

  12. Effects of Ouabain on Proliferation of Human Endothelial Cells Correlate with Na+,K+-ATPase Activity and Intracellular Ratio of Na+ and K.

    PubMed

    Tverskoi, A M; Sidorenko, S V; Klimanova, E A; Akimova, O A; Smolyaninova, L V; Lopina, O D; Orlov, S N

    2016-08-01

    Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+]i/[K+]i). Thus, we compared the dose- and time-dependences of the effect of ouabain on intracellular [Na+]i/[K+]i ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na+]i/[K+]i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of (86)Rb(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+]i/[K+]i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na+]i/[K+]i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na+]i/[K+]i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+]i/[K+]i ratio. PMID:27677555

  13. Exploring the role of polymer structure on intracellular nucleic acid delivery via polymeric nanoparticles.

    PubMed

    Bishop, Corey J; Kozielski, Kristen L; Green, Jordan J

    2015-12-10

    Intracellular nucleic acid delivery has the potential to treat many genetically-based diseases, however, gene delivery safety and efficacy remains a challenging obstacle. One promising approach is the use of polymers to form polymeric nanoparticles with nucleic acids that have led to exciting advances in non-viral gene delivery. Understanding the successes and failures of gene delivery polymers and structures is the key to engineering optimal polymers for gene delivery in the future. This article discusses the polymer structural features that enable effective intracellular delivery of DNA and RNA, including protection of nucleic acid cargo, cellular uptake, endosomal escape, vector unpacking, and delivery to the intracellular site of activity. The chemical properties that aid in each step of intracellular nucleic acid delivery are described and specific structures of note are highlighted. Understanding the chemical design parameters of polymeric nucleic acid delivery nanoparticles is important to achieving the goal of safe and effective non-viral genetic nanomedicine.

  14. Development and Evaluation of an Undergraduate Multidisciplinary Project Activity in Engineering and Design

    ERIC Educational Resources Information Center

    Smith, David R.; Cole, Joanne

    2012-01-01

    The School of Engineering and Design Multidisciplinary Project (MDP) at Brunel University is a one week long project based activity involving first year undergraduate students from across the School subject areas of Electronic and Computer Engineering, Mechanical Engineering, Civil Engineering and Design. This paper describes the main aims of the…

  15. Cycling of intracellular pH during cell division of Xenopus embryos is a cytoplasmic activity depending on protein synthesis and phosphorylation

    PubMed Central

    1990-01-01

    In Xenopus embryos, the successive and rapid cell divisions that follow fertilization are accompanied by periodic oscillations of intracellular pH (pHi). Cycling of pHi occurs in phase with several other oscillatory activities, namely nuclear divisions, M phase-promoting factor (MPF) activity, and surface contraction waves (SCWs). We report that treatments that abolish cycling of MPF activity and the SCWs also suppress the pHi oscillations, whereas those that block cell division without affecting neither MPF activity nor the SCWs do not suppress the pHi oscillations. Experiments on enucleated oocytes, matured in vitro and activated, demonstrated that the activity governing the rhythmicity of the pHi oscillations resided in the cytoplasm of the oocyte. In this respect, the activity responsible for the pHi oscillations was different from that which drives the SCWs, which necessitated the presence of the oocyte germinal vesicle (Ohsumi et al., 1986), but more closely resembled MPF activity that did not require the presence of the oocyte germinal vesicle (Dabauvalle et al., 1988). In mature eggs enucleated at the time of egg activation, the pHi oscillations were similar to those in control nucleated eggs, whereas the period between two peaks of SCWs was 35-60 min vs. 20-35 min in nucleated control eggs. Previous studies had shown that the periodicity of SCWs was larger in anucleate egg fragments than in their nucleate counterparts (Sakai and Kubota, 1981), the difference being on the order of 6-15 min (Shinagawa, 1983). However, in these previous studies, enucleation was performed 30-50 min after fertilization. Our results clearly demonstrate that the periodicity of the SCWs is lengthened when the interval between egg activation and enucleation is shortened, thereby providing an easier way to assess the nuclear dependency of the SCWs. Finally, the various possibilities concerning the role of pHi cycling during cell division are discussed. PMID:2116420

  16. PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

    PubMed

    Xu, Haiqing; Kim, Sooah; Sorek, Hagit; Lee, Youngsuk; Jeong, Deokyeol; Kim, Jungyeon; Oh, Eun Joong; Yun, Eun Ju; Wemmer, David E; Kim, Kyoung Heon; Kim, Soo Rin; Jin, Yong-Su

    2016-03-01

    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

  17. Intracellular Bacteria in Protozoa

    NASA Astrophysics Data System (ADS)

    Görtz, Hans-Dieter; Brigge, Theo

    Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.

  18. The effect of vitamin D₃ supplementation on intracellular calcium and plasma membrane calcium ATPase activity in early stages of chronic kidney disease.

    PubMed

    Morvová, M; Lajdová, I; Spustová, V; Zvarík, M; Šikurová, L

    2014-01-01

    Chronic kidney disease (CKD) is associated with increased concentration of intracellular calcium, which is pathological and may lead to irreversible damage of cell functions and structures. The aim of our study was to investigate the impact of 6 months vitamin D(3) supplementation (14 000 IU/week) on free cytosolic calcium concentration ([Ca(2+)](i)) and on the plasma membrane calcium ATPase (PMCA) activity of patients with CKD stage 2-3. PMCA activity of patients was also compared to that of healthy volunteers. Vitamin D(3) supplementation of CKD patients resulted in the decrease of [Ca(2+)](i) (119.79+/-5.87 nmol/l vs. 105.36+/-3.59 nmol/l, n=14, P<0.001), whereas PMCA activity of CKD patients (38.75+/-22.89 nmol P(i)/mg/h) remained unchanged after vitamin D(3) supplementation (40.96+/-17.74 nmol P(i)/mg/h, n=14). PMCA activity of early stage CKD patients before supplementation of vitamin D(3), was reduced by 34 % (42.01+/-20.64 nmol P(i)/mg/h) in comparison to healthy volunteers (63.68+/-20.32 nmol P(i)/mg/h, n=28, P<0.001). These results indicate that vitamin D(3) supplementation had a lowering effect on [Ca(2+)](i) and negligible effect on PMCA activity in CKD patients.

  19. Adhesion, invasion and intracellular replication of Salmonella typhimurium in a murine hepatocyte cell line. Effect of cytokines and LPS on antibacterial activity of hepatocytes.

    PubMed

    Lajarin, F; Rubio, G; Galvez, J; Garcia-Peñarrubia, P

    1996-11-01

    Elimination of pathogenic microorganisms in the liver may be an important effector mechanism in host defenses. In this paper we describe the adhesion, invasion and multiplication of Salmonella typhimurium in a murine embryonic hepatocyte cell line (ATCC TIB-73). Monolayers of hepatocytes treated with recombinant IFN gamma, IL1 beta, and LPS exhibit antibacterial activity against intracellular Salmonella. The dynamic of the infection process in stimulated vs unstimulated hepatocytes was determined by counting the number of survival bacteria in the cell monolayers at 4 and 28h after gentamicin was added to the infected cells. Salmonella typhimurium is able to adhere, invade and replicate inside the hepatocytes. The maximum number of cell-associated bacteria is approximately 15 bacteria per cell, whereas the invasive capacity of Salmonella is 0.003 bacteria per hepatocyte. Stimulated cultures display antibacterial activity compared to unstimulated controls. The antibacterial activity does not seem to be mediated by nitric oxide (NO) since inhibition of NO production by using NG-Monomethyl-L-Arginine did not revert the antibacterial activity. Also, high amounts of NO induced by adding L-Arginine to the cell cultures did not enhance hepatocyte antibacterial activity.

  20. Activation of ATP-sensitive potassium channels in rat pancreatic beta-cells by linoleic acid through both intracellular metabolites and membrane receptor signalling pathway.

    PubMed

    Zhao, Yu-Feng; Pei, Jianming; Chen, Chen

    2008-09-01

    ATP-sensitive potassium channels (K(ATP) channels) determine the excitability of pancreatic beta-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to beta-cells, but the effects of exogenous FFAs on K(ATP) channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and K(ATP) channels were observed in primary cultured rat pancreatic beta-cells. LA (20 microM) induced hyperpolarization of MP and opening of K(ATP) channels, which was totally reversed and inhibited by tolbutamide, a K(ATP) channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 microM), partially inhibited LA-induced opening of K(ATP) channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 microM), induced an opening of K(ATP) channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of K(ATP) channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on K(ATP) channels. The present study demonstrates that LA induces hyperpolarization of MP by activating K(ATP) channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and beta-cell dysfunction induced by FFAs.

  1. Estrogenic xenobiotics affect the intracellular activation signal in mitogen-induced human peripheral blood lymphocytes: immunotoxicological impact.

    PubMed

    Sakabe, K; Okuma, M; Kazuno, M; Yamaguchi, T; Yoshida, T; Furuya, H; Kayama, F; Suwa, Y; Fujii, W; Fresa, K L

    1998-01-01

    The present study was an attempt to elucidate the effect of estrogenic xenobiotics on the proliferation of mitogen-stimulated human peripheral blood lymphocyte (PBL). Our findings follow: (a) the proliferation of PBL in response to phytohemagglutinin (PHA) was mediated by protein kinase C activity, but estrogenic xenobiotics had a strong inhibitory effect on protein kinase C activity of PHA-stimulated PBL; (b) cytoplasmic extracts from PHA-stimulated PBL greatly activated DNA replication, but estrogenic xenobiotics had a strong inhibitory effect on these activities. The results suggest that the cytoplasmic signal-generating system in mitogen-treated PBL is inhibited by estrogenic xenobiotics, and that the defect occurs at all stages in the sequence of events leading to DNA synthesis and cell proliferation. PMID:9730256

  2. ["E" rosette formation in "active" T lymphocytes: phenomenon modulated by intracellular level of cyclic AMP and GMP (author's transl)].

    PubMed

    Machado, J A; Antunes, L J; Silva, E N

    1977-08-01

    The rosette formation envolving the binding of sheep red blood cells (SRBC) with active T lymphocytes was activated when the lymphocytes were incubated with levamisole, acetylcholine or carbamylcholine. Similar activation was seen when to the incubation medium was added substances of glucose metabolism (lactate, fumarate or succinate) or triphosphate de adenosina--ATP. The lymphocytes incubation with aminophyline, isoproterenol or 2-4-dinitrofenol--DNP--inhibited the rosette formation. The inhibition promoted by aminophyline was reversed by levamisole, acetylcholine or carbamylcholine, but not when lactate or ATP was used. When the rosette formation inhibition was caused by DNP, the reversion was only possible by ATP and no affect occurred if guanil cyclase activators were added to the incubation medium.

  3. Rapid burst of H2O2 by plant growth regulators increases intracellular Ca2+ amounts and modulates CD4+ T cell activation.

    PubMed

    Ahmed, Asma; Mukherjee, Sambuddho; Deobagkar, Mukta; Naik, Tanushree; Nandi, Dipankar

    2010-11-01

    The identification of small molecules that affect T cell activation is an important area of research. Three molecules that regulate plant growth and differentiation, but not their structurally similar analogs, were identified to enhance primary mouse CD4(+) T cell activation in conjunction with soluble anti-CD3 stimulation: Indoleacetic acid (natural plant auxin), 1-Napthaleneacetic acid (synthetic plant auxin) and 2,4-Dichlorophenoxyacetic acid (synthetic plant auxin and herbicide). These effects are distinct in comparison to Curcumin, the well known phenolic immunomodulator, which lowers T cell activation. An investigation into the mechanisms of action of the three plant growth regulators revealed a rapid induction of reactive oxygen species (ROS), mainly comprising H(2)O(2). In addition, these three molecules synergize with soluble anti-CD3 signaling to enhance intracellular Ca(2+) concentrations [Ca(2+)](i), leading to greater T cell activation, e.g. induction of CD25 and IL-2. Enhanced production of TNFα and IFNγ by CD4(+) T cells is also observed upon plant growth regulator treatment with soluble anti-CD3. Interestingly, maximal IL-2 production and CD4(+) T cell cycle progression are observed upon activation with soluble anti-CD3 and phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Additionally, stimulation with PMA and Ionomcyin (a Ca(2+) ionophore), which activates T cells by circumventing the TCR, and plant growth regulators also demonstrated the role of the strength of signal (SOS): T cell cycle progression is enhanced with gentle activation conditions but decreased with strong activation conditions. This study demonstrates the direct effects of three plant growth regulators on CD4(+) T cell activation and cycling.

  4. Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload.

