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Sample records for active cellular processes

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

    NASA Astrophysics Data System (ADS)

    Rao, M.

    2014-07-01

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

  2. Genetic Dominance & Cellular Processes

    ERIC Educational Resources Information Center

    Seager, Robert D.

    2014-01-01

    In learning genetics, many students misunderstand and misinterpret what "dominance" means. Understanding is easier if students realize that dominance is not a mechanism, but rather a consequence of underlying cellular processes. For example, metabolic pathways are often little affected by changes in enzyme concentration. This means that…

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

    ERIC Educational Resources Information Center

    Johnson, Margaret (Peg)

    1998-01-01

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

  4. Active Cellular Nematics

    NASA Astrophysics Data System (ADS)

    Duclos, Guillaume; Erlenkaemper, Christoph; Garcia, Simon; Yevick, Hannah; Joanny, Jean-François; Silberzan, Pascal; Biology inspired physics at mesoscales Team; Physical approach of biological problems Team

    We study the emergence of a nematic order in a two-dimensional tissue of apolar elongated fibroblast cells. Initially, these cells are very motile and the monolayer is characterized by giant density fluctuations, a signature of far-from-equilibrium systems. As the cell density increases because of proliferation, the cells align with each other forming large perfectly oriented domains while the cellular movements slow down and eventually freeze. Therefore topological defects characteristic of nematic phases remain trapped at long times, preventing the development of infinite domains. By analogy with classical non-active nematics, we have investigated the role of boundaries and we have shown that cells confined in stripes of width smaller than typically 500 µm are perfectly aligned in the stripe direction. Experiments performed in cross-shaped patterns show that both the number of cells and the degree of alignment impact the final orientation. Reference: Duclos G., Garcia S., Yevick H.G. and Silberzan P., ''Perfect nematic order in confined monolayers of spindle-shaped cells'', Soft Matter, 10, 14, 2014

  5. Cellular and molecular mechanisms activating the cell death processes by chalcones: Critical structural effects.

    PubMed

    Champelovier, Pierre; Chauchet, Xavier; Hazane-Puch, Florence; Vergnaud, Sabrina; Garrel, Catherine; Laporte, François; Boutonnat, Jean; Boumendjel, Ahcène

    2013-12-01

    Chalcones are naturally occurring compounds with diverse pharmacological activities. Chalcones derive from the common structure: 1,3-diphenylpropenone. The present study aims to better understand the mechanistic pathways triggering chalcones anticancer effects and providing evidences that minor structural difference could lead to important difference in mechanistic effect. We selected two recently investigated chalcones (A and B) and investigated them on glioblastoma cell lines. It was found that chalcone A induced an apoptotic process (type I PCD), via the activation of caspase-3, -8 and -9. Chalcone A also increased CDK1/cyclin B ratios and decreased the mitochondrial transmembrane potential (ΔΨm). Chalcone B induced an autophagic cell death process (type II PCD), ROS-related but independent of both caspases and protein synthesis. Both chalcones increased Bax/Bcl2 ratios and decreased Ki67 and CD71 antigen expressions. The present investigation reveals that despite the close structure of chalcones A and B, significant differences in mechanism of effect were found.

  6. Cellular and molecular mechanisms activating the cell death processes by chalcones: Critical structural effects.

    PubMed

    Champelovier, Pierre; Chauchet, Xavier; Hazane-Puch, Florence; Vergnaud, Sabrina; Garrel, Catherine; Laporte, François; Boutonnat, Jean; Boumendjel, Ahcène

    2013-12-01

    Chalcones are naturally occurring compounds with diverse pharmacological activities. Chalcones derive from the common structure: 1,3-diphenylpropenone. The present study aims to better understand the mechanistic pathways triggering chalcones anticancer effects and providing evidences that minor structural difference could lead to important difference in mechanistic effect. We selected two recently investigated chalcones (A and B) and investigated them on glioblastoma cell lines. It was found that chalcone A induced an apoptotic process (type I PCD), via the activation of caspase-3, -8 and -9. Chalcone A also increased CDK1/cyclin B ratios and decreased the mitochondrial transmembrane potential (ΔΨm). Chalcone B induced an autophagic cell death process (type II PCD), ROS-related but independent of both caspases and protein synthesis. Both chalcones increased Bax/Bcl2 ratios and decreased Ki67 and CD71 antigen expressions. The present investigation reveals that despite the close structure of chalcones A and B, significant differences in mechanism of effect were found. PMID:24134853

  7. Signal processing in cellular clocks.

    PubMed

    Forger, Daniel B

    2011-03-15

    Many biochemical events within a cell need to be timed properly to occur at specific times of day, after other events have happened within the cell or in response to environmental signals. The cellular biochemical feedback loops that time these events have already received much recent attention in the experimental and modeling communities. Here, we show how ideas from signal processing can be applied to understand the function of these clocks. Consider two signals from the network s(t) and r(t), either two variables of a model or two experimentally measured time courses. We show how s(t) can be decomposed into two parts, the first being a function of r(t), and the second the derivative of a function of r(t). Geometric principles are then derived that can be used to understand when oscillations appear in biochemical feedback loops, the period of these oscillations, and their time course. Specific examples of this theory are provided that show how certain networks are prone or not prone to oscillate, how individual biochemical processes affect the period, and how oscillations in one chemical species can be deduced from oscillations in other parts of the network.

  8. Interplay between cellular activity and three-dimensional scaffold-cell constructs with different foam structure processed by electron beam melting.

    PubMed

    Nune, Krishna C; Misra, R Devesh K; Gaytan, Sara M; Murr, Lawrence E

    2015-05-01

    The cellular activity, biological response, and consequent integration of scaffold-cell construct in the physiological system are governed by the ability of cells to adhere, proliferate, and biomineralize. In this regard, we combine cellular biology and materials science and engineering to fundamentally elucidate the interplay between cellular activity and interconnected three-dimensional foamed architecture obtained by a novel process of electron beam melting and computational tools. Furthermore, the organization of key proteins, notably, actin, vinclulin, and fibronectin, involved in cellular activity and biological functions and relationship with the structure was explored. The interconnected foamed structure with ligaments was favorable to cellular activity that includes cell attachment, proliferation, and differentiation. The primary rationale for favorable modulation of cellular functions is that the foamed structure provided a channel for migration and communication between cells leading to highly mineralized extracellular matrix (ECM) by the differentiating osteoblasts. The filopodial interaction amongst cells on the ligaments was a governing factor in the secretion of ECM, with consequent influence on maturation and mineralization.

  9. Coordination of autophagy with other cellular activities

    PubMed Central

    Wang, Yan; Qin, Zheng-hong

    2013-01-01

    The cell biological phenomenon of autophagy has attracted increasing attention in recent years, partly as a consequence of the discovery of key components of its cellular machinery. Autophagy plays a crucial role in a myriad of cellular functions. Autophagy has its own regulatory mechanisms, but this process is not isolated. Autophagy is coordinated with other cellular activities to maintain cell homeostasis. Autophagy is critical for a range of human physiological processes. The multifunctional roles of autophagy are explained by its ability to interact with several key components of various cell pathways. In this review, we focus on the coordination between autophagy and other physiological processes, including the ubiquitin-proteasome system (UPS), energy homeostasis, aging, programmed cell death, the immune responses, microbial invasion and inflammation. The insights gained from investigating autophagic networks should increase our understanding of their roles in human diseases and their potential as targets for therapeutic intervention. PMID:23474706

  10. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

  11. System and method for monitoring cellular activity

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  12. Molecular processes in cellular arsenic metabolism

    SciTech Connect

    Thomas, David J.

    2007-08-01

    Elucidating molecular processes that underlie accumulation, metabolism and binding of iAs and its methylated metabolites provides a basis for understanding the modes of action by which iAs acts as a toxin and a carcinogen. One approach to this problem is to construct a conceptual model that incorporates available information on molecular processes involved in the influx, metabolism, binding and efflux of arsenicals in cells. This conceptual model is initially conceived as a non-quantitative representation of critical molecular processes that can be used as a framework for experimental design and prediction. However, with refinement and incorporation of additional data, the conceptual model can be expressed in mathematical terms and should be useful for quantitative estimates of the kinetic and dynamic behavior of iAs and its methylated metabolites in cells. Development of a quantitative model will be facilitated by the availability of tools and techniques to manipulate molecular processes underlying transport of arsenicals across cell membranes or expression and activity of enzymes involved in methylation of arsenicals. This model of cellular metabolism might be integrated into more complex pharmacokinetic models for systemic metabolism of iAs and its methylated metabolites. It may also be useful in development of biologically based dose-response models describing the toxic and carcinogenic actions of arsenicals.

  13. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    PubMed

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells. PMID:27406324

  14. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    PubMed

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  15. Dynamics of active cellular response under stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Zemel, Assaf; Safran, Samuel

    2008-03-01

    Forces exerted by and on adherent cells are important for many physiological processes such as wound healing and tissue formation. In addition, recent experiments have shown that stem cell differentiation is controlled, at least in part, by the elasticity of the surrounding matrix. Using a simple theoretical model that includes the forces due to both the mechanosensitive nature of cells and the elastic response of the matrix, we predict the dynamics of orientation of cells. The model predicts many features observed in measurements of cellular forces and orientation including the increase with time of the forces generated by cells in the absence of applied stress and the consequent decrease of the force in the presence of quasi-static stresses. We also explain the puzzling observation of parallel alignment of cells for static and quasi-static stresses and of nearly perpendicular alignment for dynamically varying stresses. In addition, we predict the response of the cellular orientation to a sinusoidally varying applied stress as a function of frequency. The dependence of the cell orientation angle on the Poisson ratio of the surrounding material can be used to distinguish systems in which cell activity is controlled by stress from those where cell activity is controlled by strain. Reference: Nature Physics, vol. 3, pp 655 (2007).

  16. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  17. Mechanically improved electrospun PCL biocomposites reinforced with a collagen coating process: preparation, physical properties, and cellular activity.

    PubMed

    Hong, Soon Gee; Kim, Geun Hyung

    2013-02-01

    In this work, we fabricated highly aligned electrospun poly(ε-caprolactone)(PCL)/collagen biocomposites, which were consisted of multi-layered structure. The aligned directions of the composites were controlled with two rotating collectors, and various weight fractions (1, 2, 3 wt%) of collagen were embedded between the mat of PCL microfibers to improve the mechanical property and biological activities of osteoblast-like cells (MG63). The PCL/collagen biocomposite showed nine times of increment in mechanical strength of random PCL/collagen composite. An increase in collagen content in the biocomposites displayed significant increase of mechanical properties, hydrophilic property, water-absorption ability, and even cell viability of osteoblast-like cells (MG63). PMID:22763777

  18. Electrohydrodynamic jet process for pore-structure-controlled 3D fibrous architecture as a tissue regenerative material: fabrication and cellular activities.

    PubMed

    Kim, Min Seong; Kim, GeunHyung

    2014-07-22

    In this study, we propose a new scaffold fabrication method, "direct electro-hydrodynamic jet process," using the initial jet of an electrospinning process and ethanol media as a target. The fabricated three-dimensional (3D) fibrous structure was configured with multilayered microsized struts consisting of randomly entangled micro/nanofibrous architecture, similar to that of native extracellular matrixes. The fabrication of the structure was highly dependent on various processing parameters, such as the surface tension of the target media, and the flow rate and weight fraction of the polymer solution. As a tissue regenerative material, the 3D fibrous scaffold was cultured with preosteoblasts to observe the initial cellular activities in comparison with a solid-freeform fabricated 3D scaffold sharing a similar structural geometry. The cell-culture results showed that the newly developed scaffold provided outstanding microcellular environmental conditions to the seeded cells (about 3.5-fold better initial cell attachment and 2.1-fold better cell proliferation).

  19. [Cellular model of blood coagulation process].

    PubMed

    Bijak, Michał; Rzeźnicka, Paulina; Saluk, Joanna; Nowak, Paweł

    2015-07-01

    Blood coagulation is a process which main objective is the prevention of blood loss when the integrity of the blood vessel is damaged. Over the years, have been presented a number of concepts characterizing the mechanism of thrombus formation. Since the 60s of last century was current cascade model of the coagulation wherein forming of the fibrin clot is determined by two pathways called extrinsic and intrinsic pathways. In the nineties of the last century Monroe and Hoffman presented his concept of blood coagulation process which complement the currently valid model of cells participation especially of blood platelets which aim is to provide a negatively charged phospholipid surface and thereby allow the coagulation enzymatic complexes formation. Developed conception they called cellular model of coagulation. The aim of this work was to present in details of this blood coagulation, including descriptions of its various phases.

  20. Modeling Cellular Processes in 3-D

    PubMed Central

    Mogilner, Alex; Odde, David

    2011-01-01

    Summary Recent advances in photonic imaging and fluorescent protein technology offer unprecedented views of molecular space-time dynamics in living cells. At the same time, advances in computing hardware and software enable modeling of ever more complex systems, from global climate to cell division. As modeling and experiment become more closely integrated, we must address the issue of modeling cellular processes in 3-D. Here, we highlight recent advances related to 3-D modeling in cell biology. While some processes require full 3-D analysis, we suggest that others are more naturally described in 2-D or 1-D. Keeping the dimensionality as low as possible reduces computational time and makes models more intuitively comprehensible; however, the ability to test full 3-D models will build greater confidence in models generally and remains an important emerging area of cell biological modeling. PMID:22036197

  1. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  2. Viral capsid assembly as a model for protein aggregation diseases: Active processes catalyzed by cellular assembly machines comprising novel drug targets.

    PubMed

    Marreiros, Rita; Müller-Schiffmann, Andreas; Bader, Verian; Selvarajah, Suganya; Dey, Debendranath; Lingappa, Vishwanath R; Korth, Carsten

    2015-09-01

    Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid β peptide (Aβ) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation

  3. Transcriptional Activation of Lysosomal Exocytosis Promotes Cellular Clearance

    PubMed Central

    Medina, Diego L.; Fraldi, Alessandro; Bouche, Valentina; Annunziata, Fabio; Mansueto, Gelsomina; Spampanato, Carmine; Puri, Claudia; Pignata, Antonella; Martina, Jose A.; Sardiello, Marco; Palmieri, Michela; Polishchuk, Roman; Puertollano, Rosa; Ballabio, Andrea

    2011-01-01

    Summary Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage. PMID:21889421

  4. Cellular antioxidant activity of common vegetables.

    PubMed

    Song, Wei; Derito, Christopher M; Liu, M Keshu; He, Xiangjiu; Dong, Mei; Liu, Rui Hai

    2010-06-01

    The measurement of antioxidant activity using biologically relevant assays is important to screen fruits, vegetables, natural products, and dietary supplements for potential health benefits. The cellular antioxidant activity (CAA) assay quantifies antioxidant activity using a cell culture model and was developed to meet the need for a more biologically representative method than the popular chemistry antioxidant capacity measures. The objective of the study was to determine the CAA, total phenolic contents, and oxygen radical absorbance capacity (ORAC) values of 27 vegetables commonly consumed in the United States. Beets, broccoli, and red pepper had the highest CAA values, whereas cucumber had the lowest. CAA values were significantly correlated to total phenolic content. Potatoes were found to be the largest contributors of vegetable phenolics and CAA to the American diet. Increased fruit and vegetable consumption is an effective strategy to increase antioxidant intake and decrease oxidative stress and may lead to reduced risk of developing chronic diseases, such as cancer and cardiovascular disease.

  5. Minimal model for complex dynamics in cellular processes.

    PubMed

    Suguna, C; Chowdhury, K K; Sinha, S

    1999-11-01

    Cellular functions are controlled and coordinated by the complex circuitry of biochemical pathways regulated by genetic and metabolic feedback processes. This paper aims to show, with the help of a minimal model of a regulated biochemical pathway, that the common nonlinearities and control structures present in biomolecular interactions are capable of eliciting a variety of functional dynamics, such as homeostasis, periodic, complex, and chaotic oscillations, including transients, that are observed in various cellular processes.

  6. Mechanical and cellular processes driving cervical myelopathy

    PubMed Central

    Dolan, Roisin T; Butler, Joseph S; O’Byrne, John M; Poynton, Ashley R

    2016-01-01

    Cervical myelopathy is a well-described clinical syndrome that may evolve from a combination of etiological mechanisms. It is traditionally classified by cervical spinal cord and/or nerve root compression which varies in severity and number of levels involved. The vast array of clinical manifestations of cervical myelopathy cannot fully be explained by the simple concept that a narrowed spinal canal causes compression of the cord, local tissue ischemia, injury and neurological impairment. Despite advances in surgical technology and treatment innovations, there are limited neuro-protective treatments for cervical myelopathy, which reflects an incomplete understanding of the pathophysiological processes involved in this disease. The aim of this review is to provide a comprehensive overview of the key pathophysiological processes at play in the development of cervical myelopathy. PMID:26807352

  7. Infrared ATR: a probe for cellular activation

    NASA Astrophysics Data System (ADS)

    Timlin, Jerilyn A.; Martin, Laura E.; Alam, M. Kathleen; Haaland, David M.; Garrison, Kristen; Lyons, C. Richard; Hjelle, Brian

    2002-02-01

    We employ infrared spectroscopy (IR) with attenuated total reflectance (ATR) as a sampling technique to monitor live and dried RAW cells (a murine macrophage cell line) during activation with g-interferon and lipopolysaccharide. By comparing the spectra of activated cells at various time points to the spectra of healthy control cells, we identify spectral bands associated with nucleic acids that are markers for the cell activation process. These spectral changes are slight and can be complicated with the normal metabolic changes that occur within cells. We will discuss the use of data pretreatment strategies to accurately correct for the contribution of the buffer to the live cell spectra. We find the standard background correction method inadequate for concentrated solutions of cells. Data presented shows the severe effect incorrect background subtraction has on the cell spectra. We report a more accurate correction for phosphate buffer spectral contribution using an interactive subtraction of the buffer spectrum. We will show classification of dried control and activated macrophage cell spectra using partial-least squares analysis with multiplicative scatter correction.