    PubMed

    Huang, Su-Hua; Lien, Jin-Cherng; Chen, Chao-Jung; Liu, Yu-Ching; Wang, Ching-Ying; Ping, Chia-Fong; Lin, Yu-Fong; Huang, An-Cheng; Lin, Cheng-Wen

    2016-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca(2+) overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents. PMID:27563890

  5. Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

    PubMed Central

    Huang, Su-Hua; Lien, Jin-Cherng; Chen, Chao-Jung; Liu, Yu-Ching; Wang, Ching-Ying; Ping, Chia-Fong; Lin, Yu-Fong; Huang, An-Cheng; Lin, Cheng-Wen

    2016-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents. PMID:27563890

  6. NLRP3 Inflammasome Activation Induced by Engineered Nanomaterials

    PubMed Central

    Sun, Bingbing; Wang, Xiang; Ji, Zhaoxia; Li, Ruibin

    2014-01-01

    Engineered nanomaterials (ENMs) continue to attract significant attentions because they have novel physicochemical properties that can improve the functions of products that will benefit human lives. However, the physicochemical properties that make ENMs attractive could interact with biological systems and induce cascades of events that cause toxicological effects. Recently, there are more studies suggesting inflammasome activation may play an important role in ENMs-induced biological responses. Inflammasomes are a family of multi-protein complexes and are increasingly recognized as major mediators of host immune system. Among these, NLRP3 inflammasome is the most studied one that could directly interact with ENMs to generate inflammatory responses. In this review, we aim to link the ENM physicochemical properties to NLRP3 inflammasome activation. The understanding of the mechanisms of ENMs-NLRP3 inflammasome interaction will provide us strategies for safer nanomaterial design and therapy. PMID:23180683

  7. Kresoxim methyl dissipation kinetics and its residue effect on soil extra-cellular and intra-cellular enzymatic activity in four different soils of India.

    PubMed

    Sabale, Rupali P; Shabeer T P, Ahammed; Utture, Sagar C; Banerjee, Kaushik; Oulkar, Dasharath P; Adsule, Pandurang G; Deshmukh, Madhukar B

    2015-01-01

    The rate of degradation of kresoxim methyl and its effect on soil extra-cellular (acid phosphatase, alkaline phosphatase and β-glucosidase) and intra-cellular (dehydrogenase) enzymes were explored in four different soils of India. In all the tested soils, the degradation rate was faster at the beginning, which slowed down with time indicating a non-linear pattern of degradation. Rate of degradation in black soil was fastest followed by saline, brown and red soils, respectively and followed 1st or 1st + 1st order kinetics with half-life ranging between 1-6 days for natural soil and 1-19 days for sterile soils. The rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Although small changes in enzyme activities were observed, kresoxim methyl did not have any significant deleterious effect on the enzymatic activity of the various test soils in long run. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily due to the effect of the incubation period rather than the effect of kresoxim methyl itself. PMID:25587778

  8. Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization.

    PubMed

    Stefani, Christian; Al-Eisawi, Zaynab; Jansson, Patric J; Kalinowski, Danuta S; Richardson, Des R

    2015-11-01

    Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular (64)Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones).

  9. Kresoxim methyl dissipation kinetics and its residue effect on soil extra-cellular and intra-cellular enzymatic activity in four different soils of India.

    PubMed

    Sabale, Rupali P; Shabeer T P, Ahammed; Utture, Sagar C; Banerjee, Kaushik; Oulkar, Dasharath P; Adsule, Pandurang G; Deshmukh, Madhukar B

    2015-01-01

    The rate of degradation of kresoxim methyl and its effect on soil extra-cellular (acid phosphatase, alkaline phosphatase and β-glucosidase) and intra-cellular (dehydrogenase) enzymes were explored in four different soils of India. In all the tested soils, the degradation rate was faster at the beginning, which slowed down with time indicating a non-linear pattern of degradation. Rate of degradation in black soil was fastest followed by saline, brown and red soils, respectively and followed 1st or 1st + 1st order kinetics with half-life ranging between 1-6 days for natural soil and 1-19 days for sterile soils. The rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Although small changes in enzyme activities were observed, kresoxim methyl did not have any significant deleterious effect on the enzymatic activity of the various test soils in long run. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily due to the effect of the incubation period rather than the effect of kresoxim methyl itself.

  10. Auto Mechanics I. Learning Activity Packets (LAPs). Section C--Engine.

    ERIC Educational Resources Information Center

    Oklahoma State Board of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

    This document contains five learning activity packets (LAPs) that outline the study activities for the "engine" instructional area for an Auto Mechanics I course. The five LAPs cover the following topics: basic engine principles, cooling system, engine lubrication system, exhaust system, and fuel system. Each LAP contains a cover sheet that…

  11. Learning Activity Packets for Auto Mechanics II. Section A--Engine Rebuilding.

    ERIC Educational Resources Information Center

    Oklahoma State Board of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

    Eight learning activity packets (LAPs) are provided for the instructional area of engine rebuilding in the auto mechanics II program. They accompany an instructor's guide available separately. The LAPs outline the study activities and performance tasks for these eight units: (1) engine condition evaluation; (2) engine removal; (3) engine…

  12. 34 CFR 350.32 - What activities must a Rehabilitation Engineering Research Center conduct?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 2 2011-07-01 2010-07-01 true What activities must a Rehabilitation Engineering... DISABILITY AND REHABILITATION RESEARCH PROJECTS AND CENTERS PROGRAM What Rehabilitation Engineering Research Centers Does the Secretary Assist? § 350.32 What activities must a Rehabilitation Engineering...

  13. 34 CFR 350.32 - What activities must a Rehabilitation Engineering Research Center conduct?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 2 2012-07-01 2012-07-01 false What activities must a Rehabilitation Engineering... DISABILITY AND REHABILITATION RESEARCH PROJECTS AND CENTERS PROGRAM What Rehabilitation Engineering Research Centers Does the Secretary Assist? § 350.32 What activities must a Rehabilitation Engineering...

  14. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    SciTech Connect

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  15. Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

    Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.

  16. Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

    SciTech Connect

    Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

    2011-10-01

    We had earlier shown that exposure to arsenic (0.50 {mu}M) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca{sup 2+}) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca{sup 2+} homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca{sup 2+} levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: > Altered Ca{sup 2+} homeostasis leads to arsenic-induced HKM apoptosis. > Calpain-2 plays a critical role in the process. > ERK is pro-apoptotic in arsenic-induced HKM apoptosis. > Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

  17. Active learning increases student performance in science, engineering, and mathematics.

    PubMed

    Freeman, Scott; Eddy, Sarah L; McDonough, Miles; Smith, Michelle K; Okoroafor, Nnadozie; Jordt, Hannah; Wenderoth, Mary Pat

    2014-06-10

    To test the hypothesis that lecturing maximizes learning and course performance, we metaanalyzed 225 studies that reported data on examination scores or failure rates when comparing student performance in undergraduate science, technology, engineering, and mathematics (STEM) courses under traditional lecturing versus active learning. The effect sizes indicate that on average, student performance on examinations and concept inventories increased by 0.47 SDs under active learning (n = 158 studies), and that the odds ratio for failing was 1.95 under traditional lecturing (n = 67 studies). These results indicate that average examination scores improved by about 6% in active learning sections, and that students in classes with traditional lecturing were 1.5 times more likely to fail than were students in classes with active learning. Heterogeneity analyses indicated that both results hold across the STEM disciplines, that active learning increases scores on concept inventories more than on course examinations, and that active learning appears effective across all class sizes--although the greatest effects are in small (n ≤ 50) classes. Trim and fill analyses and fail-safe n calculations suggest that the results are not due to publication bias. The results also appear robust to variation in the methodological rigor of the included studies, based on the quality of controls over student quality and instructor identity. This is the largest and most comprehensive metaanalysis of undergraduate STEM education published to date. The results raise questions about the continued use of traditional lecturing as a control in research studies, and support active learning as the preferred, empirically validated teaching practice in regular classrooms.

  18. Active learning increases student performance in science, engineering, and mathematics.

    PubMed

    Freeman, Scott; Eddy, Sarah L; McDonough, Miles; Smith, Michelle K; Okoroafor, Nnadozie; Jordt, Hannah; Wenderoth, Mary Pat

    2014-06-10

    To test the hypothesis that lecturing maximizes learning and course performance, we metaanalyzed 225 studies that reported data on examination scores or failure rates when comparing student performance in undergraduate science, technology, engineering, and mathematics (STEM) courses under traditional lecturing versus active learning. The effect sizes indicate that on average, student performance on examinations and concept inventories increased by 0.47 SDs under active learning (n = 158 studies), and that the odds ratio for failing was 1.95 under traditional lecturing (n = 67 studies). These results indicate that average examination scores improved by about 6% in active learning sections, and that students in classes with traditional lecturing were 1.5 times more likely to fail than were students in classes with active learning. Heterogeneity analyses indicated that both results hold across the STEM disciplines, that active learning increases scores on concept inventories more than on course examinations, and that active learning appears effective across all class sizes--although the greatest effects are in small (n ≤ 50) classes. Trim and fill analyses and fail-safe n calculations suggest that the results are not due to publication bias. The results also appear robust to variation in the methodological rigor of the included studies, based on the quality of controls over student quality and instructor identity. This is the largest and most comprehensive metaanalysis of undergraduate STEM education published to date. The results raise questions about the continued use of traditional lecturing as a control in research studies, and support active learning as the preferred, empirically validated teaching practice in regular classrooms. PMID:24821756

  19. Active learning increases student performance in science, engineering, and mathematics

    PubMed Central

    Freeman, Scott; Eddy, Sarah L.; McDonough, Miles; Smith, Michelle K.; Okoroafor, Nnadozie; Jordt, Hannah; Wenderoth, Mary Pat

    2014-01-01

    To test the hypothesis that lecturing maximizes learning and course performance, we metaanalyzed 225 studies that reported data on examination scores or failure rates when comparing student performance in undergraduate science, technology, engineering, and mathematics (STEM) courses under traditional lecturing versus active learning. The effect sizes indicate that on average, student performance on examinations and concept inventories increased by 0.47 SDs under active learning (n = 158 studies), and that the odds ratio for failing was 1.95 under traditional lecturing (n = 67 studies). These results indicate that average examination scores improved by about 6% in active learning sections, and that students in classes with traditional lecturing were 1.5 times more likely to fail than were students in classes with active learning. Heterogeneity analyses indicated that both results hold across the STEM disciplines, that active learning increases scores on concept inventories more than on course examinations, and that active learning appears effective across all class sizes—although the greatest effects are in small (n ≤ 50) classes. Trim and fill analyses and fail-safe n calculations suggest that the results are not due to publication bias. The results also appear robust to variation in the methodological rigor of the included studies, based on the quality of controls over student quality and instructor identity. This is the largest and most comprehensive metaanalysis of undergraduate STEM education published to date. The results raise questions about the continued use of traditional lecturing as a control in research studies, and support active learning as the preferred, empirically validated teaching practice in regular classrooms. PMID:24821756

  20. Hybrid Active/Passive Jet Engine Noise Suppression System

    NASA Technical Reports Server (NTRS)

    Parente, C. A.; Arcas, N.; Walker, B. E.; Hersh, A. S.; Rice, E. J.

    1999-01-01

    A novel adaptive segmented liner concept has been developed that employs active control elements to modify the in-duct sound field to enhance the tone-suppressing performance of passive liner elements. This could potentially allow engine designs that inherently produce more tone noise but less broadband noise, or could allow passive liner designs to more optimally address high frequency broadband noise. A proof-of-concept validation program was undertaken, consisting of the development of an adaptive segmented liner that would maximize attenuation of two radial modes in a circular or annular duct. The liner consisted of a leading active segment with dual annuli of axially spaced active Helmholtz resonators, followed by an optimized passive liner and then an array of sensing microphones. Three successively complex versions of the adaptive liner were constructed and their performances tested relative to the performance of optimized uniform passive and segmented passive liners. The salient results of the tests were: The adaptive segmented liner performed well in a high flow speed model fan inlet environment, was successfully scaled to a high sound frequency and successfully attenuated three radial modes using sensor and active resonator arrays that were designed for a two mode, lower frequency environment.

  1. Optimization, Composition, and Antioxidant Activities of Exo- and Intracellular Polysaccharides in Submerged Culture of Cordyceps gracilis (Grev.) Durieu & Mont.

    PubMed Central

    Sharma, Sapan Kumar; Atri, Narender Singh

    2015-01-01

    Under present experiments, EPS and IPS production, monosaccharide composition, and antioxidant activities of C. gracilis were studied for the first time under submerged culture conditions. Effect of different factors on polysaccharides production was studied by orthogonal experiments using one-factor-at-a-time method. Incubation of culture in the medium with capacity 200 mL (675.12 ± 5.01 and 385.20 ± 5.01 mg/L), rotation speed 150 rpm (324.62 ± 3.32 and 254.62 ± 4.62 mg/L), 6-day culture incubation time (445.24 ± 1.11, 216.60 ± 1.71 mg/L), pH 6.0 (374.81 ± 2.52 and 219.45 ± 2.59 mg/L), and temperature 23°C (405.24 ± 1.11 and 215.60 ± 1.71 mg/L) produced higher EPS and IPS, respectively. Maximum EPS and IPS production was observed in the medium supplemented with glucose as a carbon source (464.82 ± 2.12 and 264.42 ± 2.62 mg/L) and yeast extract as a nitrogen source (465.21 ± 3.11 and 245.17 ± 3.24 mg/L), respectively. Carbon to nitrogen ratio for maximum EPS and IPS production was observed as 10 : 1 (395.29 ± 2.15 and 235.62 ± 1.40 mg/L), respectively. Glucose was found to be the major monosaccharide (62.15 ± 7.33%). Both EPS and IPS of C. gracilis showed significant DPPH radical scavenging activity, ABTS radical scavenging activity, reducing power, and iron chelating activity. PMID:25878715

  2. Compound profiling and 3D-QSAR studies of hydrazone derivatives with activity against intracellular Trypanosoma cruzi.