  8. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

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

    2015-01-01

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

  9. Regulation of targeted gene repair by intrinsic cellular processes.

    PubMed

    Engstrom, Julia U; Suzuki, Takayuki; Kmiec, Eric B

    2009-02-01

    Targeted gene alteration (TGA) is a strategy for correcting single base mutations in the DNA of human cells that cause inherited disorders. TGA aims to reverse a phenotype by repairing the mutant base within the chromosome itself, avoiding the introduction of exogenous genes. The process of how to accurately repair a genetic mutation is elucidated through the use of single-stranded DNA oligonucleotides (ODNs) that can enter the cell and migrate to the nucleus. These specifically designed ODNs hybridize to the target sequence and act as a beacon for nucleotide exchange. The key to this reaction is the frequency with which the base is corrected; this will determine whether the approach becomes clinically relevant or not. Over the course of the last five years, workers have been uncovering the role played by the cells in regulating the gene repair process. In this essay, we discuss how the impact of the cell on TGA has evolved through the years and illustrate ways that inherent cellular pathways could be used to enhance TGA activity. We also describe the cost to cell metabolism and survival when certain processes are altered to achieve a higher frequency of repair.

  10. Piezo proteins: regulators of mechanosensation and other cellular processes.

    PubMed

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature.

  11. Reporters to monitor cellular MMP12 activity

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  12. A synthetic biology approach to understanding cellular information processing

    PubMed Central

    Riccione, Katherine A; Smith, Robert P; Lee, Anna J; You, Lingchong

    2012-01-01

    The survival of cells and organisms requires proper responses to environmental signals. These responses are governed by cellular networks, which serve to process diverse environmental cues. Biological networks often contain recurring network topologies called ‘motifs’. It has been recognized that the study of such motifs allows one to predict the response of a biological network, and thus cellular behavior. However, studying a single motif in complete isolation of all other network motifs in a natural setting is difficult. Synthetic biology has emerged as a powerful approach to understanding the dynamic properties of network motifs. In addition to testing existing theoretical predictions, construction and analysis of synthetic gene circuits has led to the discovery of novel motif dynamics such as how the combination of simple motifs can lead to autonomous dynamics or how noise in transcription and translation can affect the dynamics of a motif. Here, we review developments in synthetic biology as they pertain to increasing our understanding of cellular information processing. We highlight several types of dynamic behaviors that diverse motifs can generate, including the control of input/output responses, the generation of autonomous spatial and temporal dynamics, as well as the influence of noise in motif dynamics and cellular behavior. PMID:23411668

  13. Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes.

    PubMed

    Badia, Mariana B; Arias, Cintia L; Tronconi, Marcos A; Maurino, Verónica G; Andreo, Carlos S; Drincovich, María F; Wheeler, Mariel C Gerrard

    2015-11-01

    Arabidopsis thaliana has four NADP-dependent malic enzymes (NADP-ME 1-4) for reversible malate decarboxylation, with NADP-ME2 being the only cytosolic isoform ubiquitously expressed and responsible for most of the total activity. In this work, we further investigated its physiological function by characterizing Arabidopsis plants over-expressing NADP-ME2 constitutively. In comparison to wild type, these plants exhibited reduced rosette and root sizes, delayed flowering time and increased sensitivity to mannitol and polyethylene glycol. The increased NADP-ME2 activity led to a decreased expression of other ME and malate dehydrogenase isoforms and generated a redox imbalance with opposite characteristics depending on the time point of the day analyzed. The over-expressing plants also presented a higher content of C4 organic acids and sugars under normal growth conditions. However, the accumulation of these metabolites in the over-expressing plants was substantially less pronounced after osmotic stress exposure compared to wild type. Also, a lower level of several amino acids and osmoprotector compounds was observed in transgenic plants. Thus, the gain of NADP-ME2 expression has profound consequences in the modulation of primary metabolism in A. thaliana, which reflect the relevance of this enzyme and its substrates and products in plant homeostasis.

  14. Sleep Loss Activates Cellular Inflammatory Signaling

    PubMed Central

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Vourkas, Ioannis; Sirakoulis, Georgios Ch.

    2013-04-01

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

  16. Parallelizing the Cellular Potts Model on graphics processing units

    NASA Astrophysics Data System (ADS)

    Tapia, José Juan; D'Souza, Roshan M.

    2011-04-01

    The Cellular Potts Model (CPM) is a lattice based modeling technique used for simulating cellular structures in computational biology. The computational complexity of the model means that current serial implementations restrict the size of simulation to a level well below biological relevance. Parallelization on computing clusters enables scaling the size of the simulation but marginally addresses computational speed due to the limited memory bandwidth between nodes. In this paper we present new data-parallel algorithms and data structures for simulating the Cellular Potts Model on graphics processing units. Our implementations handle most terms in the Hamiltonian, including cell-cell adhesion constraint, cell volume constraint, cell surface area constraint, and cell haptotaxis. We use fine level checkerboards with lock mechanisms using atomic operations to enable consistent updates while maintaining a high level of parallelism. A new data-parallel memory allocation algorithm has been developed to handle cell division. Tests show that our implementation enables simulations of >10 cells with lattice sizes of up to 256 3 on a single graphics card. Benchmarks show that our implementation runs ˜80× faster than serial implementations, and ˜5× faster than previous parallel implementations on computing clusters consisting of 25 nodes. The wide availability and economy of graphics cards mean that our techniques will enable simulation of realistically sized models at a fraction of the time and cost of previous implementations and are expected to greatly broaden the scope of CPM applications.

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

    PubMed Central

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

    2015-01-01

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

  18. Emergence of tissue mechanics from cellular processes: shaping a fly wing

    NASA Astrophysics Data System (ADS)

    Merkel, Matthias; Etournay, Raphael; Popovic, Marko; Nandi, Amitabha; Brandl, Holger; Salbreux, Guillaume; Eaton, Suzanne; Jülicher, Frank

    Nowadays, biologistsare able to image biological tissueswith up to 10,000 cells in vivowhere the behavior of each individual cell can be followed in detail.However, how precisely large-scale tissue deformation and stresses emerge from cellular behavior remains elusive. Here, we study this question in the developing wing of the fruit fly. To this end, we first establish a geometrical framework that exactly decomposes tissue deformation into contributions by different kinds of cellular processes. These processes comprise cell shape changes, cell neighbor exchanges, cell divisions, and cell extrusions. As the key idea, we introduce a tiling of the cellular network into triangles. This approach also reveals that tissue deformation can also be created by correlated cellular motion. Based on quantifications using these concepts, we developed a novel continuum mechanical model for the fly wing. In particular, our model includes active anisotropic stresses and a delay in the response of cell rearrangements to material stresses. A different approach to study the emergence of tissue mechanics from cellular behavior are cell-based models. We characterize the properties of a cell-based model for 3D tissues that is a hybrid between single particle models and the so-called vertex models.

  19. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    PubMed Central

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  20. Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers

    PubMed Central

    Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

    2013-01-01

    Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry–based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell–specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1–60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs. PMID:23345451

  1. Cellular processing and destinies of artificial DNA nanostructures.

    PubMed

    Lee, Di Sheng; Qian, Hang; Tay, Chor Yong; Leong, David Tai

    2016-08-01

    Since many bionanotechnologies are targeted at cells, understanding how and where their interactions occur and the subsequent results of these interactions is important. Changing the intrinsic properties of DNA nanostructures and linking them with interactions presents a holistic and powerful strategy for understanding dual nanostructure-biological systems. With the recent advances in DNA nanotechnology, DNA nanostructures present a great opportunity to understand the often convoluted mass of information pertaining to nanoparticle-biological interactions due to the more precise control over their chemistry, sizes, and shapes. Coupling just some of these designs with an understanding of biological processes is both a challenge and a source of opportunities. Despite continuous advances in the field of DNA nanotechnology, the intracellular fate of DNA nanostructures has remained unclear and controversial. Because understanding its cellular processing and destiny is a necessary prelude to any rational design of exciting and innovative bionanotechnology, in this review, we will discuss and provide a comprehensive picture relevant to the intracellular processing and the fate of various DNA nanostructures which have been remained elusive for some time. We will also link the unique capabilities of DNA to some novel ideas for developing next-generation bionanotechnologies. PMID:27119124

  2. Dynamical theory of active cellular response to external stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Safran, Samuel A.

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response.

  3. Dynamical theory of active cellular response to external stress.

    PubMed

    De, Rumi; Safran, Samuel A

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response.

  4. Cellular and Molecular Mechanisms Underpinning Macrophage Activation during Remyelination

    PubMed Central

    Lloyd, Amy F.; Miron, Veronique E.

    2016-01-01

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

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

    PubMed

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

    2015-01-01

    The aim of our study was to determine the antioxidant activities, cytotoxicity and proliferative properties in Stevia rebaudiana leaves and stems. Leaves extracts exhibited a higher antioxidant activity than stems extract, through oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Stevioside and rebaudioside A, the main sweetening metabolites in stevia leaves, exhibited a low ORAC value in comparison with plant extracts, while did not elicit any CAA. Stevia rebaudiana did not exhibit toxicity against HepG2 (hepatocellular carcinoma) human cells. No proliferative nor catalase modulations were observed in cells treated with such extracts. Our findings support the promising role of stevia that, apart from its sweetness, can act as a source of antioxidants, even at the intracellular level. This activity makes S. rebaudiana crude extract an interesting resource of natural sweetness with antioxidant properties which may find numerous applications in foods and nutritional supplements industries. PMID:26008718

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

    PubMed

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

    2015-01-01

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

  7. Activation of cellular immune response in acute pancreatitis.

    PubMed Central

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

    1997-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  9. Soil restoration with organic amendments: linking cellular functionality and ecosystem processes

    NASA Astrophysics Data System (ADS)

    Bastida, F.; Selevsek, N.; Torres, I. F.; Hernández, T.; García, C.

    2015-10-01

    A hot topic in recent decades, the application of organic amendments to arid-degraded soils has been shown to benefit microbially-mediated processes. However, despite the importance of soils for global sustainability, a gap has not been addressed yet in soil science: is there any connection between ecosystem-community processes, cellular functionality, and microbial lifestyles (i.e. oligotrophy-copiotrophy) in restored soils? Together with classical ecosystem indicators (fatty-acids, extracellular-enzyme activities, basal respiration), state-of-the-art metaproteomics was applied to fill this gap in a model-restoration experiment initiated 10-years ago by the addition of sewage-sludge and compost. Organic amendment strongly impacted ecosystem processes. Furthermore, the type of material used induced differences in the cellular functionalities through variations in the percentages of proteins involved in translation, transcription, energy production and C-fixation. We conclude that the long-term impact of organic restoration goes beyond ecosystem processes and affects cellular functionalities and phyla-lifestyles coupled with differences in microbial-community structures.

  10. Soil restoration with organic amendments: linking cellular functionality and ecosystem processes.

    PubMed

    Bastida, F; Selevsek, N; Torres, I F; Hernández, T; García, C

    2015-10-27

    A hot topic in recent decades, the application of organic amendments to arid-degraded soils has been shown to benefit microbially-mediated processes. However, despite the importance of soils for global sustainability, a gap has not been addressed yet in soil science: is there any connection between ecosystem-community processes, cellular functionality, and microbial lifestyles (i.e. oligotrophy-copiotrophy) in restored soils? Together with classical ecosystem indicators (fatty-acids, extracellular-enzyme activities, basal respiration), state-of-the-art metaproteomics was applied to fill this gap in a model-restoration experiment initiated 10-years ago by the addition of sewage-sludge and compost. Organic amendment strongly impacted ecosystem processes. Furthermore, the type of material used induced differences in the cellular functionalities through variations in the percentages of proteins involved in translation, transcription, energy production and C-fixation. We conclude that the long-term impact of organic restoration goes beyond ecosystem processes and affects cellular functionalities and phyla-lifestyles coupled with differences in microbial-community structures.

  11. Soil restoration with organic amendments: linking cellular functionality and ecosystem processes

    PubMed Central

    Bastida, F.; Selevsek, N.; Torres, I. F.; Hernández, T.; García, C.

    2015-01-01

    A hot topic in recent decades, the application of organic amendments to arid-degraded soils has been shown to benefit microbially-mediated processes. However, despite the importance of soils for global sustainability, a gap has not been addressed yet in soil science: is there any connection between ecosystem-community processes, cellular functionality, and microbial lifestyles (i.e. oligotrophy-copiotrophy) in restored soils? Together with classical ecosystem indicators (fatty-acids, extracellular-enzyme activities, basal respiration), state-of-the-art metaproteomics was applied to fill this gap in a model-restoration experiment initiated 10-years ago by the addition of sewage-sludge and compost. Organic amendment strongly impacted ecosystem processes. Furthermore, the type of material used induced differences in the cellular functionalities through variations in the percentages of proteins involved in translation, transcription, energy production and C-fixation. We conclude that the long-term impact of organic restoration goes beyond ecosystem processes and affects cellular functionalities and phyla-lifestyles coupled with differences in microbial-community structures. PMID:26503516

  12. Soil restoration with organic amendments: linking cellular functionality and ecosystem processes.

    PubMed

    Bastida, F; Selevsek, N; Torres, I F; Hernández, T; García, C

    2015-01-01

    A hot topic in recent decades, the application of organic amendments to arid-degraded soils has been shown to benefit microbially-mediated processes. However, despite the importance of soils for global sustainability, a gap has not been addressed yet in soil science: is there any connection between ecosystem-community processes, cellular functionality, and microbial lifestyles (i.e. oligotrophy-copiotrophy) in restored soils? Together with classical ecosystem indicators (fatty-acids, extracellular-enzyme activities, basal respiration), state-of-the-art metaproteomics was applied to fill this gap in a model-restoration experiment initiated 10-years ago by the addition of sewage-sludge and compost. Organic amendment strongly impacted ecosystem processes. Furthermore, the type of material used induced differences in the cellular functionalities through variations in the percentages of proteins involved in translation, transcription, energy production and C-fixation. We conclude that the long-term impact of organic restoration goes beyond ecosystem processes and affects cellular functionalities and phyla-lifestyles coupled with differences in microbial-community structures. PMID:26503516

  13. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS.

    PubMed

    Noah, Diana L; Twu, Karen Y; Krug, Robert M

    2003-03-15

    The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation of the transcription of cellular antiviral genes, which requires the interferon regulatory factor-3 (IRF-3) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

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

    PubMed

    Sirotek, K; Marounek, M; Suchorská, O

    2006-01-01

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

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

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  17. Vanderbilt University Study Creates New Roadmap for Cellular Activity - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    Scientists studying cellular processes have long sought to measure redox modifications because they provide one of the normal layers of cell control. But redox disruption or oxidative stress at the cellular level can also create a pathway to diseases like

  18. The SAGA continues: expanding the cellular role of a transcriptional co-activator complex

    PubMed Central

    Baker, SP; Grant, PA

    2009-01-01

    Throughout the last decade, great advances have been made in our understanding of how DNA-templated cellular processes occur in the native chromatin environment. Proteins that regulate transcription, replication, DNA repair, mitosis and other processes must be targeted to specific regions of the genome and granted access to DNA, which is normally tightly packaged in the higher-order chromatin structure of eukaryotic nuclei. Massive multiprotein complexes have been discovered, which facilitate access to DNA and recruitment of downstream effectors through three distinct mechanisms: chemical modification of histone amino-acid residues, ATP-dependent chromatin remodeling and histone exchange. The yeast Spt-Ada-Gcn5-Acetyl transferase (SAGA) transcriptional co-activator complex regulates numerous cellular processes through coordination of multiple histone post-translational modifications. SAGA is known to generate and interact with a number of histone modifications, including acetylation, methylation, ubiquitylation and phosphorylation. Although best characterized for its role in regulating transcriptional activation, SAGA is also required for optimal transcription elongation, mRNA export and perhaps nucleotide excision repair. Here, we discuss findings from recent years that have elucidated the function of this 1.8-MDa complex in multiple cellular processes, and how misregulation of the homologous complexes in humans may ultimately play a role in development of disease. PMID:17694076

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  20. Embryo as an active granular fluid: stress-coordinated cellular constriction chains

    NASA Astrophysics Data System (ADS)

    Gao, Guo-Jie Jason; Holcomb, Michael C.; Thomas, Jeffrey H.; Blawzdziewicz, Jerzy

    2016-10-01

    Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. However, systematic methods of studying how mechanical stress and feedback help to harmonize cellular activities within a tissue have yet to be developed. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity. Our particle-based AGF model will be useful in analyzing mechanical feedback effects in a wide variety of morphogenesis and organogenesis phenomena.

  1. Embryo as an active granular fluid: stress-coordinated cellular constriction chains.

    PubMed

    Jason Gao, Guo-Jie; Holcomb, Michael C; Thomas, Jeffrey H; Blawzdziewicz, Jerzy

    2016-10-19

    Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. However, systematic methods of studying how mechanical stress and feedback help to harmonize cellular activities within a tissue have yet to be developed. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity. Our particle-based AGF model will be useful in analyzing mechanical feedback effects in a wide variety of morphogenesis and organogenesis phenomena. PMID:27545101

  2. Ultrasmall integrin-targeted silica nanoparticles modulate signaling events and cellular processes in a concentration-dependent manner.

    PubMed

    Benezra, Miriam; Phillips, Evan; Overholtzer, Michael; Zanzonico, Pat B; Tuominen, Esa; Wiesner, Ulrich; Bradbury, Michelle S

    2015-04-01

    Cellular and molecular-level interactions of nanoparticles with biological systems are a rapidly evolving field requiring an improved understanding of endocytic trafficking as the principal driver and regulator of signaling events and cellular responses. An understanding of these processes is vital to nanomedicine applications. Studies investigating the complex interplay of these processes and their relationship to targeted nanoparticles exploiting endocytic pathways are notably lacking. It is known that integrins traffic through the endosomal pathway and participate in diverse roles controlling signal transduction, cell migration, and proliferation. Here, it is shown that ultrasmall, nontoxic, core-shell silica nanoparticles (C-dots), surface-functionalized with cRGDY peptides, modestly activate integrin-signaling pathways, in turn, promoting the enhancement of cellular functions. First, nanomolar concentrations, two orders of magnitude higher than clinical trial doses, internalize within αvβ3 integrin-expressing melanoma and endothelial cells, predominantly through an integrin receptor-dependent endocytic route. Second, integrin-mediated activation of focal adhesion kinase (FAK) and downstream signaling pathways occurs, in turn, upregulating phosphorylated protein expression levels and promoting concentration-dependent cellular migration and proliferative activity. Inhibiting FAK catalytic activity leads to decreased phosphorylation levels and cellular migration rates. These findings may inform the design of more effectively-targeted nanomedicines and provide insights into endocytic regulation of signal transduction.

  3. Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

    NASA Technical Reports Server (NTRS)

    Baldwin, Kenneth M.; Haddad, Fadia

    2002-01-01

    The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

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

    PubMed

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

    2016-02-01

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

  5. Mobile Phone Service Process Hiccups at Cellular Inc.

    ERIC Educational Resources Information Center

    Edgington, Theresa M.

    2010-01-01

    This teaching case documents an actual case of process execution and failure. The case is useful in MIS introductory courses seeking to demonstrate the interdependencies within a business process, and the concept of cascading failure at the process level. This case demonstrates benefits and potential problems with information technology systems,…

  6. [Monocyte functional activity and nonspecific antiviral cellular resistance in adaptation to the conditions of eastern Siberia].

    PubMed

    Zhiburt, E B; Filev, L V; Boĭchak, M P; Volchek, I V; Iakovlev, G P

    1993-01-01

    Monocyte functional activity and antiviral cellular resistance were studied in the newcomers to the Baikal region from the European Russia. A total of 105 patients with acute respiratory diseases and 30 normal subjects were examined. 46 healthy residents of St. Petersburg made up the reference group. The process of adaptation was found to be associated with changes in the functional activity of the immunocompetent cells. The authors came to a conclusion on the principal role of viral injury of the monocytes in the development of acute bronchitis or pneumonia.

  7. Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many pathogenic fungi are becoming resistant to currently available drugs. Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. The aim of this study was to identify benzaldehydes that...

  8. Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

    NASA Astrophysics Data System (ADS)

    Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

    2009-11-01

    Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

  9. Process maturity progress at Motorola Cellular Systems Division

    NASA Technical Reports Server (NTRS)

    Borgstahl, Ron; Criscione, Mark; Dobson, Kim; Willey, Allan

    1994-01-01

    We believe that the key success elements are related to our recognition that Software Process Improvement (SPI) can and should be organized, planned, managed, and measured as if it were a project to develop a new process, analogous to a software product. We believe that our process improvements have come as the result of these key elements: use of a rigorous, detailed requirements set (Capability Maturity Model, CMM); use of a robust, yet flexible architecture (IEEE 1074); use of a SPI project, resourced and managed like other work, to produce the specifications and implement them; and development of both internal and external goals, with metrics to support them.

  10. Particle acceleration in a complex solar active region modelled by a Cellular automata model

    NASA Astrophysics Data System (ADS)

    Dauphin, C.; Vilmer, N.; Anastasiadis, A.

    2004-12-01

    The models of cellular automat allowed to reproduce successfully several statistical properties of the solar flares. We use a cellular automat model based on the concept of self-organised critical system to model the evolution of the magnetic energy released in an eruptive active area. Each burst of magnetic energy released is assimilated to a process of magnetic reconnection. We will thus generate several current layers (RCS) where the particles are accelerated by a direct electric field. We calculate the energy gain of the particles (ions and electrons) for various types of magnetic configuration. We calculate the distribution function of the kinetic energy of the particles after their interactions with a given number of RCS for each type of configurations. We show that the relative efficiency of the acceleration of the electrons and the ions depends on the selected configuration.

  11. Processing Characteristics and Properties of the Cellular Products Made by Using Special Foaming Agents

    NASA Astrophysics Data System (ADS)

    Garbacz, Tomasz; Dulebova, Ludmila

    2012-12-01

    The paper describes the manufacturing process of extruded products by the cellular extrusion method, and presents specifications of the blowing agents used in the extrusion process as well as process conditions. The process of cellular extrusion of thermoplastic materials is aimed at obtaining cellular shapes and coats with reduced density, presenting no hollows on the surface of extruder product and displaying minimal contraction under concurrent maintenance of properties similar to properties of products extruded by means of the conventional method. In order to obtain cellular structure, the properties of extruded product are modified by applying suitable plastic or inserting auxiliary agents.

  12. Exposure to high static or pulsed magnetic fields does not affect cellular processes in the yeast Saccharomyces cerevisiae.

    PubMed

    Anton-Leberre, Veronique; Haanappel, Evert; Marsaud, Nathalie; Trouilh, Lidwine; Benbadis, Laurent; Boucherie, Helian; Massou, Sophie; François, Jean M

    2010-01-01

    We report results of a study of the effects of strong static (up to 16 T for 8 h) and pulsed (up to 55 T single-shot and 4 x 20 T repeated shots) magnetic fields on Saccharomyces cerevisiae cultures in the exponential phase of growth. In contrast to previous reports restricted to only a limited number of cellular parameters, we have examined a wide variety of cellular processes: genome-scale gene expression, proteome profile, cell viability, morphology, and growth, metabolic and fermentation activity after magnetic field exposure. None of these cellular activities were impaired in response to static or pulsed magnetic field exposure. Our results confirm and extend previous reports on the absence of magnetic field effects on yeast and support the hypothesis that magnetic fields have no impact on the transcriptional machinery and on the integrity of unicellular biological systems.

  13. Spontaneous Motion in Hierarchically Assembled Active Cellular Materials

    NASA Astrophysics Data System (ADS)

    Chen, Daniel

    2013-03-01

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

  14. Imaging large-scale cellular activity in spinal cord of freely behaving mice

    PubMed Central

    Sekiguchi, Kohei J.; Shekhtmeyster, Pavel; Merten, Katharina; Arena, Alexander; Cook, Daniela; Hoffman, Elizabeth; Ngo, Alexander; Nimmerjahn, Axel

    2016-01-01

    Sensory information from mechanoreceptors and nociceptors in the skin plays key roles in adaptive and protective motor behaviours. To date, very little is known about how this information is encoded by spinal cord cell types and their activity patterns, particularly under freely behaving conditions. To enable stable measurement of neuronal and glial cell activity in behaving mice, we have developed fluorescence imaging approaches based on two- and miniaturized one-photon microscopy. We show that distinct cutaneous stimuli activate overlapping ensembles of dorsal horn neurons, and that stimulus type and intensity is encoded at the single-cell level. In contrast, astrocytes show large-scale coordinated calcium responses to intense but not weak sensory inputs. Sensory-evoked activity is potently suppressed by anaesthesia. By revealing the cellular and computational logic of spinal cord networks under behaving conditions, our approach holds promise for better understanding of healthy and aberrant spinal cord processes. PMID:27121084

  15. Studying cellular processes and detecting disease with protein microarrays

    SciTech Connect

    Zangar, Richard C.; Varnum, Susan M.; Bollinger, Nikki

    2005-10-31

    Protein microarrays are a rapidly developing analytic tool with diverse applications in biomedical research. These applications include profiling of disease markers or autoimmune responses, understanding molecular pathways, protein modifications and protein activities. One factor that is driving this expanding usage is the wide variety of experimental formats that protein microarrays can take. In this review, we provide a short, conceptual overview of the different approaches for protein microarray. We then examine some of the most significant applications of these microarrays to date, with an emphasis on how global protein analyses can be used to facilitate biomedical research.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Rogerson, Thomas; Jayaprakash, Balaji; Cai, Denise J; Sano, Yoshitake; Lee, Yong-Seok; Zhou, Yu; Bekal, Pallavi; Deisseroth, Karl; Silva, Alcino J

    2016-01-01

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

  18. γ-Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and Biochemical Properties1

    PubMed Central

    Storozhenko, Sergei; Belles-Boix, Enric; Babiychuk, Elena; Hérouart, Didier; Davey, Mark W.; Slooten, Luit; Van Montagu, Marc; Inzé, Dirk; Kushnir, Sergei

    2002-01-01

    γ-Glutamyl transpeptidase (γ-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the γ-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for γ-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound γ-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis γ-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH. PMID:11891265

  19. Cellular Localization of Dieldrin and Structure–Activity Relationship of Dieldrin Analogues in Dopaminergic Cells

    PubMed Central

    Allen, Erin M. G.; Florang, Virginia R.; Davenport, Laurie L.; Jinsmaa, Yunden; Doorn, Jonathan A.

    2015-01-01

    The incidence of Parkinson’s disease (PD) correlates with environmental exposure to pesticides, such as the organochlorine insecticide, dieldrin. Previous studies found an increased concentration of the pesticide in the striatal region of the brains of PD patients and also that dieldrin adversely affects cellular processes associated with PD. These processes include mitochondrial function and reactive oxygen species production. However, the mechanism and specific cellular targets responsible for dieldrin-mediated cellular dysfunction and the structural components of dieldrin contributing to its toxicity (toxicophore) have not been fully defined. In order to identify the toxicophore of dieldrin, a structure–activity approach was used, with the toxicity profiles of numerous analogues of dieldrin (including aldrin, endrin, and cis-aldrin diol) assessed in PC6-3 cells. The MTT and lactate dehydrogenase (LDH) assays were used to monitor cell viability and membrane permeability after treatment with each compound. Cellular assays monitoring ROS production and extracellular dopamine metabolite levels were also used. Structure and stereochemistry for dieldrin were found to be very important for toxicity and other end points measured. Small changes in structure for dieldrin (e.g., comparison to the stereoisomer endrin) yielded significant differences in toxicity. Interestingly, the cis-diol metabolite of dieldrin was found to be significantly more toxic than the parent compound. Disruption of dopamine catabolism yielded elevated levels of the neurotoxin, 3,4-dihydroxyphenylacetaldehyde, for many organochlorines. Comparisons of the toxicity profiles for each dieldrin analogue indicated a structure-specific effect important for elucidating the mechanisms of dieldrin neurotoxicity. PMID:23763672

  20. Tensegrity II. How structural networks influence cellular information processing networks

    NASA Technical Reports Server (NTRS)

    Ingber, Donald E.

    2003-01-01

    The major challenge in biology today is biocomplexity: the need to explain how cell and tissue behaviors emerge from collective interactions within complex molecular networks. Part I of this two-part article, described a mechanical model of cell structure based on tensegrity architecture that explains how the mechanical behavior of the cell emerges from physical interactions among the different molecular filament systems that form the cytoskeleton. Recent work shows that the cytoskeleton also orients much of the cell's metabolic and signal transduction machinery and that mechanical distortion of cells and the cytoskeleton through cell surface integrin receptors can profoundly affect cell behavior. In particular, gradual variations in this single physical control parameter (cell shape distortion) can switch cells between distinct gene programs (e.g. growth, differentiation and apoptosis), and this process can be viewed as a biological phase transition. Part II of this article covers how combined use of tensegrity and solid-state mechanochemistry by cells may mediate mechanotransduction and facilitate integration of chemical and physical signals that are responsible for control of cell behavior. In addition, it examines how cell structural networks affect gene and protein signaling networks to produce characteristic phenotypes and cell fate transitions during tissue development.

  1. Vascular Ageing and Exercise: Focus on Cellular Reparative Processes

    PubMed Central

    Ross, Mark D.; Malone, Eva; Florida-James, Geraint

    2016-01-01

    Ageing is associated with an increased risk of developing noncommunicable diseases (NCDs), such as diabetes and cardiovascular disease (CVD). The increased risk can be attributable to increased prolonged exposure to oxidative stress. Often, CVD is preceded by endothelial dysfunction, which carries with it a proatherothrombotic phenotype. Endothelial senescence and reduced production and release of nitric oxide (NO) are associated with “vascular ageing” and are often accompanied by a reduced ability for the body to repair vascular damage, termed “reendothelialization.” Exercise has been repeatedly shown to confer protection against CVD and diabetes risk and incidence. Regular exercise promotes endothelial function and can prevent endothelial senescence, often through a reduction in oxidative stress. Recently, endothelial precursors, endothelial progenitor cells (EPC), have been shown to repair damaged endothelium, and reduced circulating number and/or function of these cells is associated with ageing. Exercise can modulate both number and function of these cells to promote endothelial homeostasis. In this review we look at the effects of advancing age on the endothelium and these endothelial precursors and how exercise appears to offset this “vascular ageing” process. PMID:26697131

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

    USGS Publications Warehouse

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

    2003-01-01

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

  3. Dissecting the dynamics of dysregulation of cellular processes in mouse mammary gland tumor

    PubMed Central

    2009-01-01

    Background Elucidating the sequence of molecular events underlying breast cancer formation is of enormous value for understanding this disease and for design of an effective treatment. Gene expression measurements have enabled the study of transcriptome-wide changes involved in tumorigenesis. This usually occurs through identification of differentially expressed genes or pathways. Results We propose a novel approach that is able to delineate new cancer-related cellular processes and the nature of their involvement in tumorigenesis. First, we define modules as densely interconnected and functionally enriched areas of a Protein Interaction Network. Second, 'differential expression' and 'differential co-expression' analyses are applied to the genes in these network modules, allowing for identification of processes that are up- or down-regulated, as well as processes disrupted (low co-expression) or invoked (high co-expression) in different tumor stages. Finally, we propose a strategy to identify regulatory miRNAs potentially responsible for the observed changes in module activities. We demonstrate the potential of this analysis on expression data from a mouse model of mammary gland tumor, monitored over three stages of tumorigenesis. Network modules enriched in adhesion and metabolic processes were found to be inactivated in tumor cells through the combination of dysregulation and down-regulation, whereas the activation of the integrin complex and immune system response modules is achieved through increased co-regulation and up-regulation. Additionally, we confirmed a known miRNA involved in mammary gland tumorigenesis, and present several new candidates for this function. Conclusions Understanding complex diseases requires studying them by integrative approaches that combine data sources and different analysis methods. The integration of methods and data sources proposed here yields a sensitive tool, able to pinpoint new processes with a role in cancer, dissect

  4. Copper enhances cellular and network excitabilities, and improves temporal processing in the rat hippocampus.

    PubMed

    Maureira, Carlos; Letelier, Juan Carlos; Alvarez, Osvaldo; Delgado, Ricardo; Vergara, Cecilia

    2015-12-01

    Copper, an ion with many important metabolic functions, has also been proposed to have a role as modulator on neuronal function, mostly based on its effects on voltage- and neurotransmitter-gated conductance as well as on neurological symptoms of patients with altered copper homeostasis. Nevertheless, the mechanisms by which copper exerts its neuromodulatory effects have not been clearly established in a functional neuronal network. Using rat hippocampus slices as a neuronal network model, the effects of copper in the range of 10-100 nm were tested on the intrinsic, synaptic and network properties of the CA1 region. Most of the previously described effects of this cation were in the micromolar range of copper concentrations. The current results indicate that copper is a multifaceted neuromodulator, having effects that may be grouped into two categories: (i) activity enhancement, by modulating synaptic communication and action potential (AP) conductances; and (ii) temporal processing and correlation extraction, by improving reliability and depressing inhibition. Specifically it was found that copper hyperpolarizes AP firing threshold, enhances neuronal and network excitability, modifies CA3-CA1 pathway gain, enhances the frequency of spontaneous synaptic events, decreases inhibitory network activity, and improves AP timing reliability. Moreover, copper chelation by bathocuproine decreases spontaneous network spiking activity. These results allow the proposal that copper affects the network activity from cellular to circuit levels on a moment-by-moment basis, and should be considered a crucial functional component of hippocampal neuronal circuitry.