    PubMed

    Costa, Lívia Bandeira; Cardoso, Marcos Veríssimo de Oliveira; de Oliveira Filho, Gevanio Bezerra; de Moraes Gomes, Paulo André Teixeira; Espíndola, José Wanderlan Pontes; de Jesus Silva, Thays Gabrielle; Torres, Pedro Henrique Monteiro; Silva Junior, Floriano Paes; Martin, Julio; de Figueiredo, Regina Célia Bressan Queiroz; Leite, Ana Cristina Lima

    2016-04-15

    Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi, which is endemic in Central and South America. Few treatments are available with effectiveness limited to the early (acute) stage of disease, significant toxicity and widespread drug resistance. In this work we report the outcome of a HTS-ready assay chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T. cruzi. We have selected 50 compounds that possess hydrazone as a common group. The compounds were screened using recombinant T. cruzi (Tulahuen strain) expressing beta-galactosidase. A 3D quantitative structure-activity relationship (QSAR) analysis was performed using descriptors calculated from comparative molecular field analysis (CoMFA). Our findings show that of the fifty selected hydrazones, compounds LpQM-19, 28 and 31 displayed the highest activity against T. cruzi, leading to a selectivity index (SI) of 20-fold. The 3D-QSAR analysis indicates that a particular electrostatic arrangement, where electron-deficient atoms are aligned along the molecule main axis positively correlates with compound biological activity. These results provide new candidate molecules for the development of treatments against Chagas disease. PMID:26964673

  3. DMSO Enhances TGF-β Activity by Recruiting the Type II TGF-β Receptor From Intracellular Vesicles to the Plasma Membrane.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Hou, Wei-Hsien; Huang, Jung San

    2016-07-01

    Dimethyl sulfoxide (DMSO) is used to treat many diseases/symptoms. The molecular basis of the pharmacological actions of DMSO has been unclear. We hypothesized that DMSO exerts some of these actions by enhancing TGF-β activity. Here we show that DMSO enhances TGF-β activity by ∼3-4-fold in Mv1Lu and NMuMG cells expressing Smad-dependent luciferase reporters. In Mv1Lu cells, DMSO enhances TGF-β-stimulated expression of P-Smad2 and PAI-1. It increases cell-surface expression of TGF-β receptors (TβR-I and/or TβR-II) by ∼3-4-fold without altering their cellular levels as determined by (125) I-labeled TGF-β-cross-linking/Western blot analysis, suggesting the presence of large intracellular pools in these cells. Sucrose density gradient ultracentrifugation/Western blot analysis reveals that DMSO induces recruitment of TβR-II (but not TβR-I) from its intracellular pool to plasma-membrane microdomains. It induces more recruitment of TβR-II to non-lipid raft microdomains than to lipid rafts/caveolae. Mv1Lu cells transiently transfected with TβR-II-HA plasmid were treated with DMSO and analyzed by indirect immunofluoresence staining using anti-HA antibody. In these cells, TβR-II-HA is present as a vesicle-like network in the cytoplasm as well as in the plasma membrane. DMSO causes depletion of TβR-II-HA-containing vesicles from the cytoplasm and co-localization of TβR-II-HA and cveolin-1 at the plasma membrane. These results suggest that DMSO, a fusogenic substance, enhances TGF-β activity presumably by inducing fusion of cytoplasmic vesicles (containing TβR-II) and the plasma membrane, resulting in increased localization of TβR-II to non-lipid raft microdomains where canonical signaling occurs. Fusogenic activity of DMSO may play a pivotal role in its pharmacological actions involving membrane proteins with large cytoplasmic pools. J. Cell. Biochem. 117: 1568-1579, 2016. © 2015 Wiley Periodicals, Inc.

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

    PubMed

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

    2015-01-01

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

  5. Overview of additive manufacturing activities at MTU aero engines

    NASA Astrophysics Data System (ADS)

    Bamberg, Joachim; Dusel, Karl-Heinz; Satzger, Wilhelm

    2015-03-01

    Additive Manufacturing (AM) is a promising technology to produce parts easily and effectively, just by using metallic powder or wire as starting material and a sophisticated melting process. In contrast to milling or turning technologies complex shaped and hollow parts can be built up in one step. That reduces the production costs and allows the implementation of complete new 3D designs. Therefore AM is also of great interest for aerospace and aero engine industry. MTU Aero Engines has focused its AM activities to the selective laser melting technique (SLM). This technique uses metallic powder and a laser for melting and building up the part layer by layer. It is shown which lead part was selected for AM and how the first production line was established. A special focus is set on the quality assurance of the selective laser melting process. In addition to standard non-destructive inspection techniques a new online monitoring tool was developed and integrated into the SLM machines. The basics of this technique is presented.

  6. eIF4E-binding proteins are differentially modified after ammonia versus intracellular calcium activation of sea urchin unfertilized eggs.

    PubMed

    Oulhen, Nathalie; Mulner-Lorillon, Odile; Cormier, Patrick

    2010-01-01

    Fertilization of sea urchin eggs triggers a rise of protein synthesis mainly dependent on the cap-binding protein eIF4E, which is released from its repressor 4E-BP and associates with eIF4G. Association of eIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization. Artificial activation of unfertilized eggs with the calcium ionophore A23187 results in the activation of protein synthesis comparable to the one triggered by fertilization, while increasing the intracellular pH by ammonia treatment results in partial activation of protein synthesis. Nevertheless, artificial activation does not induce the mitotic division. Here we investigate the effect of calcium ionophore and ammonia treatment of unfertilized eggs on eIF4E and its two antagonist partners, 4E-BP and eIF4G. We show that the addition of calcium ionophore to unfertilized eggs induces permanent dissociation between eIF4E and 4E-BP, whereas a reversible dissociation of the complex occurs after ammonia treatment. The regulation of the complex correlates with permanent or reversible 4E-BP disappearance depending on the treatment used to trigger artificial activation. Furthermore, while calcium ionophore treatment of unfertilized eggs induces eIF4G modifications comparable to those observed following fertilization, ammonia treatment does not. These results suggest that ionophore and ammonia treatments of unfertilized eggs induce differential protein synthesis activation by targeting eIF4E availability and specific regulation through its two partners 4E-BP and eIF4G.

  7. Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

    PubMed Central

    Lansley, A B; Sanderson, M J

    1999-01-01

    Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM. PMID:10388787

  8. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    SciTech Connect

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna . E-mail: anna.salvetti@univ-nantes.fr

    2005-05-10

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68.

  9. Engineering titania nanostructure to tune and improve its photocatalytic activity.

    PubMed

    Cargnello, Matteo; Montini, Tiziano; Smolin, Sergey Y; Priebe, Jacqueline B; Delgado Jaén, Juan J; Doan-Nguyen, Vicky V T; McKay, Ian S; Schwalbe, Jay A; Pohl, Marga-Martina; Gordon, Thomas R; Lu, Yupeng; Baxter, Jason B; Brückner, Angelika; Fornasiero, Paolo; Murray, Christopher B

    2016-04-12

    Photocatalytic pathways could prove crucial to the sustainable production of fuels and chemicals required for a carbon-neutral society. Electron-hole recombination is a critical problem that has, so far, limited the efficiency of the most promising photocatalytic materials. Here, we show the efficacy of anisotropy in improving charge separation and thereby boosting the activity of a titania (TiO2) photocatalytic system. Specifically, we show that H2 production in uniform, one-dimensional brookite titania nanorods is highly enhanced by engineering their length. By using complimentary characterization techniques to separately probe excited electrons and holes, we link the high observed reaction rates to the anisotropic structure, which favors efficient carrier utilization. Quantum yield values for hydrogen production from ethanol, glycerol, and glucose as high as 65%, 35%, and 6%, respectively, demonstrate the promise and generality of this approach for improving the photoactivity of semiconducting nanostructures for a wide range of reacting systems.

  10. Human factors in remote control engineering development activities

    SciTech Connect

    Clarke, M.M.; Hamel, W.R.; Draper, J.V.

    1983-01-01

    Human factors engineering, which is an integral part of the advanced remote control development activities at the Oak Ridge National Laboratory, is described. First, work at the Remote Systems Development Facility (RSDF) has shown that operators can perform a wide variety of tasks, some of which were not specifically designed for remote systems, with a dextrous electronic force-reflecting servomanipulator and good television remote viewing capabilities. Second, the data collected during mock-up remote maintenance experiments at the RSDF have been analyzed to provide guidelines for the design of human interfaces with an integrated advanced remote maintenance system currently under development. Guidelines have been provided for task allocation between operators, remote viewing systems, and operator controls. 6 references, 5 figures, 2 tables.

  11. Engineering titania nanostructure to tune and improve its photocatalytic activity

    PubMed Central

    Cargnello, Matteo; Montini, Tiziano; Smolin, Sergey Y.; Priebe, Jacqueline B.; Delgado Jaén, Juan J.; Doan-Nguyen, Vicky V. T.; McKay, Ian S.; Schwalbe, Jay A.; Pohl, Marga-Martina; Gordon, Thomas R.; Lu, Yupeng; Baxter, Jason B.; Brückner, Angelika; Murray, Christopher B.

    2016-01-01

    Photocatalytic pathways could prove crucial to the sustainable production of fuels and chemicals required for a carbon-neutral society. Electron−hole recombination is a critical problem that has, so far, limited the efficiency of the most promising photocatalytic materials. Here, we show the efficacy of anisotropy in improving charge separation and thereby boosting the activity of a titania (TiO2) photocatalytic system. Specifically, we show that H2 production in uniform, one-dimensional brookite titania nanorods is highly enhanced by engineering their length. By using complimentary characterization techniques to separately probe excited electrons and holes, we link the high observed reaction rates to the anisotropic structure, which favors efficient carrier utilization. Quantum yield values for hydrogen production from ethanol, glycerol, and glucose as high as 65%, 35%, and 6%, respectively, demonstrate the promise and generality of this approach for improving the photoactivity of semiconducting nanostructures for a wide range of reacting systems. PMID:27035977

  12. Passive and active structural monitoring experience: Civil engineering applications

    NASA Astrophysics Data System (ADS)

    Thompson, L. D.; Westermo, B. D.; Crum, D. B.; Law, W. R.; Trombi, R. G.

    2000-05-01

    State Departments of Transportation and regional city government officials are beginning to view the long-term monitoring of infrastructure as being beneficial for structural damage accumulation assessment, condition based maintenance, life extension, and post-earthquake or -hurricane (-tornado, -typhoon, etc.) damage assessment. Active and passive structural monitoring systems were installed over the last few years to monitor concerns in a wide range of civil infrastructure applications. This paper describes the monitoring technologies and systems employed for such applications. Bridge system applications were directed at monitoring corrosion damage accumulation, composite reinforcements for life extension, general service cracking damage related to fatigue and overloads, and post-earthquake damage. Residential system applications were directed primarily at identifying damage accumulation and post-earthquake damage assessment. A professional sports stadium was monitored for isolated ground instability problems and for post-earthquake damage assessment. Internet-based, remote, data acquisition system experience is discussed with examples of long-term passive and active system data collected from many of the individual sites to illustrate the potential for both passive and active structural health monitoring. A summary of system-based operating characteristics and key engineering recommendations are provided to achieve specific structural monitoring objectives for a wide range of civil infrastructure applications.

  13. Heparin-disaccharide affects T cells: inhibition of NF-kappaB activation, cell migration, and modulation of intracellular signaling.

    PubMed

    Hecht, Iris; Hershkoviz, Rami; Shivtiel, Shoham; Lapidot, Tzvi; Cohen, Irun R; Lider, Ofer; Cahalon, Liora

    2004-06-01

    We previously reported that disaccharides (DS), generated by enzymatic degradation of heparin or heparan sulfate, inhibit T cell-mediated immune reactions in rodents and regulate cytokine [tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and IL-1beta] secretion by T cells, macrophages, or intestinal epithelial cells. Here, we investigated the effects of a trisulfated heparin DS (3S-DS) on two aspects of T cell function: secretion of proinflammatory cytokines and migration to an inflamed site. 3S-DS down-regulated nuclear factor-kappaB activity and reduced the secretion of TNF-alpha and interferon-gamma (IFN-gamma) by anti-CD3-activated T cells. In addition, 3S-DS inhibited CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor-1alpha)-dependent migration in vitro and in vivo and decreased CXCL12-induced T cell adhesion to the extracellular matrix glycoprotein, fibronectin (FN). This inhibition was accompanied by attenuation of CXCL12-induced Pyk2 phosphorylation but did not involve internalization of the CXCL12 receptor, CXCR4, or phosphorylation of extracellular-regulated kinase. Despite inhibiting CXCL12-induced adhesion, 3S-DS, on its own, induced T cell adhesion to FN, which was accompanied by phosphorylation of Pyk2. A monosulfated DS showed no effect. Taken together, these data provide evidence that 3S-DS can regulate inflammation by inducing and modulating T cell-signaling events, desensitizing CXCR4, and modulating T cell receptor-induced responses. PMID:15020655

  14. Dysregulation of Macrophage Activation Profiles by Engineered Nanoparticles

    SciTech Connect

    Kodali, Vamsi; Littke, Matthew H.; Tilton, Susan C.; Teeguarden, Justin G.; Shi, Liang; Frevert, Charles W.; Wang, Wei; Pounds, Joel G.; Thrall, Brian D.

    2013-08-27

    Although the potential human health impacts from exposure to engineered nanoparticles (ENPs) are uncertain, past epidemiological studies have established correlations between exposure to ambient air pollution particulates and the incidence of pneumonia and lung infections. Using amorphous silica and superparamagnetic iron oxide (SPIO) as model high production volume ENPs, we examined how macrophage activation by bacterial lipopolysaccharide (LPS) or the lung pathogen Streptococcus pneumoniae is altered by ENP pretreatment. Neither silica nor SPIO treatment elicited direct cytotoxic or pro-inflammatory effects in bone marrow-derived macrophages. However, pretreatment of macrophages with SPIO caused extensive reprogramming of nearly 500 genes regulated in response to LPS challenge, hallmarked by exaggerated activation of oxidative stress response pathways and suppressed activation of both pro- and anti-inflammatory pathways. Silica pretreatment altered regulation of only 67 genes, but there was strong correlation with gene sets affected by SPIO. Macrophages exposed to SPIO displayed a phenotype suggesting an impaired ability to transition from an M1 to M2-like activation state, characterized by suppressed IL-10 induction, enhanced TNFα production, and diminished phagocytic activity toward S. pneumoniae. Studies in macrophages deficient in scavenger receptor A (SR-A) showed SR-A participates in cell uptake of both the ENPs and S. pneumonia and co-regulates the anti-inflammatory IL-10 pathway. Thus, mechanisms for dysregulation of innate immunity exist by virtue that common receptor recognition pathways are used by some ENPs and pathogenic bacteria, although the extent of transcriptional reprogramming of macrophage function depends on the physicochemical properties of the ENP after internalization. Our results also illustrate that biological effects of ENPs may be indirectly manifested only after challenging normal cell function. Finally, nanotoxicology screening

  15. Multidisciplinary and Active/Collaborative Approaches in Teaching Requirements Engineering

    ERIC Educational Resources Information Center

    Rosca, Daniela

    2005-01-01

    The requirements engineering course is a core component of the curriculum for the Master's in Software Engineering programme, at Monmouth University (MU). It covers the process, methods and tools specific to this area, together with the corresponding software quality issues. The need to produce software engineers with strong teamwork and…

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

    PubMed

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

    2016-09-01

    The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein. PMID:26250617

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

    PubMed

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

    2016-09-01

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

  18. Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

    PubMed

    Nishiguchi, Tomoki; Cho, Kichul; Yasutomi, Masumi; Ueno, Mikinori; Yamaguchi, Kenichi; Basti, Leila; Yamasaki, Yasuhiro; Takeshita, Satoshi; Kim, Daekyung; Oda, Tatsuya

    2016-10-01

    A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both

  19. Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

    PubMed

    Nishiguchi, Tomoki; Cho, Kichul; Yasutomi, Masumi; Ueno, Mikinori; Yamaguchi, Kenichi; Basti, Leila; Yamasaki, Yasuhiro; Takeshita, Satoshi; Kim, Daekyung; Oda, Tatsuya

    2016-10-01

    A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both

  20. Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity

    PubMed Central

    Kulkarni, Gauri; Xu, Zhennan; Mohamed, Ahmed M.; Li, Haichang; Tang, Xia; Limerick, Gerard; Wadsworth, William G.

    2013-01-01

    Summary How the direction of axon guidance is determined is not understood. In Caenorhabditis elegans the UNC-40 (DCC) receptor mediates a response to the UNC-6 (netrin) guidance cue that directs HSN axon development. UNC-40 becomes asymmetrically localized within the HSN neuron to the site of axon outgrowth. Here we provide experimental evidence that the direction of guidance can be explained by the stochastic fluctuations of UNC-40 asymmetric outgrowth activity. We find that the UNC-5 (UNC5) receptor and the cytoskeletal binding protein UNC-53 (NAV2) regulate the induction of UNC-40 localization by UNC-6. If UNC-40 localization is induced without UNC-6 by using an unc-53 mutation, the direction of UNC-40 localization undergoes random fluctuations. Random walk models describe the path made by a succession of randomly directed movement. This model was experimentally tested using mutations that affect Wnt/PCP signaling. These mutations inhibit UNC-40 localization in the anterior and posterior directions. As the axon forms in Wnt/PCP mutants, the direction of UNC-40 localization randomly fluctuates; it can localize in either the anterior, posterior, or ventral direction. Consistent with a biased random walk, over time the axon will develop ventrally in response to UNC-6, even though at a discrete time UNC-40 localization and outgrowth can be observed anterior or posterior. Also, axon formation is slower in the mutants than in wild-type animals. This is also consistent with a random walk since this model predicts that the mean square displacement (msd) will increase only linearly with time, whereas the msd increases quadratically with time for straight-line motion. PMID:24337114

  1. Antiandrogenic activity of extracts of diesel exhaust particles emitted from diesel-engine truck under different engine loads and speeds.

    PubMed

    Okamura, Kazumasa; Kizu, Ryoichi; Toriba, Akira; Murahashi, Tsuyoshi; Mizokami, Atsushi; Burnstein, Kerry L; Klinge, Carolyn M; Hayakawa, Kazuichi

    2004-02-15

    To clarify the alteration of androgenic and antiandrogenic activities by diesel engine conditions, we collected diesel exhaust particles (DEP) samples emitted from a diesel-engine truck under different conditions of engine loads and vehicle speeds, and DEP extract (DEPE) samples were prepared from each. The androgenic and antiandrogenic activities of the DEPE samples were examined using a prostate specific antigen (PSA) promoter-luciferase reporter gene assay in PC3/AR human prostate cancer cells. While all DEPE samples did not exhibit androgenic effects, the antiandrogenic effects were enhanced by higher engine load but not by higher vehicle speed. In this study, significant correlations between antiandrogenic and aryl hydrocarbon receptor (AhR) agonistic activities were demonstrated in PC3/AR cells by 16 polycyclic aromatic compounds and beta-naphthoflavone. Yeast two-hybrid assay and cytochrome P450 (CYP) 1A1 promoter-luciferase reporter gene assay showed that the antiandrogenic constituents acting as androgen receptor (AR) antagonists and AhR agonists were increased by only the higher engine load. In conclusion, the antiandrogenic effects of DEPE samples were enhanced by a higher engine load which resulted in DEPC samples with elevated AhR agonistic and AR antagonistic activities.

  2. Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions.

    PubMed

    Danielsen, Andreas A; Parker, Mark D; Lee, Soojung; Boron, Walter F; Aalkjaer, Christian; Boedtkjer, Ebbe

    2013-03-22

    Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.

  3. Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells.

    PubMed

    Xue, Zhongfeng; Guo, Yalei; Fang, Yongqi

    2016-04-01

    Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aβ) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aβ1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aβ1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aβ1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aβ1-42. When 3-MA was applied this exacerbated the effect of Aβ1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aβ1-42 induced cytotoxicity and so may have a preventive function in AD. PMID:26923671

  4. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    PubMed

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points. PMID:27020293

  5. Expression and activation of intracellular receptors TLR7, TLR8 and TLR9 in peripheral blood monocytes from HIV-infected patients

    PubMed Central

    Pinzón Herrera, Francisc; Cruz López, Juan J; Vera Gamboa, Ligia del Carmen; Pavía Ruiz, Norma; Santos Rivero, Adrián; Sánchez Lugo, Saulo; Puerto, Fernando

    2013-01-01

    Introduction: TLR´s play a role in host defense in HIV infection recognizing the viral DNA or RNA. Their activation induces a signaling pathway that includes the proteins MyD88, IRAK4, TRAF6 and the transcription factor NF-kBp65. Objective: To determine the expression of TLR7, TLR8 and TLR9, and activation of its signaling pathway in monocytes from patients infected with HIV. Methods. Expression of TLR7, TLR8 and TLR9 was determined in monocytes from HIV-infected patients (n= 13) and control subjects (n= 13), which were activated with specific ligands. The expression of MyD88 and NF-kBp65 were determined by flow cytometry; IRAK4 and TRAF6 were studied by immunoblotting. Results: No statistical difference was found in the expression of TLR7, 8 and 9 in monocytes from patients compared to controls, but we observed the non-significant increased expression of TLR9 in patients. The activation showed no significant difference in the expression of MyD88 and NF-kBp65 in patients when compared to controls, but were decreased in stimulated cells over non-stimulated cells. IRAK4 and TRAF6 were not detected. Conclusions: No statistical difference was observed in the expression of intracellular TLRs, MyD88 and NFkBp65 in monocytes from patients compared to controls. This was probably due to effective antiretroviral therapy being received at the time of study entry. Additional studies are needed under controlled conditions that include infected patients with and without ARVT, responders and non-responders, and work with different cell populations. PMID:24892454

  6. Hydrogen sulfide and hypoxia-induced changes in TASK (K2P3/9) activity and intracellular Ca2+ concentration in rat carotid body glomus cells

    PubMed Central

    Kim, Donghee; Kim, Insook; Wang, Jiaju; White, Carl; Carroll, John L.

    2015-01-01

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that hydrogen sulfide (H2S) may serve as an oxygen sensor/signal in the carotid body during acute hypoxia. To further test such a role for H2S, we studied the effects of H2S on the activity of TASK channel and [Ca2+]i, which are considered important for mediating the glomus cell response to hypoxia. Like hypoxia, NaHS (a H2S donor) inhibited TASK activity and elevated [Ca2+]i. To inhibit the production of H2S, glomus cells were incubated (3 hr) with inhibitors of cystathionine-β-synthase and cystathionine-γ-lyase (DL-propargylglycine, aminooxyacetic acid, β-cyano-L-alanine; 0.3 mM). SF7 fluorescence was used to assess the level of H2S production. The inhibitors blocked L-cysteine- and hypoxia-induced elevation of SF7 fluorescence intensity. In cells treated with the inhibitors, hypoxia produced an inhibition of TASK activity and a rise in [Ca2+]i, similar in magnitude to those observed in control cells. L-cysteine produced no effect on TASK activity or [Ca2+]i and did not affect hypoxia-induced inhibition of TASK and elevation of [Ca2+]i. These findings suggest that under normal conditions, H2S is not a major signal in hypoxia-induced modulation of TASK channels and [Ca2+]i in isolated glomus cells. PMID:25956223

  7. Hydrogen sulfide and hypoxia-induced changes in TASK (K2P3/9) activity and intracellular Ca(2+) concentration in rat carotid body glomus cells.

    PubMed

    Kim, Donghee; Kim, Insook; Wang, Jiaju; White, Carl; Carroll, John L

    2015-08-15

    Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca(2+) concentration ([Ca(2+)]i). Recent studies suggest that hydrogen sulfide (H2S) may serve as an oxygen sensor/signal in the carotid body during acute hypoxia. To further test such a role for H2S, we studied the effects of H2S on the activity of TASK channel and [Ca(2+)]i, which are considered important for mediating the glomus cell response to hypoxia. Like hypoxia, NaHS (a H2S donor) inhibited TASK activity and elevated [Ca(2+)]i. To inhibit the production of H2S, glomus cells were incubated (3h) with inhibitors of cystathionine-β-synthase and cystathionine-γ-lyase (DL-propargylglycine, aminooxyacetic acid, β-cyano-L-alanine; 0.3 mM). SF7 fluorescence was used to assess the level of H2S production. The inhibitors blocked L-cysteine- and hypoxia-induced elevation of SF7 fluorescence intensity. In cells treated with the inhibitors, hypoxia produced an inhibition of TASK activity and a rise in [Ca(2+)]i, similar in magnitude to those observed in control cells. L-cysteine produced no effect on TASK activity or [Ca(2+)]i and did not affect hypoxia-induced inhibition of TASK and elevation of [Ca(2+)]i. These findings suggest that under normal conditions, H2S is not a major signal in hypoxia-induced modulation of TASK channels and [Ca(2+)]i in isolated glomus cells.

  8. Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella

    PubMed Central

    Jones-Carson, Jessica; Zweifel, Adrienne E.; Tapscott, Timothy; Austin, Chad; Brown, Joseph M.; Jones, Kenneth L.; Voskuil, Martin I.; Vázquez-Torres, Andrés

    2014-01-01

    Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics. PMID:25121731

  9. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    PubMed

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points.