  5. p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

    PubMed Central

    Fackler, Oliver T.; Lu, Xiaobin; Frost, Jeffrey A.; Geyer, Matthias; Jiang, Bing; Luo, Wen; Abo, Arie; Alberts, Arthur S.; Peterlin, B. Matija

    2000-01-01

    The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef. PMID:10713183

  6. Cellular activation in limbic brain systems during social play behaviour in rats

    PubMed Central

    van Kerkhof, Linda W.M.; Trezza, Viviana; Mulder, Tessa; Gao, Ping; Voorn, Pieter; Vanderschuren, Louk J.M.J.

    2013-01-01

    Positive social interactions during the juvenile and adolescent phases of life are essential for proper social and cognitive development in mammals, including humans. During this developmental period, there is a marked increase in peer-peer interactions, signified by the abundance of social play behaviour. Despite its importance for behavioural development, our knowledge of the neural underpinnings of social play behaviour is limited. Therefore, the purpose of this study was to map the neural circuits involved in social play behaviour in rats. This was achieved by examining cellular activity after social play using the immediate early gene c-fos as a marker. After a session of social play behaviour, pronounced increases in c-fos expression were observed in the medial prefrontal cortex, medial and ventral orbitofrontal cortex, dorsal striatum, nucleus accumbens core and shell, lateral amygdala, several thalamic nuclei, dorsal raphe and the pedunculopontine tegmental nucleus. Importantly, the cellular activity patterns after social play were topographically organised in this network, as indicated by play-specific correlations in c-fos activity between regions with known direct connections. These correlations suggest involvement in social play behaviour of the projections from the medial prefrontal cortex to the striatum, and of amygdala and monoaminergic inputs to frontal cortex and striatum. The analyses presented here outline a topographically organised neural network implicated in processes such as reward, motivation and cognitive control over behaviour, which mediates social play behaviour in rats. PMID:23670540

  7. Cellular activation in limbic brain systems during social play behaviour in rats.

    PubMed

    van Kerkhof, Linda W M; Trezza, Viviana; Mulder, Tessa; Gao, Ping; Voorn, Pieter; Vanderschuren, Louk J M J

    2014-07-01

    Positive social interactions during the juvenile and adolescent phases of life are essential for proper social and cognitive development in mammals, including humans. During this developmental period, there is a marked increase in peer-peer interactions, signified by the abundance of social play behaviour. Despite its importance for behavioural development, our knowledge of the neural underpinnings of social play behaviour is limited. Therefore, the purpose of this study was to map the neural circuits involved in social play behaviour in rats. This was achieved by examining cellular activity after social play using the immediate early gene c-Fos as a marker. After a session of social play behaviour, pronounced increases in c-Fos expression were observed in the medial prefrontal cortex, medial and ventral orbitofrontal cortex, dorsal striatum, nucleus accumbens core and shell, lateral amygdala, several thalamic nuclei, dorsal raphe and the pedunculopontine tegmental nucleus. Importantly, the cellular activity patterns after social play were topographically organized in this network, as indicated by play-specific correlations in c-Fos activity between regions with known direct connections. These correlations suggest involvement in social play behaviour of the projections from the medial prefrontal cortex to the striatum, and of amygdala and monoaminergic inputs to frontal cortex and striatum. The analyses presented here outline a topographically organized neural network implicated in processes such as reward, motivation and cognitive control over behaviour, which mediates social play behaviour in rats. PMID:23670540

  8. Cellular activation in limbic brain systems during social play behaviour in rats.

    PubMed

    van Kerkhof, Linda W M; Trezza, Viviana; Mulder, Tessa; Gao, Ping; Voorn, Pieter; Vanderschuren, Louk J M J

    2014-07-01

    Positive social interactions during the juvenile and adolescent phases of life are essential for proper social and cognitive development in mammals, including humans. During this developmental period, there is a marked increase in peer-peer interactions, signified by the abundance of social play behaviour. Despite its importance for behavioural development, our knowledge of the neural underpinnings of social play behaviour is limited. Therefore, the purpose of this study was to map the neural circuits involved in social play behaviour in rats. This was achieved by examining cellular activity after social play using the immediate early gene c-Fos as a marker. After a session of social play behaviour, pronounced increases in c-Fos expression were observed in the medial prefrontal cortex, medial and ventral orbitofrontal cortex, dorsal striatum, nucleus accumbens core and shell, lateral amygdala, several thalamic nuclei, dorsal raphe and the pedunculopontine tegmental nucleus. Importantly, the cellular activity patterns after social play were topographically organized in this network, as indicated by play-specific correlations in c-Fos activity between regions with known direct connections. These correlations suggest involvement in social play behaviour of the projections from the medial prefrontal cortex to the striatum, and of amygdala and monoaminergic inputs to frontal cortex and striatum. The analyses presented here outline a topographically organized neural network implicated in processes such as reward, motivation and cognitive control over behaviour, which mediates social play behaviour in rats.

  9. Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

    PubMed

    Kikuchi, Haruhisa; Kubohara, Yuzuru; Nguyen, Van Hai; Katou, Yasuhiro; Oshima, Yoshiteru

    2013-08-01

    Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster.

  10. Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

    PubMed

    Kikuchi, Haruhisa; Kubohara, Yuzuru; Nguyen, Van Hai; Katou, Yasuhiro; Oshima, Yoshiteru

    2013-08-01

    Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster. PMID:23746784

  11. Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

    PubMed Central

    2011-01-01

    Background Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy. Methods Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI). Results Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective

  12. Cellular redox status influences both cytotoxic and NF-kappa B activation in natural killer cells.

    PubMed Central

    Valle Blázquez, M; Luque, I; Collantes, E; Aranda, E; Solana, R; Peña, J; Muñoz, E

    1997-01-01

    The role of cellular redox status in both cytotoxic activity and NF-kappa B activation in natural killer (NK) cells was investigated. The results indicate that stimulation of NK cells, either freshly isolated from peripheral blood lymphocytes (PBL) or long-term cultured NK clones, with specific cell targets results in an increased binding activity of NF-kappa B and AP-1 transcription factors measured by gel retardation. Pretreatment of NK cells with the antioxidant pyrrolidine dithiocarbarmate (PDTC) leads to the inhibition of NF-kappa B activation but the AP-1 binding to DNA was superinduced. The inhibition of NF-kappa B by PDTC paralleled with an inhibition of spontaneous cytotoxicity mediated by NK cells. Moreover, the inhibitors of serine proteases, N-alpha-tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone, also blocked the cytolytic activity of NK cells against the sensitive target K562. In contrast, NK activity was not affected by pretreatment of the effector cells with the proteasome inhibitor N-acetyl-leu-leu-norleucinal which selectively inhibits NF-kappa B activation. Altogether, these results support the hypothesis that the activation of NK cells involved transcriptional and post-transcriptional events, and that reactive intermediates may play an important role in the molecular processes related with the generation of a cytotoxic response by NK cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9155655

  13. Pomegranate Extracts and Cancer Prevention: Molecular and Cellular Activities

    PubMed Central

    Syed, Deeba N.; Chamcheu, Jean-Christopher; Adhami, Vaqar M.; Mukhtar, Hasan

    2014-01-01

    There is increased appreciation by the scientific community that dietary phytochemicals can be potential weapons in the fight against cancer. Emerging data has provided new insights into the molecular and cellular framework needed to establish novel mechanism-based strategies for cancer prevention by selective bioactive food components. The unique chemical composition of the pomegranate fruit, rich in antioxidant tannins and flavonoids has drawn the attention of many investigators. Polyphenol rich fractions derived from the pomegranate fruit have been studied for their potential chemopreventive and/or cancer therapeutic effects in several animal models. Although data from in vitro and in vivo studies look convincing, well designed clinical trials in humans are needed to ascertain whether pomegranate can become part of our armamentarium against cancer. This review summarizes the available literature on the effects of pomegranate against various cancers. PMID:23094914

  14. ENHANCING FUNGICIDAL ACTIVITY OF FLUDIOXONIL BY DISRUPTING CELLULAR GLUTATHIONE HOMEOSTASIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungicidal activity of fludioxonil, a phenylpyrrole fungicide, is elevated by co-application with the aspirin/salicylic acid metabolite, 2,5-dihydroxybenzoic acid (2,5-DHBA). Fludioxonil fungicidal activity is potentiated through the mitogen-activated protein kinase (MAPK) pathway regulating osmotic...

  15. Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling.

    PubMed

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

    2016-08-25

    In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol. PMID:27545348

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

    ERIC Educational Resources Information Center

    Etchison, Cindy; Deal, Walter F., III

    1992-01-01

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

  17. Cellular antioxidative, cytotoxic, and antileishmanial activities of Homalium letestui

    PubMed Central

    Efiom Okokon, Jude; Dar Farooq, Ahsana; Choudhary, Mohammed Iqbal

    2013-01-01

    Objective: Homalium letestui Pellegr (Flacourtiaceae) is used in traditional medicine in parts of Nigeria for the treatment of malaria, ulcer, and inflammatory diseases and as an aphrodisiac. This investigation was aimed to evaluate the cytotoxic, immunomodulatory, and antileishmanial properties of stem extract and fractions of Homalium letestui (H. letestui). Materials and Methods: Cytotoxic activity against HeLa cells was done using sulphorhodamine (SRB) method and DNA interaction activity using gel electrophoresis. Immunomodulatory activity of the extract in whole blood, neutrophils, and macrophages was also investigated using luminol/lucigenin-based chemiluminescence assay. The extract and fractions were similarly screened for antileishmanial activity against promastigotes of Leishmania major in vitro. The GCMS analysis of the most active fraction against HeLa cells was carried out. Results: The stem extract exerted prominent cytotoxic activity with the dichloromethane fraction exhibiting the most pronounced effect (GI50 -5.12±1.45 µg/ml, LC50- 57.3±2.33 µg/ml, TGI -12.6±0.87 µg/ml). The crude extract and the fractions did not interact with DNA when investigated using electrophoresis. The extract significantly ((p<0.05 – 0.001) inhibited oxidative burst activity in whole blood (–27.90-66.90%), isolated polymorphonuclear cells (PMNs) (16.50-67.0%), and mononuclear cells (MNCs) (4.31-98.50%) when two different phagocytosis activators (serum opsonizing zymosan-A and PMA) were used. The extract also exhibited moderate antileishmanial activity against promastigotes of Leishmania major in vitro. GCMS analysis of active fraction revealed pharmacologically active compounds. Conclusion: These results suggest that the stem extract/fractions of H. letestui possess cytotoxic, immunomodulatory, and antileishmanial activities. PMID:25050257

  18. Cellular growth defects triggered by an overload of protein localization processes

    PubMed Central

    Kintaka, Reiko; Makanae, Koji; Moriya, Hisao

    2016-01-01

    High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes. PMID:27538565

  19. Cellular growth defects triggered by an overload of protein localization processes.

    PubMed

    Kintaka, Reiko; Makanae, Koji; Moriya, Hisao

    2016-01-01

    High-level expression of a protein localized to an intracellular compartment is expected to cause cellular defects because it overloads localization processes. However, overloads of localization processes have never been studied systematically. Here, we show that the expression levels of green fluorescent proteins (GFPs) with localization signals were limited to the same degree as a toxic misfolded GFP in budding yeast cells, and that their high-level expression caused cellular defects associated with localization processes. We further show that limitation of the exportin Crm1 determined the expression limit of GFP with a nuclear export signal. Although misfolding of GFP with a vesicle-mediated transport signal triggered endoplasmic reticulum stress, it was not the primary determinant of its expression limit. The precursor of GFP with a mitochondrial targeting signal caused a cellular defect. Finally, we estimated the residual capacities of localization processes. High-level expression of a localized protein thus causes cellular defects by overloading the capacities of localization processes. PMID:27538565

  20. Phosphorylation Hypothesis: A Fourth Sink of ATP for Cellular Information Processing?

    NASA Astrophysics Data System (ADS)

    Qian, Hong

    2015-03-01

    Adenosine triphosphate (ATP) molecule is used in living cells as a universal ``energy currency.'' The Gibbs free energy liberated from hydrolysis reaction of ATP to ADP + Pi is used for (a) biosynthesis, (b) ionic and neutral molecular pumping, and (c) mechanical movement. They are known collectively as the three major energy sinks at the cellular level. Using biochemical activities of various enzymes, a cell carries out information processing, known as signal transduction. Essentially all signal transduction reactions also require ATP (or GTP) hydrolysis. In the past, such energy dissipative reactions are considered as ``futile.'' However, it is clear that the free energy derived from a futile cycle is used to correct errors in biomolecular recognition, improve robustness in cell development, overcome Boltzmann's equilibrium law of probability, and drive Maxwell's demons (one notes that Gibbs' chemical potential is a thermodynamic force without mechanical interpretation). The free energy involved in processing information will be explained in terms of stochastic entropy production -- the central concept in irreversible and nonequilibrium steady-state (NESS) thermodynamics.

  1. Using Primary Literature in an Undergraduate Assignment: Demonstrating Connections among Cellular Processes

    ERIC Educational Resources Information Center

    Yeong, Foong May

    2015-01-01

    Learning basic cell biology in an essential module can be daunting to second-year undergraduates, given the depth of information that is provided in major molecular and cell biology textbooks. Moreover, lectures on cellular pathways are organised into sections, such that at the end of lectures, students might not see how various processes are…

  2. Cellular Links between Neuronal Activity and Energy Homeostasis

    PubMed Central

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

    2012-01-01

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

  3. A Fluorescent Reporter of AMPK activity and Cellular Energy Stress

    PubMed Central

    Tsou, Peiling; Zheng, Bin; Hsu, Chia-Hsien; Sasaki, Atsuo T; Cantley, Lewis C.

    2011-01-01

    SUMMARY AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here we describe a biosensor, AMPKAR, which exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatio-temporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 & α2 and on the upstream kinase, LKB1. Interestingly we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidics device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation. PMID:21459332

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

    PubMed Central

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

    2015-01-01

    The cell-based antioxidant activity assay as more biological relevant assay was considered to be more accurate to predict antioxidant activity in vivo than chemical activity assays. In the present study, the five main Phyllanthus emblica L. cultivars in China were subjected for cellular antioxidant activity based on HepG2 cells as well as antiproliferative activity. Total phenolics, total flavonoids and oxygen radical absorbance capacity were also measured. The results showed that Qingyougan, Binggan and Boligan (832±100, 774±52 and 704±28 μmol of quercetin equivalents/100 g) had higher cellular antioxidant activity than Tianyougan and Yougan (553±50 and 457±24 μmol of quercetin equivalents/100 g) in phosphate buffered saline wash protocol whereas, Boligan (3735±217 μmol of quercetin equivalents/100 g) had the highest cellular antioxidant activity and Tianyougan (2025±171 μmol of quercetin equivalents/100 g) had the lowest cellular antioxidant activity in no phosphate buffered saline wash protocol. The highest and lowest antiproliferative activities were observed in Binggan and Tianyougan (median effective dose: 6.95±0.11 and 14.03±0.10 mg/ml), respectively. The significant correlation was only observed between total flavonoids and cellular antioxidant activity from no phosphate buffered saline wash protocol (R2 =0.908, P<0.05), and total flavonoids and antiproliferative activity (R2 =0.887, P<0.05), suggesting the major contribution of flavonoids to the bioactivities of emblica. Overall, the data obtained revealed that different Phyllanthus emblica L. cultivars had strong cellular antioxidant and antiproliferative activities, thus should be recommended to increase consumption for health. PMID:26180272

  5. Prebiotic activation processes.

    NASA Technical Reports Server (NTRS)

    Lohrmann, R.; Orgel, L. E.

    1973-01-01

    Questions regarding the combination of amino acids and ribonucleotides to polypeptides and polynucleotides are investigated. Each of the reactions considered occurs in the solid state in plausible prebiotic conditions. Together they provide the basis for a unified scheme of amino acid and nucleotide activation. Urea, imidazole and Mg(++) are essential catalytic components of the reaction mixtures. However, these compounds could probably be replaced by other organic molecules.

  6. Transition from a planar interface to cellular and dendritic structures during rapid solidification processing

    NASA Technical Reports Server (NTRS)

    Laxmanan, V.