  10. H⁺-activated Na⁺ influx in the ventricular myocyte couples Ca²⁺-signalling to intracellular pH.

    PubMed

    Garciarena, Carolina D; Youm, Jae Boum; Swietach, Pawel; Vaughan-Jones, Richard D

    2013-08-01

    Acid extrusion on Na(+)-coupled pH-regulatory proteins (pH-transporters), Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC), drives Na(+) influx into the ventricular myocyte. This H(+)-activated Na(+)-influx is acutely up-regulated at pHi<7.2, greatly exceeding Na(+)-efflux on the Na(+)/K(+) ATPase. It is spatially heterogeneous, due to the co-localisation of NHE1 protein (the dominant pH-transporter) with gap-junctions at intercalated discs. Overall Na(+)-influx via NBC is considerably lower, but much is co-localised with L-type Ca(2+)-channels in transverse-tubules. Through a functional coupling with Na(+)/Ca(2+) exchange (NCX), H(+)-activated Na(+)-influx increases sarcoplasmic-reticular Ca(2+)-loading and release during intracellular acidosis. This raises Ca(2+)-transient amplitude, rescuing it from direct H(+)-inhibition. Functional coupling is biochemically regulated and linked to membrane receptors, through effects on NHE1 and NBC. It requires adequate cytoplasmic Na(+)-mobility, as NHE1 and NCX are spatially separated (up to 60μm). The relevant functional NCX activity must be close to dyads, as it exerts no effect on bulk diastolic Ca(2+). H(+)-activated Na(+)-influx is up-regulated during ischaemia-reperfusion and some forms of maladaptive hypertrophy and heart failure. It is thus an attractive system for therapeutic manipulation. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

  11. Non-transferrin bound iron, cytokine activation and intracellular reactive oxygen species generation in hemodialysis patients receiving intravenous iron dextran or iron sucrose.

    PubMed

    Pai, Amy Barton; Conner, Todd; McQuade, Charles R; Olp, Jonathan; Hicks, Paul

    2011-08-01

    Intravenous (IV) iron supplementation is widely used to support erythropoeisis in hemodialysis patients. IV iron products are associated with oxidative stress that has been measured principally by circulating biomarkers such as products of lipid peroxidation. The pro-oxidant effects of IV iron are presumed to be due at least in part, by free or non-transferrin bound iron (NTBI). However, the effects of IV iron on intracellular redox status and downstream effectors is not known. This prospective, crossover study compared cytokine activation, reactive oxygen species generation and oxidative stress after single IV doses of iron sucrose and iron dextran. This was a prospective, open-label, crossover study. Ten patients with end-stage renal disease (ESRD) on hemodialysis and four age and sex-matched healthy were assigned to receive 100 mg of each IV iron product over 5 min in random sequence with a 2 week washout between products. Subjects were fasted and fed a low iron diet in the General Clinical Research Center at the University of New Mexico. Serum and plasma samples for IL-1, IL-6, TNF-α and IL-10 and NTBI were obtained at baseline, 60 and 240 min after iron infusion. Peripheral blood mononuclear cells (PBMC) were isolated at the same time points and stained with fluorescent probes to identify intracellular reactive oxygen species and mitochondrial membrane potential (Δψm) by flow cytometry. Lipid peroxidation was assessed by plasma F(2) isoprostane concentration. Mean ± SEM maximum serum NTBI values were significantly higher among patients receiving IS compared to ID (2.59 ± 0.31 and 1.0 ± 0.36 µM, respectively, P = 0.005 IS vs. ID) Mean ± SEM NTBI area under the serum concentration-time curve (AUC) was 3-fold higher after IS versus ID (202 ± 53 vs. 74 ± 23 µM*min/l, P = 0.04) in ESRD patients, indicating increased exposure to NTBI. IV iron administration was associated with increased pro-inflammatory cytokines. Serum IL-6 concentrations increased most

  12. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  13. Diverse age-related effects of Bacopa monnieri and donepezil in vitro on cytokine production, antioxidant enzyme activities, and intracellular targets in splenocytes of F344 male rats.

    PubMed

    Priyanka, Hannah P; Singh, Ran Vijay; Mishra, Miti; ThyagaRajan, Srinivasan

    2013-02-01

    Aged people are more prone to developing neurodegenerative and infectious diseases, autoimmune disorders, and cancer due to impairment of neuroendocrine-immune functions. Neuronal degeneration and immunosuppression aided by increased generation of reactive oxygen species combined with loss of antioxidant enzyme activities promote the aging process. Bacopa monnieri (brahmi), an Ayurvedic herb, and donepezil, a reversible acetylcholinesterase inhibitor, have been used to reverse cognitive dysfunctions in several neurodegenerative diseases. The aim of this study was to investigate the effects of in vitro incubation of lymphocytes from spleens of young (3-month-old), early middle-aged (8- to 9-month-old), and old (18-month-old) F344 rats with brahmi (0.001%, 0.01%, 0.05%, 0.1%, and 1%) and donepezil (5, 10, 25, 50, and 100 μg/ml) on Concanavalin (Con A)-induced proliferation of T lymphocytes and cytokine production, and the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)]. In addition, the effects of these compounds on the expression of intracellular signaling pathway markers (ERK, p-ERK, CREB, p-CREB, Akt and p-Akt), nitric oxide (NO) production, and the extent of lipid peroxidation were measured in the splenocytes. Age-related decline in Con A-induced proliferation of T lymphocytes was not reversed by treatment with brahmi and donepezil but donepezil alone further reduced the lymphocyte proliferation in young rats. Lower doses of brahmi treatment reversed the age-related decrease in Con A-induced IL-2 and IFN-γ production by the splenocytes while their production by splenocytes was suppressed by treatment with donepezil in the young and early middle-aged rats. An age-associated decline in the activities of SOD, CAT, GPx, and GST was evident in the lymphocytes of spleen. Brahmi enhanced CAT activity of lymphocytes in all the age groups while donepezil increased SOD

  14. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

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

    2011-12-01

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

  15. Enhancing Human Spermine Synthase Activity by Engineered Mutations

    PubMed Central

    Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

    2013-01-01

    Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. PMID:23468611

  16. Sequence, transcription activity, and evolutionary origin of the R-body coding plasmid pKAP298 from the intracellular parasitic bacterium Caedibacter taeniospiralis.

    PubMed

    Jeblick, Jörn; Kusch, Jürgen

    2005-02-01

    We isolated the intracellular parasitic bacterium Caedibacter taeniospiralis from cultures of the freshwater ciliate Paramecium tetraurelia strain 298. Plasmid pKAP298 as well as the total RNA were isolated from the bacteria. pKAP298 was totally sequenced (49.1 kb; NCBI accession number AY422720). From southern blots of pKAP-fragments and Digoxigenin-labeled cDNA of the Caedibacter-RNA, we generated transcription maps of pKAP298. The observed transcription activity indicated functions of the plasmid besides the synthesis of the R-body, a complex protein inclusion associated with toxic effects of Caedibacter cells on host paramecia. We identified 63 potential protein coding regions on pKAP298, and a novel transposon as well as known transposons were characterized. A group II intron was identified. Homologies with putative phage genes were detected on pKAP298 that direct to the evolution of pKAP298 from a bacteriophage. This original phage most probably belonged to the Caudovirales. Hints on a toxin coding region of pKAP298 are given: a protein with homology to the Soj-/ParA-family also has homologies to a membrane associated ATPase, which is involved in eukaryotic ATPase dependent ion carriers and may be associated with toxic effects on paramecia ingesting this protein.

  17. Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.

    PubMed

    Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C; Fan, Jian-Qiang

    2007-09-01

    Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

  18. Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

    PubMed Central

    Ishii, Satoshi; Chang, Hui-Hwa; Kawasaki, Kunito; Yasuda, Kayo; Wu, Hui-Li; Garman, Scott C.; Fan, Jian-Qiang

    2007-01-01

    Fabry disease is a lysosomal storage disorder caused by the deficiency of α-Gal A (α-galactosidase A) activity. In order to understand the molecular mechanism underlying α-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal Km and Vmax values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) α-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q α-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant α-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant α-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. PMID:17555407

  19. Guidelines for Engineering Teachers Concerning Educating the Engineer for Innovative and Entrepreneurial Activity.

    ERIC Educational Resources Information Center

    Eekels, J.

    1987-01-01

    Emphasizes that the concept of design is fundamental in innovation. Outlines the work of the European Society for Engineering Education-Working group on Innovation. Describes the innovation-management stream in the curriculum of the faculty of Industrial Design Engineering at Delft University of Technology, Netherlands. (CW)

  20. Energetic Beam Processing of Silicon to Engineer Optoelectronically Active Defects

    NASA Astrophysics Data System (ADS)

    Recht, Daniel

    This thesis explores ways to use ion implantation and nanosecond pulsed laser melting, both energetic beam techniques, to engineer defects in silicon. These defects are chosen to facilitate the use of silicon in optoelectronic applications for which its indirect bandgap is not ideal. Chapter 2 develops a kinetic model for the use of point defects as luminescence centers for light-emitting diodes and demonstrates an experimental procedure capable of high-throughput screening of the electroluminescent properties of such defects. Chapter 3 discusses the dramatic change in optical absorption observed in silicon highly supersaturated (i.e., hyperdoped) with the chalcogens sulfur, selenium, and tellurium and reports the first measurements of the optical absorption of such materials for photon energies greater than the bandgap of silicon. Chapter 3 examines the use of silicon hyperdoped with chalcogens in light detectors and concludes that while these devices display strong internal gain that is coupled to a particular type of surface defect, hyperdoping with chalcogens does not lead directly to measurable sub-bandgap photoconductivity. Chapter 4 considers the potential for Silicon to serve as the active material in an intermediate-band solar cell and reports experimental progress on two proposed approaches for hyperdoping silicon for this application. The main results of this chapter are the use of native-oxide etching to control the surface evaporation rate of sulfur from silicon and the first synthesis of monocrystalline silicon hyperdoped with gold.

  1. Effect of the ostreolysin A/pleurotolysin B pore-forming complex on neuroblastoma cell morphology and intracellular Ca²⁺ activity.

    PubMed

    Vrecl, Milka; Babnik, Monika; Diacci, Uroš; Benoit, Evelyne; Frangež, Robert

    2015-04-01

    Ostreolysin A (OlyA) and pleurotolysin B (PlyB), isolated from edible oyster mushrooms, form a cytolytic complex (OlyA/PlyB) in membrane cells that causes respiratory arrest. This study evaluated the mechanisms underlying cytotoxic OlyA/PlyB activity in neuroblastoma NG108-15 cells. Confocal microscopy with morphometric analysis revealed that OlyA/PlyB increased the 3-dimensional projected area of differentiated cells. Iso-osmotic replacement of NaCl by sucrose or Na-isethionate prevented the cellular swelling. This suggests that formation of cellular edema requires the presence of Na(+) and/or Cl(-) in the extracellular space and may be related to an influx of Na(+) and/or a shift in Cl(-), which induce a marked influx of water that is ultimately responsible for cellular swelling. In addition, extracellular Ca(2+) moderately contributed to the swelling because benzamil (10 µM), a 3Na(+)/Ca(2+) exchange (NCX) inhibitor, and Ca(2+)-free medium partially prevented this response. Fluorometric measurements revealed that OlyA/PlyB, at approximately 15-fold higher concentrations, increased the intracellular Ca(2+) activity [Ca(2+)]i. This increase was dependent on the presence of Na(+) and Ca(2+) in the external medium and was sensitive to benzamil. It is thus likely that a switch in the NCX mode, associated with the de novo formation of non-selective ion pores by OlyA/PlyB in cellular plasma membranes, plays an important role in this effect. Overall, OlyA/PlyB affects neuroblastoma cell morphology and Ca(2+) homeostasis to influence the toxin-induced respiratory arrest. PMID:25556216

  2. MiRImpact, a new bioinformatic method using complete microRNA expression profiles to assess their overall influence on the activity of intracellular molecular pathways

    PubMed Central

    Artcibasova, Alina V.; Korzinkin, Mikhail B.; Sorokin, Maksim I.; Shegay, Peter V.; Zhavoronkov, Alex A.; Gaifullin, Nurshat; Alekseev, Boris Y.; Vorobyev, Nikolay V.; Kuzmin, Denis V.; Kaprin, Аndrey D.; Borisov, Nikolay M.; Buzdin, Anton A.

    2016-01-01

    ABSTRACT MicroRNAs (miRs) are short noncoding RNA molecules that regulate expression of target mRNAs. Many published sources provide information about miRs and their targets. However, bioinformatic tools elucidating higher level impact of the established total miR profiles, are still largely missing. Recently, we developed a method termed OncoFinder enabling quantification of the activities of intracellular molecular pathways basing on gene expression data. Here we propose a new technique, MiRImpact, which enables to link miR expression data with its estimated outcome on the regulation of molecular pathways, like signaling, metabolic, cytoskeleton rearrangement, and DNA repair pathways. MiRImpact uses OncoFinder rationale for pathway activity calculations, with the major distinctions that (i) it deals with the concentrations of miRs - known regulators of gene products participating in molecular pathways, and (ii) miRs are considered as negative regulators of target molecules, if other is not specified. MiRImpact operates with 2 types of databases: for molecular targets of miRs and for gene products participating in molecular pathways. We applied MiRImpact to compare regulation of human bladder cancer-specific signaling pathways at the levels of mRNA and miR expression. We took 2 most complete alternative databases of experimentally validated miR targets – miRTarBase and DianaTarBase, and an OncoFinder database featuring 2725 gene products and 271 signaling pathways. We showed that the impact of miRs is orthogonal to pathway regulation at the mRNA level, which stresses the importance of studying posttranscriptional regulation of gene expression. We also report characteristic set of miR and mRNA regulation features linked with bladder cancer. PMID:27027999

  3. Ethanol Enhances TGF-β Activity by Recruiting TGF-β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Johnson, Frank E; Huang, Jung San

    2016-04-01

    Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼ 2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼ 2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues.

  4. Engineering and Technology Challenges for Active Debris Removal

    NASA Technical Reports Server (NTRS)

    Liou, Jer-Chyi

    2011-01-01

    After more than fifty years of space activities, the near-Earth environment is polluted with man-made orbital debris. The collision between Cosmos 2251 and the operational Iridium 33 in 2009 signaled a potential collision cascade effect, also known as the "Kessler Syndrome", in the environment. Various modelling studies have suggested that the commonly-adopted mitigation measures will not be sufficient to stabilize the future debris population. Active debris removal must be considered to remediate the environment. This paper summarizes the key issues associated with debris removal and describes the technology and engineering challenges to move forward. Fifty-four years after the launch of Sputnik 1, satellites have become an integral part of human society. Unfortunately, the ongoing space activities have left behind an undesirable byproduct orbital debris. This environment problem is threatening the current and future space activities. On average, two Shuttle window panels are replaced after every mission due to damage by micrometeoroid or orbital debris impacts. More than 100 collision avoidance maneuvers were conducted by satellite operators in 2010 to reduce the impact risks of their satellites with respect to objects in the U.S. Space Surveillance Network (SSN) catalog. Of the four known accident collisions between objects in the SSN catalog, the last one, collision between Cosmos 2251 and the operational Iridium 33 in 2009, was the most significant. It was the first ever accidental catastrophic destruction of an operational satellite by another satellite. It also signaled the potential collision cascade effect in the environment, commonly known as the "Kessler Syndrome," predicted by Kessler and Cour-Palais in 1978 [1]. Figure 1 shows the historical increase of objects in the SSN catalog. The majority of the catalog objects are 10 cm and larger. As of April 2011, the total objects tracked by the SSN sensors were more than 22,000. However, approximately 6000 of

  5. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  6. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits.

  7. Intracellular shunting of O{sub 2}{sup −} contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

    SciTech Connect

    Decleva, Eva; Menegazzi, Renzo; Fasolo, Alba; Defendi, Federica

    2013-07-15

    Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn{sup 2+}, we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O{sub 2}{sup −} and H{sub 2}O{sub 2}, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H{sub 2}O{sub 2} production. Furthermore, we show that inhibiting Hv1 with Zn{sup 2+} results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H{sub 2}O{sub 2}. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O{sub 2}{sup −} generated within gp91{sup phox} in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux. Highlights: • Neutrophils’ respiratory burst is not inhibited by the H{sup +} channel inhibitor Zn{sup 2+}. • Intracellular production of O{sub 2}{sup −} and H{sub 2}O{sub 2} is increased in the presence of Zn{sup 2+}. • Intracellular H{sub 2}O{sub 2} production is increased in H{sup +} channels knock-down cells. • Zn{sup 2+} increases the rate but not the extent of

  8. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei.

    PubMed

    Abdelbaqi, Suha; Deslouches, Berthony; Steckbeck, Jonathan; Montelaro, Ronald; Reed, Douglas S

    2016-02-01

    Broad-spectrum antimicrobials are needed to effectively treat patients infected in the event of a pandemic or intentional release of a pathogen prior to confirmation of the pathogen's identity. Engineered cationic antimicrobial peptides (eCAPs) display activity against a number of bacterial pathogens including multi-drug-resistant strains. Two lead eCAPs, WLBU2 and WR12, were compared with human cathelicidin (LL-37) against three highly pathogenic bacteria: Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. Both WLBU2 and WR12 demonstrated bactericidal activity greater than that of LL-37, particularly against F. tularensis and Y. pestis. Only WLBU2 had bactericidal activity against B. pseudomallei. WLBU2, WR12 and LL-37 were all able to inhibit the growth of the three bacteria in vitro. Because these bacteria can be facultative intracellular pathogens, preferentially infecting macrophages and dendritic cells, we evaluated the activity of WLBU2 against F. tularensis in an ex vivo infection model with J774 cells, a mouse macrophage cell line. In that model WLBU2 was able to achieve greater than 50% killing of F. tularensis at a concentration of 12.5 μM. These data show the therapeutic potential of eCAPs, particularly WLBU2, as a broad-spectrum antimicrobial for treating highly pathogenic bacterial infections. PMID:26673248

  9. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  10. Active Class Composed with Mechanical Engineering and Environment

    NASA Astrophysics Data System (ADS)

    Torii, Shuichi; Imamura, Yasuhiro; Ohshima, Yasutaka; Ariyoshi, Kouji

    This paper reports that the learning experience for a group working disassembling and assembling an internal combustion engine is constructed for a group working. At the same time, the environment education combined with basic studies is introduced with the aid of a Diesel engine running with biofuel. It is found that (1) the leaning experience enhances the leaning motivation and problem solution ability for students, (2) a group working disassembling and assembling an internal combustion engine is effective for greediness for learning, and (3) the environmental study attracts student.

  11. Anti-plasmodial activity of aroylhydrazone and thiosemicarbazone iron chelators: effect on erythrocyte membrane integrity, parasite development and the intracellular labile iron pool.

    PubMed

    Walcourt, Asikiya; Kurantsin-Mills, Joseph; Kwagyan, John; Adenuga, Babafemi B; Kalinowski, Danuta S; Lovejoy, David B; Lane, Darius J R; Richardson, Des R

    2013-12-01

    Iron chelators inhibit the growth of the malaria parasite, Plasmodium falciparum, in culture and in animal and human studies. We previously reported the anti-plasmodial activity of the chelators, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), 2-hydroxy-1-naphthylaldehyde 4-methyl-3-thiosemicarbazone (N4mT), and 2-hydroxy-1-naphthylaldehyde 4-phenyl-3-thiosemicarbazone (N4pT). In fact, these ligands showed greater growth inhibition of chloroquine-sensitive (3D7) and chloroquine-resistant (7G8) strains of P. falciparum in culture compared to desferrioxamine (DFO). The present study examined the effects of 311, N4mT and N4pT on erythrocyte membrane integrity and asexual parasite development. While the characteristic biconcave disk shape of the erythrocytes was unaffected, the chelators caused very slight hemolysis at IC50 values that inhibited parasite growth. The chelators 311, N4mT and N4pT affected all stages of the intra-erythrocytic development cycle (IDC) of P. falciparum in culture. However, while these ligands primarily affected the ring-stage, DFO inhibited primarily trophozoite and schizont-stages. Ring, trophozoite and schizont-stages of the IDC were inhibited by significantly lower concentrations of 311, N4mT, and N4pT (IC50=4.45±1.70, 10.30±4.40, and 3.64±2.00μM, respectively) than DFO (IC50=23.43±3.40μM). Complexation of 311, N4mT and N4pT with iron reduced their anti-plasmodial activity. Estimation of the intracellular labile iron pool (LIP) in erythrocytes showed that the chelation efficacy of 311, N4mT and N4pT corresponded to their anti-plasmodial activities, suggesting that the LIP may be a potential source of non-heme iron for parasite metabolism within the erythrocyte. This study has implications for malaria chemotherapy that specifically disrupts parasite iron utilization.

  12. Analyzing the Function of Cartilage Replacements: A Laboratory Activity to Teach High School Students Chemical and Tissue Engineering Concepts

    ERIC Educational Resources Information Center

    Renner, Julie N.; Emady, Heather N.; Galas, Richards J., Jr.; Zhange, Rong; Baertsch, Chelsey D.; Liu, Julie C.

    2013-01-01

    A cartilage tissue engineering laboratory activity was developed as part of the Exciting Discoveries for Girls in Engineering (EDGE) Summer Camp sponsored by the Women In Engineering Program (WIEP) at Purdue University. Our goal was to increase awareness of chemical engineering and tissue engineering in female high school students through a…

  13. Biomolecular engineering of intracellular switches in eukaryotes

    PubMed Central

    Pastuszka, M.K.; Mackay, J.A.

    2010-01-01

    Tools to selectively and reversibly control gene expression are useful to study and model cellular functions. When optimized, these cellular switches can turn a protein's function “on” and “off” based on cues designated by the researcher. These cues include small molecules, drugs, hormones, and even temperature variations. Here we review three distinct areas in gene expression that are commonly targeted when designing cellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, with examples including the tetracycline gene induction system as well as nuclear receptors. Translational switches target the process of turning the mRNA signal into protein, with examples including riboswitches and RNA interference. Post-translational switches control how proteins interact with one another to attenuate or relay signals. Examples of post-translational modification include dimerization and intein splicing. In general, the delay times between switch and effect decreases from transcription to translation to post-translation; furthermore, the fastest switches may offer the most elegant opportunities to influence and study cell behavior. We discuss the pros and cons of these strategies, which directly influence their usefulness to study and implement drug targeting at the tissue and cellular level. PMID:21209849

  14. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  15. Synthetic nanocarriers for intracellular protein delivery.

    PubMed

    Du, Juanjuan; Jin, Jing; Yan, Ming; Lu, Yunfeng

    2012-01-01

    Introducing exogenous proteins intracellularly presents tremendous chances in scientific research and clinical applications. The effectiveness of this method, however, has been limited by lack of efficient ways to achieve intracellular protein delivery and poor stability of the delivered proteins. Over the years, a variety of nanomaterials have been explored as intracellular protein delivery vectors, including liposomes, polymers, gold nanoparticles, mesoporous silica particles, and carbon nanotubes. Nanomaterials stand out in various protein delivery systems due to various advantages, such as efficient intracellular delivery, long circulation time, and passive tumor targeting. Additionally, chemistry behind these nanomaterials provides readily engineered materials, enabling versatile designs of delivery agents. Intracellular delivery mediated by such nanocarriers achieved varying degrees of success. Different problems associated with these nanocarriers, however, still hamper their real-world applications. Developing new delivery methods or vectors remains essential but challenging. This review surveys the current developments in protein delivery based on synthetic nanocarriers, including liposomes, polymers and inorganic nanocarriers; Prospects for future development of protein delivery nanocarriers are also provided.

  16. Methodology for the systems engineering process. Volume 1: System functional activities

    NASA Technical Reports Server (NTRS)

    Nelson, J. H.

    1972-01-01

    Systems engineering is examined in terms of functional activities that are performed in the conduct of a system definition/design, and system development is described in a parametric analysis that combines functions, performance, and design variables. Emphasis is placed on identification of activities performed by design organizations, design specialty groups, as well as a central systems engineering organizational element. Identification of specific roles and responsibilities for doing functions, and monitoring and controlling activities within the system development operation are also emphasized.

  17. Engineered enzymatically active bacteriophages and methods of uses thereof

    DOEpatents

    Collins, James J; Kobayashi, Hideki; Kearn, Mads; Araki, Michihiro; Friedland, Ari; Lu, Timothy Kuan-Ta

    2012-05-22

    The present invention provides engineered bacteriophages that express at least one biofilm degrading enzyme on their surface and uses thereof for degrading bacterial biofilms. The invention also provides genetically engineered bacteriophages expressing the biofilm degrading enzymes and proteins necessary for the phage to replicate in different naturally occurring biofilm producing bacteria. The phages of the invention allow a method of biofilm degradation by the use of one or only a few administration of the phage because the system using these phages is self perpetuating, and capable of degrading biofilm even when the concentration of bacteria within the biofilm is low.

  18. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

    PubMed Central

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A.; Schreiber, Stuart L.; Ioset, Jean-Robert; Gray, David W.

    2015-01-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. PMID:26407168

  19. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity.

    PubMed

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A; Schreiber, Stuart L; Ioset, Jean-Robert; Gray, David W

    2015-09-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. PMID:26407168

  20. Engineering as a Social Activity: Preparing Engineers to Thrive in the Changing World of Work

    ERIC Educational Resources Information Center

    Joyner, Fredricka F.; Mann, Derek T. Y.; Harris, Todd

    2012-01-01

    Key macro-trends are combining to create a new work context for the practice of engineering. Telecommuting and virtual teams create myriad possibilities and challenges related to managing work and workers. Social network technology tools allow for unprecedented global, 24/7 collaboration. Globalization has created hyper-diverse organizations,…

  1. Technology and Engineering Education Students' Perceptions of Hands-On and Hands-Off Activities

    ERIC Educational Resources Information Center

    Sianez, David M.; Fugere, Madeleine A.; Lennon, Carter A.

    2010-01-01

    Technology and engineering education students responded to a survey regarding hands-on and hands-off activities. First, the students listed hands-on and hands-off activities and what characterized the two types of activities. Activities such as building or assembling something as well as working manually with tools were viewed as hands-on. Passive…

  2. Anti-Human Immunodeficiency Virus Type 1 Activity, Intracellular Metabolism, and Pharmacokinetic Evaluation of 2′-Deoxy-3′-Oxa-4′-Thiocytidine

    PubMed Central

    de Muys, Jean-Marc; Gourdeau, Henriette; Nguyen-Ba, Nghe; Taylor, Debra L.; Ahmed, Parvin S.; Mansour, Tarek; Locas, Celine; Richard, Nathalie; Wainberg, Mark A.; Rando, Robert F.