    1986-01-01

    The development of theoretical models which characterize the planar-cellular and cell-dendrite transitions is described. The transitions are analyzed in terms of the Chalmers number, the solute Peclet number, and the tip stability parameter, which correlate microstructural features and processing conditions. The planar-cellular transition is examined using the constitutional supercooling theory of Chalmers et al., (1953) and it is observed that the Chalmers number is between 0 and 1 during dendritic and cellular growth. Analysis of cell-dendrite transition data reveal that the transition occurs when the solute Peclet number goes through a minimum, the primary arm spacings go through a maximum, and the Chalmers number is equal to 1/2. The relation between the tip stability parameter and the solute Peclet number is investigated and it is noted that the tip stability parameter is useful for studying dendritic growth in alloys.

  7. Processing and modeling of cellular solids for light-weight structures

    SciTech Connect

    Nieh, T.G.

    1997-12-01

    Cellular solids (also known as porous solids) comprise a special class of materials. Such materials are common in nature; wood, cork, sponge and coral are examples. Recently man has also made his own cellular solids. For example, many honeycomb-like materials, made up of parallel, prismatic cells, are used for lightweight aerospace structural components. Polymeric foams have been used in everything from disposable coffee cups, packaging materials, to the crash padding of an aircraft cockpit. Advanced techniques now exist for foaming not only polymers, but metals and ceramics as well. These newer foams are increasingly used for catalysts (chemical), preforms for metal-matrix composites, thermal insulators and thermal shock resistant materials (thermal), acoustic dampers (acoustic), cushions, vibration reducers, and systems for absorbing the kinetic energy from impacts (mechanical). Their uses exploit the special combination of properties offered by cellular solids, properties which, ultimately, derive from their cellular structure. The objective of this proposed research is to develop processing techniques to produce metallic foams with controlled cellular structures and to understand and model the mechanical behavior of this special class of materials.

  8. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    PubMed

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

  9. Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

    PubMed

    Bergemann, Claudia; Cornelsen, Matthias; Quade, Antje; Laube, Thorsten; Schnabelrauch, Matthias; Rebl, Henrike; Weißmann, Volker; Seitz, Hermann; Nebe, Barbara

    2016-02-01

    The generation of hybrid materials based on β-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts. PMID:26652403

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

    PubMed

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

    2011-12-01

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

  11. Path planning on cellular nonlinear network using active wave computing technique

    NASA Astrophysics Data System (ADS)

    Yeniçeri, Ramazan; Yalçın, Müstak E.

    2009-05-01

    This paper introduces a simple algorithm to solve robot path finding problem using active wave computing techniques. A two-dimensional Cellular Neural/Nonlinear Network (CNN), consist of relaxation oscillators, has been used to generate active waves and to process the visual information. The network, which has been implemented on a Field Programmable Gate Array (FPGA) chip, has the feature of being programmed, controlled and observed by a host computer. The arena of the robot is modelled as the medium of the active waves on the network. Active waves are employed to cover the whole medium with their own dynamics, by starting from an initial point. The proposed algorithm is achieved by observing the motion of the wave-front of the active waves. Host program first loads the arena model onto the active wave generator network and command to start the generation. Then periodically pulls the network image from the generator hardware to analyze evolution of the active waves. When the algorithm is completed, vectorial data image is generated. The path from any of the pixel on this image to the active wave generating pixel is drawn by the vectors on this image. The robot arena may be a complicated labyrinth or may have a simple geometry. But, the arena surface always must be flat. Our Autowave Generator CNN implementation which is settled on the Xilinx University Program Virtex-II Pro Development System is operated by a MATLAB program running on the host computer. As the active wave generator hardware has 16, 384 neurons, an arena with 128 × 128 pixels can be modeled and solved by the algorithm. The system also has a monitor and network image is depicted on the monitor simultaneously.

  12. Pharmacological Investigations of the Cellular Transduction Pathways Used by Cholecystokinin to Activate Nodose Neurons

    PubMed Central

    Zhao, Huan; Kinch, Dallas C.; Simasko, Steven M.

    2011-01-01

    Cholecystokinin (CCK) directly activates vagal afferent neurons resulting in coordinated gastrointestinal functions and satiation. In vitro, the effects of CCK on dissociated vagal afferent neurons are mediated via activation of the vanilloid family of transient receptor potential (TRPV) cation channels leading to membrane depolarization and an increase in cytosolic calcium. However, the cellular transduction pathway(s) involved in this process between CCK receptors and channel opening have not been identified. To address this question, we monitored CCK-induced cytosolic calcium responses in dissociated nodose neurons from rat in the presence or absence of reagents that interact with various intracellular signaling pathways. We found that the phospholipase C (PLC) inhibitor U-73122 significantly attenuated CCK-induced responses, whereas the inactive analog U-73433 had no effect. Responses to CCK were also cross-desensitized by a brief pretreatment with m-3M3FBS, a PLC stimulator. Together these observations strongly support the participation of PLC in the effects of CCK on vagal afferent neurons. In contrast, pharmacological antagonism of phospholipase A2, protein kinase A, and phosphatidylinositol 3-kinase revealed that they are not critical in the CCK-induced calcium response in nodose neurons. Further investigations of the cellular pathways downstream of PLC showed that neither protein kinase C (PKC) nor generation of diacylglycerol (DAG) or release of calcium from intracellular stores participates in the response to CCK. These results suggest that alteration of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) content by PLC activity mediates CCK-induced calcium response and that this pathway may underlie the vagally-mediated actions of CCK to induce satiation and alter gastrointestinal functions. PMID:21664195

  13. A new cellular nonlinear network emulation on FPGA for EEG signal processing in epilepsy

    NASA Astrophysics Data System (ADS)

    Müller, Jens; Müller, Jan; Tetzlaff, Ronald

    2011-05-01

    For processing of EEG signals, we propose a new architecture for the hardware emulation of discrete-time Cellular Nonlinear Networks (DT-CNN). Our results show the importance of a high computational accuracy in EEG signal prediction that cannot be achieved with existing analogue VLSI circuits. The refined architecture of the processing elements and its resource schedule, the cellular network structure with local couplings, the FPGA-based embedded system containing the DT-CNN, and the data flow in the entire system will be discussed in detail. The proposed DT-CNN design has been implemented and tested on an Xilinx FPGA development platform. The embedded co-processor with a multi-threading kernel is utilised for control and pre-processing tasks and data exchange to the host via Ethernet. The performance of the implemented DT-CNN has been determined for a popular example and compared to that of a conventional computer.

  14. Point process models for localization and interdependence of punctate cellular structures.

    PubMed

    Li, Ying; Majarian, Timothy D; Naik, Armaghan W; Johnson, Gregory R; Murphy, Robert F

    2016-07-01

    Accurate representations of cellular organization for multiple eukaryotic cell types are required for creating predictive models of dynamic cellular function. To this end, we have previously developed the CellOrganizer platform, an open source system for generative modeling of cellular components from microscopy images. CellOrganizer models capture the inherent heterogeneity in the spatial distribution, size, and quantity of different components among a cell population. Furthermore, CellOrganizer can generate quantitatively realistic synthetic images that reflect the underlying cell population. A current focus of the project is to model the complex, interdependent nature of organelle localization. We built upon previous work on developing multiple non-parametric models of organelles or structures that show punctate patterns. The previous models described the relationships between the subcellular localization of puncta and the positions of cell and nuclear membranes and microtubules. We extend these models to consider the relationship to the endoplasmic reticulum (ER), and to consider the relationship between the positions of different puncta of the same type. Our results do not suggest that the punctate patterns we examined are dependent on ER position or inter- and intra-class proximity. With these results, we built classifiers to update previous assignments of proteins to one of 11 patterns in three distinct cell lines. Our generative models demonstrate the ability to construct statistically accurate representations of puncta localization from simple cellular markers in distinct cell types, capturing the complex phenomena of cellular structure interaction with little human input. This protocol represents a novel approach to vesicular protein annotation, a field that is often neglected in high-throughput microscopy. These results suggest that spatial point process models provide useful insight with respect to the spatial dependence between cellular structures.

  15. Changes in cellular degradation activity in young and old worker honeybees (Apis mellifera).

    PubMed

    Hsu, Chin-Yuan; Chuang, Yu-Lung; Chan, Yu-Pei

    2014-02-01

    The trophocytes and fat cells of honeybees (Apis mellifera) have been used in cellular senescence studies, but the changes of cellular degradation activity with aging in workers are unknown. In this study, cellular degradation activity was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed the following: (1) 20S proteosome activity decreased with aging, whereas its expression increased with aging; (2) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and the 70 kD heat shock cognate protein (Hsc70) decreased with aging; (3) the size and number of autophagic vacuoles decreased with aging; (4) p62/SQSTM1 and polyubiquitin aggregate expression decreased with aging; (5) lysosomal efficiency decreased with aging; and (6) molecular target of rapamycin (mTOR) expression increased with aging. These results indicate that young workers have higher levels of cellular degradation activity than old workers and that aging results in a decline in the cellular degradation activity in worker honeybees.

  16. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

    PubMed Central

    2013-01-01

    Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

  17. Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

    PubMed Central

    Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

    2015-01-01

    ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the

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

    PubMed

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

    2016-04-19

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

  19. SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity*

    PubMed Central

    Aukrust, Ingvild; Bjørkhaug, Lise; Negahdar, Maria; Molnes, Janne; Johansson, Bente B.; Müller, Yvonne; Haas, Wilhelm; Gygi, Steven P.; Søvik, Oddmund; Flatmark, Torgeir; Kulkarni, Rohit N.; Njølstad, Pål R.

    2013-01-01

    Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 β-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic β-cells. PMID:23297408

  20. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events.

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

    Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

  1. Roles and regulation of Neutral Sphingomyelinase-2 in cellular and pathological processes

    PubMed Central

    Shamseddine, Achraf A.; Airola, Michael V.; Hannun, Yusuf A.

    2015-01-01

    Our understanding of the functions of ceramide signaling has advanced tremendously over the past decade. In this review, we focus on the roles and regulation of neutral sphingomyelinase 2 (nSMase2), an enzyme that generates the bioactive lipid ceramide through the hydrolysis of the membrane lipid sphingomyelin. A large body of work has now implicated nSMase2 in a diverse set of cellular functions, physiological processes, and disease pathologies. We discuss different aspects of this enzyme’s regulation from transcriptional, post-translational, and biochemical. Furthermore, we highlight nSMase2 involvement in cellular processes including inflammatory signaling, exosome generation, cell growth, and apoptosis, which in turn play important roles in pathologies such as cancer metastasis, Alzheimer’s disease, and other organ systems disorders. Lastly, we examine avenues where targeted nSMase2-inhibition may be clinically beneficial in disease scenarios. PMID:25465297

  2. The Sarcomeric M-Region: A Molecular Command Center for Diverse Cellular Processes

    PubMed Central

    Hu, Li-Yen R.; Ackermann, Maegen A.; Kontrogianni-Konstantopoulos, Aikaterini

    2015-01-01

    The sarcomeric M-region anchors thick filaments and withstands the mechanical stress of contractions by deformation, thus enabling distribution of physiological forces along the length of thick filaments. While the role of the M-region in supporting myofibrillar structure and contractility is well established, its role in mediating additional cellular processes has only recently started to emerge. As such, M-region is the hub of key protein players contributing to cytoskeletal remodeling, signal transduction, mechanosensing, metabolism, and proteasomal degradation. Mutations in genes encoding M-region related proteins lead to development of severe and lethal cardiac and skeletal myopathies affecting mankind. Herein, we describe the main cellular processes taking place at the M-region, other than thick filament assembly, and discuss human myopathies associated with mutant or truncated M-region proteins. PMID:25961035

  3. Quantifying colocalization of a conditionally active transcription factor FOXP3 in three-dimensional cellular space

    NASA Astrophysics Data System (ADS)

    Abraham, Thomas; Allan, Sarah E.; Levings, Megan K.

    2009-02-01

    Biological macromolecular interactions between proteins, transcription factors, DNA and other types of biomolecules, are fundamentally important to several cellular and biological processes. 3D Multi-channel confocal microscopy and colocalization analysis of fluorescent signals have proven to be invaluable tools for detecting such molecular interactions. The aim of this work was to quantify colocalization of the FOXP3 transcription factor in 3D cellular space generated from the confocal 3D image sets. 293T cells transfected with a conditionally active form of FOXP3 were stained for nuclei with Hoechst, for FOXP3 with anti-FOXP3 conjugated to PE, and 4-hydroxytamoxifen used as protein translocation and activation agent. Since the protein signal was weak and nonspecific intensity contributions were strong, it was difficult to perform colocalization analysis and estimate colocalization quantities. We performed 3D restoration by deconvolution method on the confocal images using experimentally measured point spread functions (PSFs) and subsequently a color shift correction. The deconvolution method eliminated nonspecific intensity contributions originating from PSF imposed by optical microscopy diffraction resolution limits and noise since these factors significantly affected colocalization analysis and quantification. Visual inspection of the deconvolved 3D image suggested that the FOXP3 molecules are predominantly colocalized within the nuclei although the fluorescent signals from FOXP3 molecules were also present in the cytoplasm. A close inspection of the scatter plot (colocalization map) and correlation quantities such as the Pearsons and colocalization coefficients showed that the fluorescent signals from the FOXP3 molecules and DNA are strongly correlated. In conclusion, our colocalization quantification approach confirms the preferential association of the FOXP3 molecules with the DNA despite the presence of fluorescent signals from the former one both in the

  4. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    NASA Astrophysics Data System (ADS)

    Song, Zhen

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

  5. Driving mechanisms of passive and active transport across cellular membranes as the mechanisms of cell metabolism and development as well as the mechanisms of cellular distance reactions on hormonal expression and the immune response.

    PubMed

    Ponisovskiy, M R

    2011-01-01

    The article presents mechanisms of cell metabolism, cell development, cell activity, and maintenance of cellular stability. The literature is reviewed from the point of view of these concepts. The balance between anabolic and catabolic processes induces chemical potentials in the extracellular and intracellular media. The chemical potentials of these media are defined as the driving forces of both passive and active transport of substances across cellular membranes. The driving forces of substance transport across cellular membranes as in cellular metabolism and in immune responses and hormonal expressions are considered in the biochemical and biophysical models, reflecting the mechanisms for maintenance of stability of the internal medium and internal energy of an organism. The interactions of passive transport and active transport of substances across cellular walls promote cell proliferation, as well as the mechanism of cellular capacitors, promoting remote reactions across distance for hormonal expression and immune responses. The offered concept of cellular capacitors has given the possibility to explain the mechanism of remote responses of cells to new situations, resulting in the appearance of additional agents. The biophysical model develops an explanation of some cellular functions: cellular membrane action have been identified with capacitor action, based on the similarity of the structures and as well as on similarity of biophysical properties of electric data that confirm the action of the compound-specific interactions of cells within an organism, promoting hormonal expressions and immune responses to stabilize the thermodynamic system of an organism. Comparison of a cellular membrane action to a capacitor has given the possibility for the explanations of exocytosis and endocytosis mechanisms, internalization of the receptor-ligand complex, selection as a receptor reaction to a ligand by immune responses or hormonal effects, reflecting cellular

  6. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    NASA Astrophysics Data System (ADS)

    Song, Zhen

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

  7. Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress.

    PubMed

    Ozden, Ozkan; Park, Seong-Hoon; Kim, Hyun-Seok; Jiang, Haiyan; Coleman, Mitchell C; Spitz, Douglas R; Gius, David

    2011-02-01

    A fundamental observation in biology is that mitochondrial function, as measured by increased reactive oxygen species (ROS), changes significantly with age, suggesting a potential mechanistic link between the cellular processes governing longevity and mitochondrial metabolism homeostasis. In addition, it is well established that altered ROS levels are observed in multiple age-related illnesses including carcinogenesis, neurodegenerative, fatty liver, insulin resistance, and cardiac disease, to name just a few. Manganese superoxide dismutase (MnSOD) is the primary mitochondrial ROS scavenging enzyme that converts superoxide to hydrogen peroxide, which is subsequently converted to water by catalase and other peroxidases. It has recently been shown that MnSOD enzymatic activity is regulated by the reversible acetylation of specific, evolutionarily conserved lysine(s) in the protein. These results, suggest for the first time, that the mitochondria contain bidirectional post-translational signaling networks, similar to that observed in the cytoplasm and nucleus, and that changes in lysine acetylation alter MnSOD enzymatic activity. In addition, these new results demonstrate that the mitochondrial anti-aging or fidelity / sensing protein, SIRT3, responds to changes in mitochondrial nutrient and/or redox status to alter the enzymatic activity of specific downstream targets, including MnSOD that adjusts and/or maintains ROS levels as well as metabolic homeostatic poise. PMID:21386137

  8. Activation of the hypothalamic-pituitary-adrenal stress axis induces cellular oxidative stress

    PubMed Central

    Spiers, Jereme G.; Chen, Hsiao-Jou Cortina; Sernia, Conrad; Lavidis, Nickolas A.