    1999-01-01

    The racemic nucleoside analogue 2′-deoxy-3′-oxa-4′-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5′-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The Ki values for dOTC-TP obtained against human DNA polymerases α, β, and γ were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 μM, rising to only 2.53 and 2.5 μM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [3H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 μM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 μM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability

  3. 77 FR 3844 - Agency Information Collection (Architect-Engineer Fee Proposal) Activity Under OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-25

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF VETERANS AFFAIRS Agency Information Collection (Architect--Engineer Fee Proposal) Activity Under OMB Review AGENCY...: a. Architect--Engineer Fee Proposal, VA Form 10-6298. b. Daily Log (Contract Progress...

  4. Design and Implementation of a Project-Based Active/Cooperative Engineering Design Course for Freshmen

    ERIC Educational Resources Information Center

    Abdulaal, R. M.; Al-Bahi, A. M.; Soliman, A. Y.; Iskanderani, F. I.

    2011-01-01

    A project-based active/cooperative design course is planned, implemented, assessed and evaluated to achieve several desired engineering outcomes. The course allows freshman-level students to gain professional hands-on engineering design experience through an opportunity to practise teamwork, quality principles, communication skills, life-long…

  5. The Role of Entrepreneurship Program Models and Experiential Activities on Engineering Student Outcomes

    ERIC Educational Resources Information Center

    Duval-Couetil, Nathalie; Shartrand, Angela; Reed, Teri

    2016-01-01

    Entrepreneurship education is being delivered to greater numbers of engineering students through a variety of courses, programs, and experiential learning activities. Some of these opportunities are designed primarily to serve engineering students in their departments and colleges, while others are cross-campus, university-wide efforts to serve…

  6. Manpower and Financial Resources Allocated to Academic Science and Engineering Activities, 1965-71.

    ERIC Educational Resources Information Center

    National Science Foundation, Washington, DC. Div. of Science Resources Studies.

    This report summarizes the results of the National Science Foundation's biennial survey of manpower and financial resources for scientific engineering activities at institutions of higher education, 1971. The survey was conducted by mail questionnaires sent to 2,198 universities and colleges that maintained science and engineering programs, and…

  7. The Complex Dynamics of Student Engagement in Novel Engineering Design Activities

    NASA Astrophysics Data System (ADS)

    McCormick, Mary

    In engineering design, making sense of "messy," design situations is at the heart of the discipline (Schon, 1983); engineers in practice bring structure to design situations by organizing, negotiating, and coordinating multiple aspects (Bucciarelli, 1994; Stevens, Johri, & O'Connor, 2014). In classroom settings, however, students are more often given well-defined, content-focused engineering tasks (Jonassen, 2014). These tasks are based on the assumption that elementary students are unable to grapple with the complexity or open-endedness of engineering design (Crismond & Adams, 2012). The data I present in this dissertation suggest the opposite. I show that students are not only able to make sense of, or frame (Goffman, 1974), complex design situations, but that their framings dynamically involve their nascent abilities for engineering design. The context of this work is Novel Engineering, a larger research project that explores using children's literature as an access point for engineering design. Novel Engineering activities are inherently messy: there are characters with needs, settings with implicit constraints, and rich design situations. In a series of three studies, I show how students' framings of Novel Engineering design activities involve their reasoning and acting as beginning engineers. In the first study, I show two students whose caring for the story characters contributes to their stability in framing the task: they identify the needs of their fictional clients and iteratively design a solution to meet their clients' needs. In the second, I show how students' shifting and negotiating framings influence their engineering assumptions and evaluation criteria. In the third, I show how students' coordinating framings involve navigating a design process to meet clients' needs, classroom expectations, and technical requirements. Collectively, these studies contribute to literature by documenting students' productive beginnings in engineering design. The

  8. Introducing a new semi-active engine mount using force controlled variable stiffness

    NASA Astrophysics Data System (ADS)

    Azadi, Mojtaba; Behzadipour, Saeed; Faulkner, Gary

    2013-05-01

    This work introduces a new concept in designing semi-active engine mounts. Engine mounts are under continuous development to provide better and more cost-effective engine vibration control. Passive engine mounts do not provide satisfactory solution. Available semi-active and active mounts provide better solutions but they are more complex and expensive. The variable stiffness engine mount (VSEM) is a semi-active engine mount with a simple ON-OFF control strategy. However, unlike available semi-active engine mounts that work based on damping change, the VSEM works based on the static stiffness change by using a new fast response force controlled variable spring. The VSEM is an improved version of the vibration mount introduced by the authors in their previous work. The results showed significant performance improvements over a passive rubber mount. The VSEM also provides better vibration control than a hydromount at idle speed. Low hysteresis and the ability to be modelled by a linear model in low-frequency are the advantages of the VSEM over the vibration isolator introduced earlier and available hydromounts. These specifications facilitate the use of VSEM in the automotive industry, however, further evaluation and developments are needed for this purpose.

  9. Software Engineering Support Activities for Very Small Entities

    NASA Astrophysics Data System (ADS)

    Ribaud, Vincent; Saliou, Philippe; O'Connor, Rory V.; Laporte, Claude Y.

    The emerging ISO/IEC 29110 standard Lifecycle profiles for Very Small Entities has at its core a Management and Engineering Guides which is targeted at very small entity (enterprise, organization, department or project) having up to 25 people, to assist them unlock the potential benefits of using standards which are specifically designed to address there needs. The developers of the standard, ISO/IEC JCT1/SC7 Working Group 24 (WG24), recommend the use of pilot projects as a mean to trial the adoption of the new International standard in small organisations. Accordingly an ISO/IEC 29110 pilot project has been established between the Software Engineering group of Brest University and a 14 person company with the aim of establishing an engineering discipline for a new web-based project. This paper details the lessons learned from the pilot project and based on our experiences with using ISO/IEC 29110 we identify a potential deficiency and accordingly propose new process area, "Infrastructure and Support" for include in the future evolution of ISO/IEC 29110 Process Profiles.

  10. Structural analysis of the human interferon gamma receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity.

    PubMed Central

    Cook, J R; Jung, V; Schwartz, B; Wang, P; Pestka, S

    1992-01-01

    Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor. Images PMID:1454813

  11. Three Dimensions of Learning: Experiential Activity for Engineering Innovation Education and Research

    ERIC Educational Resources Information Center

    Killen, Catherine P.

    2015-01-01

    This paper outlines a novel approach to engineering education research that provides three dimensions of learning through an experiential class activity. A simulated decision activity brought current research into the classroom, explored the effect of experiential activity on learning outcomes and contributed to the research on innovation decision…

  12. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  13. Adenylyl cyclase activation underlies intracellular cyclic AMP accumulation, cyclic AMP transport, and extracellular adenosine accumulation evoked by beta-adrenergic receptor stimulation in mixed cultures of neurons and astrocytes derived from rat cerebral cortex.

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

    We have previously shown that stimulation of cortical cultures containing both neurons and astrocytes with the beta-adrenergic agonist isoproterenol (ISO) results in transport of cAMP from astrocytes followed by extracellular hydrolysis to adenosine [Rosenberg et al. J. Neurosci. 14 (1994) 2953-2965]. In this study we found that the endogenous catecholamines epinephrine (EPI) and norepinephrine (NE), but not dopamine, serotonin, or histamine, all at 10 microM, significantly stimulated intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation in cortical cultures. Detailed dose-response experiments were performed for NE and EPI, as well as ISO. For each catecholamine, the potencies in evoking intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation were similar. These data provide additional evidence that a single common mechanism, namely beta-adrenergic mediated activation of adenylyl cyclase, underlies intracellular cAMP accumulation, cAMP transport, and extracellular adenosine accumulation. It appears that regulation of extracellular adenosine levels via cAMP transport and extracellular hydrolysis to adenosine may be a final common pathway of neuromodulation in cerebral cortex for catecholamines, and, indeed, any substance whose receptors are coupled to adenylyl cyclase.

  14. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

    PubMed

    Guthrie, H D; Welch, G R

    2006-08-01

    The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen

  15. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    SciTech Connect

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  16. Structural Engineering. Technology Learning Activity. Teacher Edition. Technology Education Series.

    ERIC Educational Resources Information Center

    Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

    This curriculum guide provides technology learning activities designed to prepare students in grades 6-10 to work in the world of the future. The 8-day course provides exploratory, hands-on learning activities and information that can enhance the education of students of all types in an integrated curriculum that provides practical applications of…

  17. Population Pharmacokinetic/Pharmacodynamic Analysis of the Bactericidal Activities of Sutezolid (PNU-100480) and Its Major Metabolite against Intracellular Mycobacterium tuberculosis in Ex Vivo Whole-Blood Cultures of Patients with Pulmonary Tuberculosis

    PubMed Central

    Zhu, Tong; Friedrich, Sven O.; Diacon, Andreas

    2014-01-01

    Sutezolid (PNU-100480 [U-480]) is an oxazolidinone antimicrobial being developed for the treatment of tuberculosis. An active sulfoxide metabolite (PNU-101603 [U-603]), which reaches concentrations in plasma several times those of the parent, has been reported to drive the killing of extracellular Mycobacterium tuberculosis by sutezolid in hollow-fiber culture. However, the relative contributions of the parent and metabolite against intracellular M. tuberculosis in vivo are not fully understood. The relationships between the plasma concentrations of U-480 and U-603 and intracellular whole-blood bactericidal activity (WBA) in ex vivo cultures were examined using a direct competitive population pharmacokinetic (PK)/pharmacodynamic 4-parameter sigmoid model. The data set included 690 PK determinations and 345 WBA determinations from 50 tuberculosis patients enrolled in a phase 2a sutezolid trial. The model parameters were solved iteratively. The median U-603/U-480 concentration ratio was 7.1 (range, 1 to 28). The apparent 50% inhibitory concentration of U-603 for intracellular M. tuberculosis was 17-fold greater than that of U-480 (90% confidence interval [CI], 9.9- to 53-fold). Model parameters were used to simulate in vivo activity after oral dosing with sutezolid at 600 mg twice a day (BID) and 1,200 mg once a day (QD). Divided dosing resulted in greater cumulative activity (−0.269 log10 per day; 90% CI, −0.237 to −0.293 log10 per day) than single daily dosing (−0.186 log10 per day; 90% CI, −0.160 to −0.208 log10 per day). U-480 accounted for 84% and 78% of the activity for BID and QD dosing, respectively, despite the higher concentrations of U-603. Killing of intracellular M. tuberculosis by orally administered sutezolid is mainly due to the activity of the parent compound. Taken together with the findings of other studies in the hollow-fiber model, these findings suggest that sutezolid and its metabolite act on different mycobacterial subpopulations

  18. Laser-activated remote phosphor light engine for projection applications

    NASA Astrophysics Data System (ADS)

    Daniels, Martin; Mehl, Oliver; Hartwig, Ulrich

    2015-09-01

    Recent developments in blue emitting laser diodes enable attractive solutions in projection applications using phosphors for efficient light conversion with very high luminance levels. Various commercially available projectors incorporating this technology have entered the market in the past years. While luminous flux levels are still comparable to lamp-based systems, lifetime expectations of classical lamp systems are exceeded by far. OSRAM GmbH has been exploring this technology for several years and has introduced the PHASER® brand name (Phosphor + laser). State-of-the-art is a rotating phosphor wheel excited by blue laser diodes to deliver the necessary primary colors, either sequentially for single-imager projection engines, or simultaneously for 3-panel systems. The PHASER® technology enables flux and luminance scaling, which allows for smaller imagers and therefore cost-efficient projection solutions. The resulting overall efficiency and ANSI lumen specification at the projection screen of these systems is significantly determined by the target color gamut and the light transmission efficiency of the projection system. With increasing power and flux level demand, thermal issues, especially phosphor conversion related, dominate the opto-mechanical system design requirements. These flux levels are a great challenge for all components of an SSL-projection system (SSL:solid-state lighting). OSRAḾs PHASER® light engine platform is constantly expanded towards higher luminous flux levels as well as higher luminance levels for various applications. Recent experiments employ blue laser pump powers of multiple 100 Watts to excite various phosphors resulting in luminous flux levels of more than 40 klm.

  19. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S.

    2010-06-01

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  20. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S

    2013-02-19

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  1. Neuronal in vitro activity is more sensitive to valproate than intracellular ATP: Considerations on conversion problems of IC50 in vitro data for animal replacement.

    PubMed

    Nissen, Matthias; Buehler, Sebastian M; Stubbe, Marco; Gimsa, Jan

    2016-06-01

    We investigated the effects of acute valproate (VPA) on mouse embryonic primary cortex cells (MEPCs). Intracellular ATP concentrations were compared with changes in the mean action potential (AP) frequencies of MEPC networks growing on microelectrode arrays. Our data implies biphasic reactions towards increasing VPA concentrations for both parameters. Intracellular ATP and mean AP frequencies increased around characteristic concentrations of 0.15 and 0.07mM to hormetic plateaus of approx. 120% and 160% of their controls, before fading around 17 and 1.7 mM, respectively. The biphasic in vitro behavior of the two parameters hinders a simple extraction of IC50 and Hillslope values. Different ways of data-fitting with single and double logistic functions are discussed. For a typical hormetic increase of 60% above control, IC50 and Hillslope were decreased by 37% and 15%, respectively. Despite these marginal effects at a logarithmic concentration scale, the hormetic and double logistic behavior of parameters may provide information on the mode of action of toxic compounds. Comparison of our values with the LD50 of mice, recalculated by normalization to body mass, suggests that a neurotoxic rather than a cytotoxic mechanism is killing the animals. The future use of cellular microsystems to replace animal experiments will motivate the development of new microsensors, as well as the consideration of newly accessible parameters in systems biology models. PMID:27091084

  2. Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody.

    PubMed Central

    Marasco, W A; Haseltine, W A; Chen, S Y

    1993-01-01

    A single-chain antibody, derived from a human monoclonal antibody that recognizes the CD4 binding region of the human immunodeficiency virus type 1 (HIV-1) envelope protein, has been designed for intracellular expression in eukaryotic cells. The single-chain antibody is composed of an immunoglobulin heavy-chain leader sequence and heavy- and light-chain variable regions that are joined by an interchain linker. The antibody is stably expressed and retained in the endoplasmic reticulum and is not toxic to the cells. The antibody binds to the envelope protein within the cell and inhibits processing of the envelope precursor and syncytia formation. The infectivity of the HIV-1 particles produced by cells that express the single-chain antibody is substantially reduced. These studies illustrate the feasibility of designing antibodies that bind and inactivate molecules intracellularly. Antibodies that act on target molecules within cells should provide a useful tool for research as well as for control of infectious and other diseases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8356098

  3. Collaborative Human Engineering Work in Space Exploration Extravehicular Activities (EVA)

    NASA Technical Reports Server (NTRS)

    DeSantis, Lena; Whitmore, Mihriban

    2007-01-01

    A viewgraph presentation on extravehicular activities in space exploration in collaboration with other NASA centers, industries, and universities is shown. The topics include: 1) Concept of Operations for Future EVA activities; 2) Desert Research and Technology Studies (RATS); 3) Advanced EVA Walkback Test; 4) Walkback Subjective Results; 5) Integrated Suit Test 1; 6) Portable Life Support Subsystem (PLSS); 7) Flex PLSS Design Process; and 8) EVA Information System; 9)

  4. Determination of intracellular nitrate.

    PubMed Central

    Romero, J M; Lara, C; Guerrero, M G

    1989-01-01

    A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans. PMID:2497740

  5. Engineering a hyper-catalytic enzyme by photo-activated conformation modulation

    SciTech Connect

    Agarwal, Pratul K

    2012-01-01

    Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in cis and trans conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8-52 fold better catalytic activity than the unmodulated enzyme.

  6. Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

    PubMed Central

    Lahti, Jennifer L.; Lui, Bertrand H.; Beck, Stayce E.; Lee, Stephen S.; Ly, Daphne P.; Longaker, Michael T.; Yang, George P.; Cochran, Jennifer R.

    2011-01-01

    Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity. PMID:21439278

  7. Engine

    SciTech Connect

    Shin, H.B.

    1984-02-28

    An internal combustion engine has a piston rack depending from each piston. This rack is connected to a power output shaft through a mechanical rectifier so that the power output shaft rotates in only one direction. A connecting rod is pivotally connected at one end to the rack and at the other end to the crank of a reduced function crankshaft so that the crankshaft rotates at the same angular velocity as the power output shaft and at the same frequency as the pistons. The crankshaft has a size, weight and shape sufficient to return the pistons back into the cylinders in position for the next power stroke.

  8. AdipoR-increased intracellular ROS promotes cPLA2 and COX-2 expressions via activation of PKC and p300 in adiponectin-stimulated human alveolar type II cells.

    PubMed

    Chen, Hsiao-Mei; Yang, Chuen-Mao; Chang, Jia-Feng; Wu, Chi-Sheng; Sia, Kee-Chin; Lin, Wei-Ning

    2016-08-01

    Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases. PMID:27288489

  9. Inhalable DNase I microparticles engineered with biologically active excipients.

    PubMed

    Osman, Rihab; Al Jamal, Khuloud T; Kan, Pei-Lee; Awad, Gehanne; Mortada, Nahed; El-Shamy, Abd-Elhameed; Alpar, Oya

    2013-12-01

    Highly viscous mucus poses a big challenge for the delivery of particulates carrying therapeutics to patients with cystic fibrosis. In this study, surface modifying DNase I loaded particles using different excipients to achieve better lung deposition, higher enzyme stability or better biological activity had been exploited. For the purpose, controlled release microparticles (MP) were prepared by co-spray drying DNase I with the polymer poly-lactic-co-glycolic acid (PLGA) and the biocompatible lipid surfactant 1,2-dipalmitoyl-Sn-phosphatidyl choline (DPPC) using various hydrophilic excipients. The effect of the included modifiers on the particle morphology, size, zeta potential as well as enzyme encapsulation efficiency, biological activity and release had been evaluated. Powder aerosolisation performance and particle phagocytosis by murine macrophages were also investigated. The results showed that more than 80% of enzyme activity was recovered after MP preparation and that selected surface modifiers greatly increased the enzyme encapsulation efficiency. The particle morphology was greatly modified altering in turn the powders inhalation indices where dextran, ovalbumin and chitosan hydrochloride increased considerably the respirable fraction compared to the normal hydrophilic carriers lactose and PVP. Despite of the improved aerosolisation caused by chitosan hydrochloride, yet retardation of chitosan coated particles in artificial mucus samples discouraged its application. On the other hand, dextran and polyanions enhanced DNase I effect in reducing cystic fibrosis mucus viscosity. DPPC proved good ability to reduce particles phagocytic uptake even in the presence of the selected adjuvants. The prepared MP systems were biocompatible with lung epithelial cells. To conclude, controlled release DNase I loaded PLGA-MP with high inhalation indices and enhanced mucolytic activity on CF sputum could be obtained by surface modifying the particles with PGA or dextran. PMID

  10. Experimental Investigation of Active Noise Controller for Internal Combustion Engine Exhaust System

    NASA Astrophysics Data System (ADS)

    Wu, Jian-Da; Chen, Chih-Keng; Lee, Chun-Ying; Lee, Tian-Hua

    2002-10-01

    Two active noise control (ANC) algorithms for internal combustion engine exhaust systems are developed and their performances are compared in various experiments. The first controller is based on the filtered-x least mean square (FXLMS) algorithm with feedback neutralization, while the second is a fixed controller with a gain-scheduled active control technique for broadband attenuation with thermal effects. Both control algorithms are implemented on a digital signal processing (DSP) platform. Experiments are carried out to evaluate the attenuation performance of the proposed active noise control systems for an engine exhaust system. The results of the experiments indicate that both the adaptive controller and the gain-scheduled controller effectively suppress the noise of engine exhaust systems. The experimental comparison and analysis of the proposed controllers are also described.

  11. Three dimensions of learning: experiential activity for engineering innovation education and research

    NASA Astrophysics Data System (ADS)

    Killen, Catherine P.

    2015-09-01

    This paper outlines a novel approach to engineering education research that provides three dimensions of learning through an experiential class activity. A simulated decision activity brought current research into the classroom, explored the effect of experiential activity on learning outcomes and contributed to the research on innovation decision making. The 'decision task' was undertaken by more than 480 engineering students. It increased their reported measures of learning and retention by an average of 0.66 on a five-point Likert scale, and revealed positive correlations between attention, enjoyment, ongoing interest and learning and retention. The study also contributed to innovation management research by revealing the influence of different data visualisation methods on decision quality, providing an example of research-integrated education that forms part of the research process. Such a dovetailing of different research studies demonstrates how engineering educators can enhance educational impact while multiplying the outcomes from their research efforts.

  12. Targeting caspases in intracellular protozoan infections.

    PubMed

    Guillermo, Landi V C; Pereira, Wânia F; De Meis, Juliana; Ribeiro-Gomes, Flavia L; Silva, Elisabeth M; Kroll-Palhares, Karina; Takiya, Christina M; Lopes, Marcela F

    2009-06-01

    Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

  13. Macrophage defense mechanisms against intracellular bacteria

    PubMed Central

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  14. Endosomal escape: a bottleneck in intracellular delivery.

    PubMed

    Shete, Harshad K; Prabhu, Rashmi H; Patravale, Vandana B

    2014-01-01

    With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives. Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives. PMID:24730275

  15. Macrophage defense mechanisms against intracellular bacteria.

    PubMed

    Weiss, Günter; Schaible, Ulrich E

    2015-03-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  16. Engineering a Therapeutic Lectin by Uncoupling Mitogenicity from Antiviral Activity

    PubMed Central

    Swanson, Michael D.; Boudreaux, Daniel M.; Salmon, Loïc; Chugh, Jeetender; Winter, Harry C.; Meagher, Jennifer L.; André, Sabine; Murphy, Paul V.; Oscarson, Stefan; Roy, René; King, Steven; Kaplan, Mark H.; Goldstein, Irwin J.; Tarbet, E. Bart; Hurst, Brett L.; Smee, Donald F.; de la Fuente, Cynthia; Hoffmann, Hans-Heinrich; Xue, Yi; Rice, Charles M.; Schols, Dominique; Garcia, J. Victor; Stuckey, Jeanne A.; Gabius, Hans-Joachim; Al-Hashimi, Hashim M.; Markovitz, David M.

    2015-01-01

    Summary A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code. PMID:26496612

  17. Preliminary experiments on active control of fan noise from a turbofan engine

    NASA Technical Reports Server (NTRS)

    Thomas, R. H.; Burdisso, R. A.; Fuller, C. R.; O'Brien, W. F.

    1993-01-01

    In the preliminary experiments reported here, active acoustic sources positioned around the circumference of a turbofan engine were used to control the fan noise radiated forward through the inlet. The main objective was to demonstrate the potential of active techniques to alleviate the noise pollution that will be produced by the next generation of larger engines. A reduction of up to 19 dB in the radiation directivity was demonstrated in a zone that encompasses a 30-deg angle, near the error sensor, while spillover effects were observed toward the lateral direction. The simultaneous control of two tones was also demonstrated using two identical controllers in a parallel control configuration.

  18. Heat-activated heat-pump development and potential application of Stirling-engine technology

    NASA Astrophysics Data System (ADS)

    Fairchild, P. D.; West, C. D.

    1982-06-01

    Presented is a brief overview of the heat-activated heat pump technology development program being carried out with emphasis on the Stirling engine technology projects. The major projects are reviewed as they were formulated and carried out under the previous product development guidelines. The revised technology development focus and current status of those major hardware projects are discussed. The key issues involved in applying Stirling engine technology to heat pump equipment are assessed. The approach and planned future activities to address those issues are described. Also included are brief descriptions of two projects in this area supported by the Gas Research Institute.

  19. Science/Engineering Education Division assessment activities: An overview. Annual report, FY 1993

    SciTech Connect

    Not Available

    1994-02-01

    Evaluation and assessment of employment trends and education programs are important functions of the Science/Engineering Education Division (SEED). Objectives of SEED`s evaluation and assessment activities are to provide quantitative measures of the impact of programs on participants; assess programmatic achievements; provide valuable information for continued program operation; ensure that the programs meet their objectives; develop and maintain data bases on scientific and engineering employment and education; provide information about trends in employment and education for energy-related scientists and engineers; and provide analyses of energy-related science and engineering employment requirements, future labor market trends, adequacy of supply of new graduates, and implications for education programs. Whenever possible, data are collected that are consistent with information obtained by other national surveys to facilitate comparisons to national norms.

  20. Large area flexible SERS active substrates using engineered nanostructures

    NASA Astrophysics Data System (ADS)

    Chung, Aram J.; Huh, Yun Suk; Erickson, David

    2011-07-01

    Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as clothing. We demonstrate the formation of four different types of nanostructure arrays (pillar, nib, ellipsoidal cylinder, and triangular tip) by controlling the evaporation angle, substrate rotation, and deposition rate of metals onto anodized alumina nanoporous membranes as large as 27 mm. In addition, we present experimental results showing how a hybrid structure comprising of gold nanospheres embedded in a silver nano-pillar structure can be used to obtain a 50× SERS enhancement over the raw nanoparticles themselves.Surface enhanced Raman scattering (SERS) is an analytical sensing method that provides label-free detection, molecularly specific information, and extremely high sensitivity. The Raman enhancement that makes this method attractive is mainly attributed to the local amplification of the incident electromagnetic field that occurs when a surface plasmon mode is excited at a metallic nanostructure. Here, we present a simple, cost effective method for creating flexible, large area SERS-active substrates using a new technique we call shadow mask assisted evaporation (SMAE). The advantage of large, flexible SERS substrates such as these is they have more area for multiplexing and can be incorporated into irregular surfaces such as