    2015-01-01

    Glucocorticoids released from the adrenal gland in response to stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis induce activity in the cellular reduction-oxidation (redox) system. The redox system is a ubiquitous chemical mechanism allowing the transfer of electrons between donor/acceptors and target molecules during oxidative phosphorylation while simultaneously maintaining the overall cellular environment in a reduced state. The objective of this review is to present an overview of the current literature discussing the link between HPA axis-derived glucocorticoids and increased oxidative stress, particularly focussing on the redox changes observed in the hippocampus following glucocorticoid exposure. PMID:25646076

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  10. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time

    PubMed Central

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C.; Gallagher, William M.; O'Shea, Donal F.

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    Kagawa, Shingo; Natsuizaka, Mitsuteru; Whelan, Kelly A.; Facompre, Nicole; Naganuma, Seiji; Ohashi, Shinya; Kinugasa, Hideaki; Egloff, Ann Marie; Basu, Devraj; Gimotty, Phyllis A.; Klein-Szanto, Andres J; Bass, Adam; Wong, Kwok-Kin; Diehl, J. Alan; Rustgi, Anil K.; Nakagawa, Hiroshi

    2014-01-01

    Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context. PMID:24931169

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

    PubMed

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

    2015-04-30

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

  14. Beta Cell Formation in vivo Through Cellular Networking, Integration and Processing (CNIP) in Wild Type Adult Mice.

    PubMed

    Doiron, Bruno; Hu, Wenchao; DeFronzo, Ralph A

    2016-01-01

    Insulin replacement therapy is essential in type 1 diabetic individuals and is required in ~40- 50% of type 2 diabetics during their lifetime. Prior attempts at beta cell regeneration have relied upon pancreatic injury to induce beta cell proliferation, dedifferentiation and activation of the embryonic pathway, or stem cell replacement. We report an alternative method to transform adult non-stem (somatic) cells into pancreatic beta cells. The Cellular Networking, Integration and Processing (CNIP) approach targets cellular mechanisms involved in pancreatic function in the organ's adult state and utilizes a synergistic mechanism that integrates three important levels of cellular regulation to induce beta cell formation: (i) glucose metabolism, (ii) membrane receptor function, and (iii) gene transcription. The aim of the present study was to induce pancreatic beta cell formation in vivo in adult animals without stem cells and without dedifferentiating cells to recapitulate the embryonic pathway as previously published (1-3). Our results employing CNIP demonstrate that: (i) insulin secreting cells can be generated in adult pancreatic tissue in vivo and circumvent the problem of generating endocrine (glucagon and somatostatin) cells that exert deleterious effects on glucose homeostasis, and (ii) longterm normalization of glucose tolerance and insulin secretion can be achieved in a wild type diabetic mouse model. The CNIP cocktail has the potential to be used as a preventative or therapeutic treatment or cure for both type 1 and type 2 diabetes. PMID:26696016

  15. Systems Glycobiology: Integrating Glycogenomics, Glycoproteomics, Glycomics, and Other 'Omics Data Sets to Characterize Cellular Glycosylation Processes.

    PubMed

    Bennun, Sandra V; Hizal, Deniz Baycin; Heffner, Kelley; Can, Ozge; Zhang, Hui; Betenbaugh, Michael J

    2016-08-14

    The number of proteins encoded in the human genome has been estimated at between 20,000 and 25,000, despite estimates that the entire proteome contains more than a million proteins. One reason for this difference is due to many post-translational modifications of protein that contribute to proteome complexity. Among these, glycosylation is of particular relevance because it serves to modify a large number of cellular proteins. Glycogenomics, glycoproteomics, glycomics, and glycoinformatics are helping to accelerate our understanding of the cellular events involved in generating the glycoproteome, the variety of glycan structures possible, and the importance of roles that glycans play in therapeutics and disease. Indeed, interest in glycosylation has expanded rapidly over the past decade, as large amounts of experimental 'omics data relevant to glycosylation processing have accumulated. Furthermore, new and more sophisticated glycoinformatics tools and databases are now available for glycan and glycosylation pathway analysis. Here, we summarize some of the recent advances in both experimental profiling and analytical methods involving N- and O-linked glycosylation processing for biotechnological and medically relevant cells together with the unique opportunities and challenges associated with interrogating and assimilating multiple, disparate high-throughput glycosylation data sets. This emerging era of advanced glycomics will lead to the discovery of key glycan biomarkers linked to diseases and help establish a better understanding of physiology and improved control of glycosylation processing in diverse cells and tissues important to disease and production of recombinant therapeutics. Furthermore, methodologies that facilitate the integration of glycomics measurements together with other 'omics data sets will lead to a deeper understanding and greater insights into the nature of glycosylation as a complex cellular process.

  16. Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

    PubMed Central

    Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

    2015-01-01

    AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this

  17. Correlation between proliferative activity and cellular thickness of human mesenchymal stem cells

    SciTech Connect

    Katsube, Yoshihiro; Hirose, Motohiro Nakamura, Chikashi; Ohgushi, Hajime

    2008-04-04

    A cell's shape is known to be related to its proliferative activity. In particular, large and flat mammalian adult stem cells seem to show slow proliferation, however using quantitative analysis to prove the phenomenon is difficult. We measured the proliferation and cellular thickness of human mesenchymal stem cells (MSCs) by atomic force microscopy and found that MSCs with high proliferative activity were thick while those with low proliferative activity were thin, even though these MSCs were early passage cells. Further, low proliferative MSCs contained many senescence-associated {beta}-galactosidase positive cells together with high senescence-associated gene expression. These findings suggest that the measurement of cellular thickness is useful for estimating the proliferative activity of human MSCs and is expected to be a practical tool for MSC applications in regenerative medicine.

  18. The inactive-active phase transition in the noisy additive (exclusive-or) probabilistic cellular automaton

    NASA Astrophysics Data System (ADS)

    Mendonça, J. Ricardo G.

    2016-07-01

    We investigate the inactive-active phase transition in an array of additive (exclusive-or) cellular automata (CA) under noise. The model is closely related with the Domany-Kinzel (DK) probabilistic cellular automaton (PCA), for which there are rigorous as well as numerical estimates on the transition probabilities. Here, we characterize the critical behavior of the noisy additive cellular automaton by mean field analysis and finite-size scaling and show that its phase transition belongs to the directed percolation universality class of critical behavior. As a by-product of our analysis, we argue that the critical behavior of the noisy elementary CA 90 and 102 (in Wolfram’s enumeration scheme) must be the same. We also perform an empirical investigation of the mean field equations to assess their quality and find that away from the critical point (but not necessarily very far away) the mean field approximations provide a reasonably good description of the dynamics of the PCA.

  19. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway.

  20. 2D-CELL: image processing software for extraction and analysis of 2-dimensional cellular structures

    NASA Astrophysics Data System (ADS)

    Righetti, F.; Telley, H.; Leibling, Th. M.; Mocellin, A.

    1992-01-01

    2D-CELL is a software package for the processing and analyzing of photographic images of cellular structures in a largely interactive way. Starting from a binary digitized image, the programs extract the line network (skeleton) of the structure and determine the graph representation that best models it. Provision is made for manually correcting defects such as incorrect node positions or dangling bonds. Then a suitable algorithm retrieves polygonal contours which define individual cells — local boundary curvatures are neglected for simplicity. Using elementary analytical geometry relations, a range of metric and topological parameters describing the population are then computed, organized into statistical distributions and graphically displayed.

  1. Structural elucidation and cellular antioxidant activity evaluation of major antioxidant phenolics in lychee pulp.

    PubMed

    Su, Dongxiao; Ti, Huihui; Zhang, Ruifen; Zhang, Mingwei; Wei, Zhengchen; Deng, Yuanyuan; Guo, Jinxin

    2014-09-01

    Lychee pulp contains phenolic compounds that are strong antioxidants, but the identities of the major antioxidants present are unknown. In the present study, the major contributors to the antioxidant activity of fresh lychee pulp were identified and their cellular antioxidant activities were investigated. Aqueous acetone extracts of lychee pulp were fractionated on polyamide resin, and those fractions with the largest antioxidant and radical scavenging activities were selected using cellular antioxidant activity and oxygen radical absorbance capacity assays. Three compounds that were major contributors to the antioxidant activity in these fractions were obtained by reverse-phase preparative HPLC and identified as quercetin 3-O-rutinoside-7-O-α-L-rhamnosidase (quercetin 3-rut-7-rha), quercetin 3-O-rutinoside (rutin) and (-)-epicatechin using NMR spectroscopy, HMBC, and ESI-MS spectrometry. The concentration of quercetin 3-rut-7-rha was 17.25mg per 100g of lychee pulp fresh weight. This is the first report of the identification and cellular antioxidant activity of quercetin 3-rut-7-rha from lychee pulp.

  2. Particle acceleration and radiation in flaring complex solar active regions modeled by cellular automata

    NASA Astrophysics Data System (ADS)

    Dauphin, C.; Vilmer, N.; Anastasiadis, A.

    2007-06-01

    Context: We study the acceleration and radiation of electrons and ions interacting with multiple small-scale dissipation regions resulting from the magnetic energy release process. Aims: We aim to calculate the distribution functions of the kinetic energy of the particles and the X-ray spectra and γ-ray fluxes produced by the accelerated particles. Methods: The evolution of the magnetic energy released in an active region is mimicked by a cellular automaton model based on the concept of self-organized criticality. Each burst of magnetic energy release is associated with a reconnecting current sheet (RCS) in which the particles are accelerated by a direct electric field. Results: We calculate the energy gain of the particles (ions and electrons) for three different magnetic configurations of the RCS after their interactions with a given number of RCS. We finally compare our results with existing observations. Conclusions: The results of our simulation can reproduce several properties of the observations such as variable electron and ion energy contents and γ-ray line ratio. Even if very flat X-ray spectra have been reported in a few events, the X-ray spectra produced in this model are too flat when compared to most X-ray observations.

  3. Evidence of parasexual activity in "asexual amoebae" Cochliopodium spp. (Amoebozoa): extensive cellular and nuclear fusion.

    PubMed

    Tekle, Yonas I; Anderson, O Roger; Lecky, Ariel F

    2014-09-01

    The majority of microbial eukaryotes have long been considered asexual, though new evidence indicates sex, or sexual-like (parasexual) behaviors that deviate from the usual union of two gametes, among other variant aspects. Over a dozen amoebozoans are implicated to have sexual stages. However, the exact mechanism by which sex occurs in these lineages remains elusive. This is mainly due to the diverse quality and cryptic nature of their life cycle. In this study we present evidence of some previously unreported aspects of the life cycle of an amoeba, Cochliopodium, that undergoes unusual intraspecific interactions using light microscopy and immunocytochemistry. Similar to other amoebozoans, Cochliopodium, is considered asexual with no published reports of sex or parasexuality. We also investigated environmental conditions that govern the observed intraspecific interactions. Both light microscopic and immunocytochemistry evidence demonstrates Cochliopodium undergoes cellular fusion (plasmogamy) and nuclear fusion (karyogamy). Large plasmodia eventually undergo karyogamy and contain large fused, polyploid, nuclei. These are observed to fragment, subsequently, by karyotomy (nuclear fission) and cytoplasmic fission to yield uninucleated amoebae. This process could lead to a non-meiotic, parasexual exchange of chromosomes in Cochliopodium. These findings strongly suggest that Cochliopodium is involved in parasexual activity and should no longer be considered strictly asexual.

  4. Activation of hERG3 channel stimulates autophagy and promotes cellular senescence in melanoma

    PubMed Central

    Perez-Neut, Mathew; Haar, Lauren; Rao, Vidhya; Santha, Sreevidya; Lansu, Katherine; Rana, Basabi; Jones, Walter K.; Gentile, Saverio

    2016-01-01

    Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma. PMID:26942884

  5. p190RhoGAP has cellular RacGAP activity regulated by a polybasic region.

    PubMed

    Lévay, Magdolna; Bartos, Balázs; Ligeti, Erzsébet

    2013-06-01

    p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac-Rho antagonism than it was realized earlier.

  6. Antioxidative Activity of Platinum Nanocolloid and Its Protective Effect Against Chemical-Induced Hepatic Cellular Damage.

    PubMed

    Choi, Mi-Ran; Do, Le Thanh; Chung, Yong-Hoon; Yoo, Hoon; Yu, Rina

    2015-08-01

    Oxidative stress, a major cause of cellular injuries, is closely associated with a variety of chronic diseases such as cancer, liver diseases, degenerative brain disease and aging. In this study, we investigated antioxidant properties of platinum nanocolloid (PNC) against various oxidative stress conditions in vitro/in vivo by treating PNC on liver cell or tissue. Antioxidant activities of the PNC were determined by measuring quenching capacity on reactive oxygen species and its protective action against hydrogen peroxide or CCl4-induced oxidative cellular damage in HepG2 cell or liver tissue of mice. In vitro study, PNC markedly suppressed the production H2O2, ·OH, α,α-diphenyl-β-picrylhydrazyl radical and nitric oxide in a dose-dependent manner. PNC also inhibited hydrogen peroxide-induced oxidative cellular damage in HepG2 hepatocytes. In vivo study with mice, PNC reduced hepatic lipid peroxidation and CCl4 induced toxicity. Our results support that platinum nanocolloid has antioxidant activities and protects hepatic cellular oxidative damage. Thus platinum nanocolloid may have a potential to be used as an antioxidant supplement.

  7. Histone H3.3 and its proteolytically processed form drive a cellular senescence program

    PubMed Central

    Duarte, Luis F.; Young, Andrew R. J.; Wang, Zichen; Wu, Hsan-Au; Panda, Taniya; Kou, Yan; Kapoor, Avnish; Hasson, Dan; Mills, Nicholas R.; Ma’ayan, Avi; Narita, Masashi; Bernstein, Emily

    2014-01-01

    The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

  8. Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans-Ignicoccus hospitalis Relationship

    PubMed Central

    Giannone, Richard J.; Huber, Harald; Karpinets, Tatiana; Heimerl, Thomas; Küper, Ulf; Rachel, Reinhard; Keller, Martin; Hettich, Robert L.; Podar, Mircea

    2011-01-01

    Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis. PMID:21826220

  9. Proteomic characterization of cellular and molecular processes that enable the Nanoarchaeum equitans-Ignicoccus hospitalis relationship

    SciTech Connect

    Giannone, Richard J; Huber, Dr. Harald; Karpinets, Tatiana V; Heimerl, Dr. Thomas; Kueper, Dr. Ulf; Rachel, Dr. Reinhard; Keller, Martin; Hettich, Robert {Bob} L; Podar, Mircea

    2011-01-01

    Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis.

  10. Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis

    PubMed Central

    Oropesa-Ávila, M; Fernández-Vega, A; de la Mata, M; Maraver, J G; Cordero, M D; Cotán, D; de Miguel, M; Calero, C P; Paz, M V; Pavón, A D; Sánchez, M A; Zaderenko, A P; Ybot-González, P; Sánchez-Alcázar, J A

    2013-01-01

    Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit β4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na+/Ca2+ exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na+/K+ pump subunit β was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential

  11. Conflict game in evacuation process: A study combining Cellular Automata model

    NASA Astrophysics Data System (ADS)

    Zheng, Xiaoping; Cheng, Yuan

    2011-03-01

    The game-theoretic approach is an essential tool in the research of conflicts of human behaviors. The aim of this study is to research crowd dynamic conflicts during evacuation processes. By combining a conflict game with a Cellular Automata model, the following factors such as rationality, herding effect and conflict cost are taken into the research on frequency of each strategy of evacuees, and evacuation time. Results from Monte Carlo simulations show that (i) in an emergency condition, rationality leads to “vying” behaviors and inhibited “polite” behavior; (ii) high herding causes a crowd of high rationality (especially in normal circumstances) to become more “vying” in behavior; (iii) the high-rationality crowd is shown to spend more evacuation time than a low-rationality crowd in emergency situations. This study provides a new perspective to understand conflicts in evacuation processes as well as the rationality of evacuees.

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

    PubMed Central

    Little, Max A.; Jones, Nick S.

    2013-01-01

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

  13. Light-activated hypericin induces cellular destruction of nasopharyngeal carcinoma cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.

    2010-01-01

    Hypericin from Hypericum perforatum plants shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that light-activated hypericin induced the cellular destruction of nasopharyngeal carcinoma cells. The result showed that hypericin resulted in a drug- and light-dose dependent cytotoxicity in the CNE-2 cells, meaning the photocytotoxicity of hypericin depends on both of the drug concentration (0 - 2.5 μM) and light-doses (1 - 8 J/cm2). We further investigated the apoptosis of the CNE-2 cells 8 hours after photosensitization of hypericin using fluorescence microscopy with Hoechst 33258 staining. Flow cytometry with annexin V-FITC and PI staining was used to analyze early and late apoptosis. These data demonstrated that light-activated hypericin could significantly lead to the cellular destruction of the CNE-2 cells and induce early apoptosis as a prominent mode of cell death.

  14. LED-activated pheophorbide a induces cellular destruction of colon cancer cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.; Liu, L.; Xia, X. S.

    2010-07-01

    Pheophorbide a (Pa) from Chinese herbal medicine Scutellaria Barbata and Silkworm Excreta shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that LED-activated Pa induced the cellular destruction of colon cancer HT-29 cells. The results showed that Pa resulted in a drug-dose dependent photocytotoxicity in the HT-29 cells, meaning the photocytotoxicity of Pa depends on the drug concentration (0 - 2 μM). We further investigated the apoptosis of the HT-29 cells 18 hours after photosensitization of Pa using a confocal laser scanning microscopy with Hoechst 33258 staining. These data demonstrated that LED-activated Pa could significantly induce the cellular destruction of the HT-29 cells.

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

    PubMed Central

    Jonoska, Nataša; Karpenko, Daria; Seki, Shinnosuke

    2016-01-01

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

  16. Preliminary cellular-automata forecast of permit activity from 1998 to 2010, Idaho and Western Montana

    USGS Publications Warehouse

    Raines, G.L.; Zientek, M.L.; Causey, J.D.; Boleneus, D.E.

    2002-01-01

    For public land management in Idaho and western Montana, the U.S. Forest Service (USFS) has requested that the U.S. Geological Survey (USGS) predict where mineral-related activity will occur in the next decade. Cellular automata provide an approach to simulation of this human activity. Cellular automata (CA) are defined by an array of cells, which evolve by a simple transition rule, the automaton. Based on exploration trends, we assume that future exploration will focus in areas of past exploration. Spatial-temporal information about mineral-related activity, that is permits issued by USFS and Bureau of Land Management (BLM) in the last decade, and spatial information about undiscovered resources, provide a basis to calibrate a CA. The CA implemented is a modified annealed voting rule that simulates mineral-related activity with spatial and temporal resolution of 1 mi2 and 1 year based on activity from 1989 to 1998. For this CA, the state of the economy and exploration technology is assumed constant for the next decade. The calibrated CA reproduces the 1989-1998-permit activity with an agreement of 94%, which increases to 98% within one year. Analysis of the confusion matrix and kappa correlation statistics indicates that the CA underestimates high activity and overestimates low activity. Spatially, the major differences between the actual and calculated activity are that the calculated activity occurs in a slightly larger number of small patches and is slightly more uneven than the actual activity. Using the calibrated CA in a Monte Carlo simulation projecting from 1998 to 2010, an estimate of the probability of mineral activity shows high levels of activity in Boise, Caribou, Elmore, Lincoln, and western Valley counties in Idaho and Beaverhead, Madison, and Stillwater counties in Montana, and generally low activity elsewhere. ?? 2002 International Association for Mathematical Geology.

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

    PubMed Central

    Tatsuo, Hironobu; Ono, Nobuyuki; Yanagi, Yusuke

    2001-01-01

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

  18. Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles.

    PubMed

    Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

    2014-01-01

    Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles - GME-EC and GME-EC/MC nanoparticles - successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

  19. Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles

    PubMed Central

    Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

    2014-01-01

    Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles – GME-EC and GME-EC/MC nanoparticles – successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

  20. Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles.

    PubMed

    Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

    2014-01-01

    Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles - GME-EC and GME-EC/MC nanoparticles - successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity.

  1. Oxidized (non)-regenerated cellulose affects fundamental cellular processes of wound healing

    PubMed Central

    Wagenhäuser, M. U.; Mulorz, J.; Ibing, W.; Simon, F.; Spin, J. M.; Schelzig, H.; Oberhuber, A.

    2016-01-01

    In this study we investigated how hemostats such as oxidized regenerated cellulose (ORC, TABOTAMP) and oxidized non-regenerated cellulose (ONRC, RESORBA CELL) influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated in vitro with ORC and ONRC. Cell proliferation was assayed over time, and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC, and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p < 0.05). When control media was pH-lowered to match the lower pH values typically seen with ORC and ONRC, significant differences in cell proliferation and migration were still observed between ONRC and ORC (p < 0.05). However, in these pH conditions, inhibition of matrix contraction was only significant for ONRC (p < 0.05). We find that ORC and ONRC inhibit fibroblast proliferation, migration and matrix contraction, and stronger inhibition of these essential cellular processes of wound healing were observed for ONRC when compared with ORC. These results will require further validation in future in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing. PMID:27557881

  2. Oxidized (non)-regenerated cellulose affects fundamental cellular processes of wound healing.

    PubMed

    Wagenhäuser, M U; Mulorz, J; Ibing, W; Simon, F; Spin, J M; Schelzig, H; Oberhuber, A

    2016-01-01

    In this study we investigated how hemostats such as oxidized regenerated cellulose (ORC, TABOTAMP) and oxidized non-regenerated cellulose (ONRC, RESORBA CELL) influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated in vitro with ORC and ONRC. Cell proliferation was assayed over time, and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC, and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p < 0.05). When control media was pH-lowered to match the lower pH values typically seen with ORC and ONRC, significant differences in cell proliferation and migration were still observed between ONRC and ORC (p < 0.05). However, in these pH conditions, inhibition of matrix contraction was only significant for ONRC (p < 0.05). We find that ORC and ONRC inhibit fibroblast proliferation, migration and matrix contraction, and stronger inhibition of these essential cellular processes of wound healing were observed for ONRC when compared with ORC. These results will require further validation in future in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing. PMID:27557881

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

    NASA Astrophysics Data System (ADS)

    Gollas, Frank; Tetzlaff, Ronald

    2009-05-01

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

  4. NMDA-R inhibition affects cellular process formation in Tilapia melanocytes; a model for pigmented adrenergic neurons in process formation and retraction.

    PubMed

    Ogundele, Olalekan Michael; Okunnuga, Adetokunbo Adedotun; Fabiyi, Temitope Deborah; Olajide, Olayemi Joseph; Akinrinade, Ibukun Dorcas; Adeniyi, Philip Adeyemi; Ojo, Abiodun Ayodele

    2014-06-01

    elongation of secondary cellular processes (highly branched) from primary major processes (Less branched); co-incubation of glutamate and ketamine induced short and highly branched process formation. Cyanide toxicity induced degeneration and reduction of cell size while co-treatment of cyanide and ketamine gave changes similar to that observed in glutamate-ketamine co-incubation. NMDA-R is present in the melanocytes. Activation of the receptor reduced elongation process, while inhibition of the receptor facilitated cell process elongation and branching. This confirms that like pigmented adrenergic cells of the nervous system, this cell contains NMDA-R and this receptor also regulates cell process elongation. The study also showed that inhibition of NMDA-R in melanocytes gave opposite outcomes to the role of the receptor in developing neurons; a function that is protective in adult neurons.

  5. Career management: an active process.

    PubMed

    Mackowiak, J; Eckel, F M

    1985-03-01

    The self-assessment, goal-setting, and career-planning techniques of career management are discussed, and the organization's role in career management is discussed. Career management is a planned process, initiated and carried out by an individual with the assistance of others. Because work and nonwork activities are so interrelated, career and life management planning can maximize a pharmacist's personal success. The career- and life-management process begins with the development of a personal definition of success. A self-assessment must be made of one's values, needs, interests, and activities. The next step of the process involves setting goals and establishing a plan or strategy to achieve them. Establishing a career path requires researching alternate career goals. Career competencies are identified that can increase an employee's chances of success. The employer shares the responsibility for career development through coaching, job structuring, and keeping the employee aware of constraints. Through the integration of the roles of the individual and the organization in the career-management process, employees can optimize their contribution to an organization. Pharmacists can successfully manage their careers by applying the techniques of self-assessment, goal setting, and career planning. PMID:3985018

  6. Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids.

    PubMed Central

    Keller, R; Fischer, W; Keist, R; Bassetti, S

    1992-01-01

    Lipoteichoic acids (LTAs) from various bacterial species, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, and Listeria monocytogenes, were examined for the ability to induce secretory and cellular responses in a pure population of bone marrow-derived mononuclear phagocytes. Some of the highly purified LTAs, in particular LTAs from Bacillus subtilis, S. pyogenes, E. faecalis, and Enterococcus hirae, were able to affect each of the macrophage parameters measured, i.e., reductive capacity, secretion of tumor necrosis factor and nitrite, and tumoricidal activity. As after stimulation with whole organisms or other bacterial products, secretion of tumor necrosis factor induced by these LTAs reached its maximum within the first few hours of the interaction, while secretion of nitrite and tumoricidal activity required 24 to 36 h for full expression. Other purified LTAs, i.e., LTAs from Streptococcus sanguis, S. pneumoniae, and L. monocytogenes, as well as lipomannan from Micrococcus luteus affected only some of these parameters, while native LTA from S. aureus was inactive. There was no obvious correlation between biological activity and chain length, kind of glycosyl substituents, glycolipid structures, or fatty acid composition of LTAs. Deacylation of LTAs resulted in a complete loss of activity, and deacylated LTAs did not impair the activity of their acylated counterparts, suggesting that acyl chains may be essential for binding of LTA to the cell surface. The results demonstrate that some LTA species are potent inducers of macrophage secretory and cellular activities. PMID:1500175

  7. Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cardiomyocyte hypertrophy.

    PubMed

    Takeishi, Yasuchika; Goto, Kaoru; Kubota, Isao

    2007-09-01

    Diacylglycerol (DAG) kinase (DGK) phosphorylates and converts DAG to phosphatidic acid. DGK regulates cellular DAG levels and attenuates DAG signaling. The 10 mammalian DGK isoforms have been identified to date. In cardiac myocytes, DGKalpha, epsilon, and zeta are expressed, and DGKzeta is the predominant isoform. DGKzeta inhibits protein kinase C (PKC) activation and subsequent hypertrophic programs in response to endothelin-1 (ET-1) in neonatal rat cardiomyocytes. DGKzeta blocks cardiac hypertrophy induced by G protein-coupled receptor agonists and pressure overload in vivo. DGKzeta attenuates ventricular remodeling and improves survival after myocardial infarction. These data provide a novel insight for subcellular mechanisms of cardiac hypertrophy and heart failure, and DGKzeta may be a new therapeutic target to prevent cardiac hypertrophy and progression to heart failure. PMID:17659347

  8. The Na+/K(+)-pump in rat peritoneal mast cells: some aspects of regulation of activity and cellular function.

    PubMed

    Knudsen, T

    1995-11-01

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also have important clinical implications. The aim of the present work has been to document the existence of the Na+/K(+)-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na+/K(+)-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with 86Rb+ as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE directed stimulation of the cell was used as a parameter for cellular degranulation. Histamine was measured by spectrofluorometry. The finding of an ouabain-sensitive uptake mechanism in the mast cell documents the presence of a functional Na+/K(+)-pump in this cell. The pump activity is inhibited by lanthanides and by the divalent cations calcium, magnesium, barium and strontium. The pump has a large reserve capacity which probably is caused by a low intracellular concentration of sodium. This enables the pump to respond to changes in the intracellular sodium concentration. The inhibitory effect of di- and trivalent ions on the pump activity is probably a result of the inhibitory effect of these ions on the cellular sodium uptake. The digitalis glycosides, ouabain and digoxin, but not the more lipophilic drug digitoxigenin, increase both IgE-directed and non-IgE-directed histamine release

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

    PubMed

    Kozma, Robert; Puljic, Marko

    2013-09-01

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

  10. Magnetic Resonance Microscopy of Human and Porcine Neurons and Cellular Processes

    PubMed Central

    Flint, Jeremy J.; Hansen, Brian; Portnoy, Sharon; Lee, Choong-Heon; King, Michael A.; Fey, Michael; Vincent, Franck; Stanisz, Greg J; Vestergaard-Poulsen, Peter; Blackband, Stephen J

    2012-01-01

    With its unparalleled ability to safely generate high-contrast images of soft tissues, magnetic resonance imaging (MRI) has remained at the forefront of diagnostic clinical medicine. Unfortunately due to resolution limitations, clinical scans are most useful for detecting macroscopic structural changes associated with a small number of pathologies. Moreover, due to a longstanding inability to directly observe magnetic resonance (MR) signal behavior at the cellular level, such information is poorly characterized and generally must be inferred. With the advent of the MR microscope in 1986 came the ability to measure MR signal properties of theretofore unobservable tissue structures. Recently, further improvements in hardware technology have made possible the ability to visualize mammalian cellular structure. In the current study, we expand upon previous work by imaging the neuronal cell bodies and processes of human and porcine α-motor neurons. Complimentary imaging studies are conducted in pig tissue in order to demonstrate qualitative similarities to human samples. Also, apparent diffusion coefficient (ADC) maps were generated inside porcine α-motor neuron cell bodies and portions of their largest processes (mean = 1.7±0.5 μm2/ms based on 53 pixels) as well as in areas containing a mixture of extracellular space, microvasculature, and neuropil (0.59±0.37 μm2/ms based on 33 pixels). Three-dimensional reconstruction of MR images containing α-motor neurons shows the spatial arrangement of neuronal projections between adjacent cells. Such advancements in imaging portend the ability to construct accurate models of MR signal behavior based on direct observation and measurement of the components which comprise functional tissues. These tools would not only be useful for improving our interpretation of macroscopic MRI performed in the clinic, but they could potentially be used to develop new methods of differential diagnosis to aid in the early detection of a

  11. Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.

    PubMed

    Haber, R S; Pressley, T A; Loeb, J N; Ismail-Beigi, F

    1987-07-01

    The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus "clamped" at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

  12. Two-Photon Enzymatic Probes Visualizing Sub-cellular/Deep-brain Caspase Activities in Neurodegenerative Models

    PubMed Central

    Qian, Linghui; Zhang, Cheng-Wu; Mao, Yanli; Li, Lin; Gao, Nengyue; Lim, Kah-Leong; Xu, Qing-Hua; Yao, Shao Q.

    2016-01-01

    Caspases work as a double-edged sword in maintaining cell homeostasis. Highly regulated caspase activities are essential during animal development, but dysregulation might lead to different diseases, e.g. extreme caspase activation is known to promote neurodegeneration. At present, visualization of caspase activation has mostly remained at the cellular level, in part due to a lack of cell-permeable imaging probes capable of direct, real-time investigations of endogenous caspase activities in deep tissues. Herein, we report a suite of two-photon, small molecule/peptide probes which enable sensitive and dynamic imaging of individual caspase activities in neurodegenerative models under physiological conditions. With no apparent toxicity and the ability of imaging endogenous caspases both in different subcellular organelles of mammalian cells and in brain tissues, these probes serve as complementary tools to conventional histological analysis. They should facilitate future explorations of caspases at molecular, cellular and organism levels and inspire development of novel two-photon probes against other enzymes. PMID:27210613

  13. Urinary neopterin, a non-invasive marker of mammalian cellular immune activation, is highly stable under field conditions.

    PubMed

    Heistermann, Michael; Higham, James P

    2015-01-01

    Studying immunity and immune function in ecology and evolution requires field studies, but there has been a dearth of non-invasive markers of immune activation available for studying large wild mammals. Recently, we analytically and biologically validated the measurement of urinary neopterin (NEO), a biomarker of cellular immune activation, in captive macaques. However, applying this to free-ranging settings is complicated by issues involving sample collection, processing, storage, and transport. Here, we collected urine samples from captive macaques and undertook experiments simulating common field issues. We tested the effects on urinary NEO sample measurements following: dirt and faecal contamination; storage at room temperature; differences in processing and long-term storage methods (freezing, lyophilising, blotting onto filter paper); and freeze-thaw cycles. Our results show that concentrations of urinary NEO are highly stable--they are not affected by soil or faecal contamination, can be collected on filter paper and stored for many months frozen or lyophilised with minimal effect, and are resistant to multiple 24 hr freeze-thaws. With the addition of a biocidal preservative, concentrations are even stable at room temperature for long periods. Urinary NEO is remarkably resilient, and is highly suitable for non-invasive field studies of cellular immune responses in wild large mammals.

  14. Urinary neopterin, a non-invasive marker of mammalian cellular immune activation, is highly stable under field conditions

    PubMed Central

    Heistermann, Michael; Higham, James P.

    2015-01-01

    Studying immunity and immune function in ecology and evolution requires field studies, but there has been a dearth of non-invasive markers of immune activation available for studying large wild mammals. Recently, we analytically and biologically validated the measurement of urinary neopterin (NEO), a biomarker of cellular immune activation, in captive macaques. However, applying this to free-ranging settings is complicated by issues involving sample collection, processing, storage, and transport. Here, we collected urine samples from captive macaques and undertook experiments simulating common field issues. We tested the effects on urinary NEO sample measurements following: dirt and faecal contamination; storage at room temperature; differences in processing and long-term storage methods (freezing, lyophilising, blotting onto filter paper); and freeze-thaw cycles. Our results show that concentrations of urinary NEO are highly stable – they are not affected by soil or faecal contamination, can be collected on filter paper and stored for many months frozen or lyophilised with minimal effect, and are resistant to multiple 24 hr freeze-thaws. With the addition of a biocidal preservative, concentrations are even stable at room temperature for long periods. Urinary NEO is remarkably resilient, and is highly suitable for non-invasive field studies of cellular immune responses in wild large mammals. PMID:26549509

  15. Comparison of phytochemical profiles, antioxidant and cellular antioxidant activities of different varieties of blueberry (Vaccinium spp.).

    PubMed

    Wang, Huailing; Guo, Xinbo; Hu, Xiaodan; Li, Tong; Fu, Xiong; Liu, Rui Hai

    2017-02-15

    Numerous reports have demonstrated that the consumption of fruits and vegetables is beneficial for the human health. Blueberries, in particular, are rich in phytochemicals including free and bound forming. Phytochemical profiles of 14 varieties of blueberry were compared in this study. 12 compounds were analyzed and had significant changes in blueberry fruits. Total antioxidant activities in different blueberry varieties varied about 2.6times by oxygen radical absorbance capacity (ORAC) assay, and 2times by peroxyl radical scavenging capacity (PSC) assay. The cellular antioxidant activities (CAA) in different varieties varied about 3.9times without phosphate buffer saline (PBS) wash, and 4.7times with PBS wash by CAA assay. Blueberry extracts had potent antiproliferative activities against HepG2 human liver cancer cells, indicating the potential protective benefits associated with their use as functional foods. The anti-proliferative activity was observed to be dose-dependent in blueberry extracts. PMID:27664697

  16. Comparison of phytochemical profiles, antioxidant and cellular antioxidant activities of different varieties of blueberry (Vaccinium spp.).

    PubMed

    Wang, Huailing; Guo, Xinbo; Hu, Xiaodan; Li, Tong; Fu, Xiong; Liu, Rui Hai

    2017-02-15

    Numerous reports have demonstrated that the consumption of fruits and vegetables is beneficial for the human health. Blueberries, in particular, are rich in phytochemicals including free and bound forming. Phytochemical profiles of 14 varieties of blueberry were compared in this study. 12 compounds were analyzed and had significant changes in blueberry fruits. Total antioxidant activities in different blueberry varieties varied about 2.6times by oxygen radical absorbance capacity (ORAC) assay, and 2times by peroxyl radical scavenging capacity (PSC) assay. The cellular antioxidant activities (CAA) in different varieties varied about 3.9times without phosphate buffer saline (PBS) wash, and 4.7times with PBS wash by CAA assay. Blueberry extracts had potent antiproliferative activities against HepG2 human liver cancer cells, indicating the potential protective benefits associated with their use as functional foods. The anti-proliferative activity was observed to be dose-dependent in blueberry extracts.

  17. Celebrating Soft Matter's 10th Anniversary: Cell division: a source of active stress in cellular monolayers.

    PubMed

    Doostmohammadi, Amin; Thampi, Sumesh P; Saw, Thuan B; Lim, Chwee T; Ladoux, Benoit; Yeomans, Julia M

    2015-10-01

    We introduce the notion of cell division-induced activity and show that the cell division generates extensile forces and drives dynamical patterns in cell assemblies. Extending the hydrodynamic models of lyotropic active nematics we describe turbulent-like velocity fields that are generated by the cell division in a confluent monolayer of cells. We show that the experimentally measured flow field of dividing Madin-Darby Canine Kidney (MDCK) cells is reproduced by our modeling approach. Division-induced activity acts together with intrinsic activity of the cells in extensile and contractile cell assemblies to change the flow and director patterns and the density of topological defects. Finally we model the evolution of the boundary of a cellular colony and compare the fingering instabilities induced by cell division to experimental observations on the expansion of MDCK cell cultures.

  18. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation.

    PubMed Central

    Stack, M S; Gately, S; Bafetti, L M; Enghild, J J; Soff, G A

    1999-01-01

    Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion. PMID:10229661

  19. Suppression of cellular proliferation and invasion by the concerted lipid and protein phosphatase activities of PTEN

    PubMed Central

    Davidson, Lindsay; Maccario, Helene; Perera, Nevin M.; Yang, Xuesong; Spinelli, Laura; Tibarewal, Priyanka; Glancy, Ben; Gray, Alex; Weijer, Cornelis J.; Downes, C. Peter; Leslie, Nick R.

    2009-01-01

    PTEN is a tumour suppressor with phosphatase activity in vitro against both lipids and proteins and other potential non-enzymatic mechanisms of action. Although the importance of PTEN’s lipid phosphatase activity in regulating the PI3K signalling pathway is recognised, the significance of PTEN’s other mechanisms of action is currently unclear. Here, we describe the systematic identification of a PTEN mutant, PTEN Y138L, with activity against lipid, but not soluble substrates. Using this mutant we provide evidence for the interfacial activation of PTEN against lipid substrates. We also show that when re-expressed at physiological levels in PTEN null U87MG glioblastoma cells the protein phosphatase activity of PTEN is not required to regulate cellular PtdInsP3 levels or the downstream protein kinase Akt/PKB. Finally, in 3D Matrigel cultures of U87MG cells similarly re-expressing PTEN mutants, both the protein and lipid phosphatase activities were required to inhibit invasion, but either activity alone significantly inhibited proliferation, albeit only weakly for the protein phosphatase activity. Our data provides a novel tool to address the significance of PTEN’s separable lipid and protein phosphatase activities and suggest that both activities act to suppress proliferation and act together to suppress invasion. PMID:19915616

  20. [Polyenzymatic activation of cellular hydrolases induced by interferon; its role in cytolysis and other cellular manifestations induced by interferon].

    PubMed

    Zagury, D; Mazière, J C; Morgan, D; Fouchard, M; Gagne, I; Hosli, P

    1982-01-25

    Interferon (IFN) treatment of human effector T cells activates CMC directed against K562 targets. An increase of the activity of the different hydrolytic enzymes tested (hexosaminidase and acid-phosphatase) measured in the effector cell population parallels cytolysis activation. IFN and IFN inducers (PIC) treatment of cultured murine L-929 cells generate molecular mediated cytolysis. An increase of hydrolytic enzyme activity (hexosaminidase; B-glucuronidase, acid and alkalin phosphatases) precedes cell lysis. Thus, in both cytolytic systems, IFN induced a polyenzymatic activation of hydrolases. This activation is similar to coordinate enzyme induction discovered by Hosli [16] in pathological and experimental instances, when absorbed substrates cannot be hydrolyzed by lack of specific enzymes. Activation of hydrolytic enzymes induced by IFN which determines its enhancing effect on CMC and on fibroblast autolysis [17] could speculatively explain other effects of IFN such as antiviral action, diminution of cell protein synthesis, inhibition of cell division or molecular alterations of cell surface. PMID:6177383

  1. [Main Cellular Redox Couples].

    PubMed

    Bilan, D S; Shokhina, A G; Lukyanov, S A; Belousov, V V

    2015-01-01

    Most of the living cells maintain the continuous flow of electrons, which provides them by energy. Many of the compounds are presented in a cell at the same time in the oxidized and reduced states, forming the active redox couples. Some of the redox couples, such as NAD+/NADH, NADP+/NADPH, oxidized/reduced glutathione (GSSG/GSH), are universal, as they participate in adjusting of many cellular reactions. Ratios of the oxidized and reduced forms of these compounds are important cellular redox parameters. Modern research approaches allow setting the new functions of the main redox couples in the complex organization of cellular processes. The following information is about the main cellular redox couples and their participation in various biological processes.

  2. The role of cellular coupling in the spontaneous generation of electrical activity in uterine tissue.

    PubMed

    Xu, Jinshan; Menon, Shakti N; Singh, Rajeev; Garnier, Nicolas B; Sinha, Sitabhra; Pumir, Alain

    2015-01-01

    The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition. PMID:25793276

  3. Concurrent measurement of cellular turbidity and hemoglobin to evaluate the antioxidant activity of plants.

    PubMed

    Bellik, Yuva; Iguer-Ouada, Mokrane

    2016-01-01

    In past decades, a multitude of analytical methods for measuring antioxidant activity of plant extracts has been developed. However, when using methods to determine hemoglobin released from human erythrocytes treated with ginger extracts, we found hemoglobin concentrations were significantly higher than in untreated control samples. This suggests in the presence of antioxidants that measuring hemoglobin alone is not sufficient to determine hemolysis. We show concurrent measurement of erythrocyte concentration and hemoglobin is essential in such assays, and describe a new protocol based on simultaneous measurement of cellular turbidity and hemoglobin.

  4. How the Venus flytrap actively snaps: hydrodynamic measurements at the cellular level

    NASA Astrophysics Data System (ADS)

    Colombani, Mathieu; Forterre, Yoel; GEP Team

    2012-11-01

    Although they lack muscle, plants have evolved a remarkable range of mechanisms to create rapid motion, from the rapid folding of sensitive plants to seed dispersal. Of these spectacular examples that have long fascinated scientists, the carnivorous plant Venus flytrap, whose leaves snap together in a fraction of second to capture insects, has long been a paradigm for study. Recently, we have shown that this motion involves a snap-buckling instability due to the shell-like geometry of the leaves of the trap. However, the origin of the movement that allows the plant to cross the instability threshold and actively bend remains largely unknown. In this study, we investigate this active motion using a micro-fluidic pressure probe that gives direct hydraulic and mechanical measurements at the cellular level (osmotic pressure, cell membrane permeability, cell wall elasticity). Our results challenge the role of osmotically-driven water flows usually put forward to explain Venus flytrap's active closure.

  5. A beacon of hope in stroke therapy-Blockade of pathologically activated cellular events in excitotoxic neuronal death as potential neuroprotective strategies.

    PubMed

    Hoque, Ashfaqul; Hossain, M Iqbal; Ameen, S Sadia; Ang, Ching-Seng; Williamson, Nicholas; Ng, Dominic C H; Chueh, Anderly C; Roulston, Carli; Cheng, Heung-Chin

    2016-04-01

    Excitotoxicity, a pathological process caused by over-stimulation of ionotropic glutamate receptors, is a major cause of neuronal loss in acute and chronic neurological conditions such as ischaemic stroke, Alzheimer's and Huntington's diseases. Effective neuroprotective drugs to reduce excitotoxic neuronal loss in patients suffering from these neurological conditions are urgently needed. One avenue to achieve this goal is to clearly define the intracellular events mediating the neurotoxic signals originating from the over-stimulated glutamate receptors in neurons. In this review, we first focus on the key cellular events directing neuronal death but not involved in normal physiological processes in the neurotoxic signalling pathways. These events, referred to as pathologically activated events, are potential targets for the development of neuroprotectant therapeutics. Inhibitors blocking some of the known pathologically activated cellular events have been proven to be effective in reducing stroke-induced brain damage in animal models. Notable examples are inhibitors suppressing the ion channel activity of neurotoxic glutamate receptors and those disrupting interactions of specific cellular proteins occurring only in neurons undergoing excitotoxic cell death. Among them, Tat-NR2B9c and memantine are clinically effective in reducing brain damage caused by some acute and chronic neurological conditions. Our second focus is evaluation of the suitability of the other inhibitors for use as neuroprotective therapeutics. We also discuss the experimental approaches suitable for bridging our knowledge gap in our current understanding of the excitotoxic signalling mechanism in neurons and discovery of new pathologically activated cellular events as potential targets for neuroprotection.

  6. Focused Metabolite Profiling for Dissecting Cellular and Molecular Processes of Living Organisms in Space Environments

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Regulatory control in biological systems is exerted at all levels within the central dogma of biology. Metabolites are the end products of all cellular regulatory processes and reflect the ultimate outcome of potential changes suggested by genomics and proteomics caused by an environmental stimulus or genetic modification. Following on the heels of genomics, transcriptomics, and proteomics, metabolomics has become an inevitable part of complete-system biology because none of the lower "-omics" alone provide direct information about how changes in mRNA or protein are coupled to changes in biological function. The challenges are much greater than those encountered in genomics because of the greater number of metabolites and the greater diversity of their chemical structures and properties. To meet these challenges, much developmental work is needed, including (1) methodologies for unbiased extraction of metabolites and subsequent quantification, (2) algorithms for systematic identification of metabolites, (3) expertise and competency in handling a large amount of information (data set), and (4) integration of metabolomics with other "omics" and data mining (implication of the information). This article reviews the project accomplishments.

  7. Multiscattering-enhanced optical biosensor: multiplexed, non-invasive and continuous measurements of cellular processes

    PubMed Central

    Koman, Volodymyr B.; Santschi, Christian; Martin, Olivier J. F.

    2015-01-01

    The continuous measurement of uptake or release of biomarkers provides invaluable information for understanding and monitoring the metabolism of cells. In this work, a multiscattering-enhanced optical biosensor for the multiplexed, non-invasive, and continuous detection of hydrogen peroxide (H2O2), lactate and glucose is presented. The sensing scheme is based on optical monitoring of the oxidation state of the metalloprotein cytochrome c (cyt c). The analyte of interest is enzymatically converted into H2O2 leading to an oxidation of the cyt c. Contact microspotting is used to prepare nanoliter-sized sensing spots containing either pure cyt c, a mixture of cyt c with glucose oxidase (GOx) to detect glucose, or a mixture of cyt c with lactate oxidase (LOx) to detect lactate. The sensing spots are embedded in a multiscattering porous medium that enhances the optical signal. We achieve limits of detection down to 240 nM and 110 nM for lactate and glucose, respectively. A microfluidic embodiment enables multiplexed and crosstalk-free experiments on living organisms. As an example, we study the uptake of exogenously supplied glucose by the green algae Chlamydomonas reinhardtii and simultaneously monitor the stress-related generation of H2O2. This multifunctional detection scheme provides a powerful tool to study biochemical processes at cellular level. PMID:26203366

  8. Multiscattering-enhanced optical biosensor: multiplexed, non-invasive and continuous measurements of cellular processes.

    PubMed

    Koman, Volodymyr B; Santschi, Christian; Martin, Olivier J F

    2015-07-01

    The continuous measurement of uptake or release of biomarkers provides invaluable information for understanding and monitoring the metabolism of cells. In this work, a multiscattering-enhanced optical biosensor for the multiplexed, non-invasive, and continuous detection of hydrogen peroxide (H2O2), lactate and glucose is presented. The sensing scheme is based on optical monitoring of the oxidation state of the metalloprotein cytochrome c (cyt c). The analyte of interest is enzymatically converted into H2O2 leading to an oxidation of the cyt c. Contact microspotting is used to prepare nanoliter-sized sensing spots containing either pure cyt c, a mixture of cyt c with glucose oxidase (GOx) to detect glucose, or a mixture of cyt c with lactate oxidase (LOx) to detect lactate. The sensing spots are embedded in a multiscattering porous medium that enhances the optical signal. We achieve limits of detection down to 240 nM and 110 nM for lactate and glucose, respectively. A microfluidic embodiment enables multiplexed and crosstalk-free experiments on living organisms. As an example, we study the uptake of exogenously supplied glucose by the green algae Chlamydomonas reinhardtii and simultaneously monitor the stress-related generation of H2O2. This multifunctional detection scheme provides a powerful tool to study biochemical processes at cellular level. PMID:26203366

  9. Human immunodeficiency virus-induced pathology favored by cellular transmission and activation

    SciTech Connect

    Lewis, D.E.; Yoffe, B.; Bosworth, C.G.; Hollinger, F.B.; Rich, R.R.

    1988-03-01

    Epidemiological data suggest that transmission of human immunodeficiency virus (HIV) occurs primarily by transference of virally infected cells. However, the efficiency of lytic productive infection induced by HIV after transmission of cell-associated virus vs. free virus is difficult to assess. The present studies compare the extent of depletion of CD4+ (helper/inducer) T cells after mixing uninfected cells with either free HIV or irradiated HIV-infected allogeneic or autologous cells in vitro. Rapid CD4+ cellular depletion occurred only in cultures containing allogeneic infected cells or after addition of a nonspecific T cell activation signal to cultures with autologous infected cells. These in vitro observations strongly support the epidemiological implication that interactions between infected and uninfected cells are the most efficient means of transmission and HIV-induced cytopathology in vivo. They also provide direct support for the concept that immunological stimulation by foreign cells infected with HIV dramatically increases the likelihood of transmission. These in vitro observations suggest a model for the acquisition of HIV in vivo and the role of cellular activation in dissemination of the virus to uninfected cells in an infected individual.

  10. Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

    SciTech Connect

    Kiran, Shashi; Oddi, Vineesha; Ramakrishna, Gayatri

    2015-02-01

    Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

  11. Sleep Deprivation and Divergent Toll-like Receptor-4 Activation of Cellular Inflammation in Aging

    PubMed Central

    Carroll, Judith E.; Carrillo, Carmen; Olmstead, Richard; Witarama, Tuff; Breen, Elizabeth C.; Yokomizo, Megumi; Seeman, Teresa E.; Irwin, Michael R.

    2015-01-01

    Objectives: Sleep disturbance and aging are associated with increases in inflammation, as well as increased risk of infectious disease. However, there is limited understanding of the role of sleep loss on age-related differences in immune responses. This study examines the effects of sleep deprivation on toll-like receptor activation of monocytic inflammation in younger compared to older adults. Design, Setting, and Participants: Community-dwelling adults (n = 70) who were categorized as younger (25–39 y old, n = 21) and older (60–84 y old, n = 49) participants, underwent a sleep laboratory-based experimental partial sleep deprivation (PSD) protocol including adaptation, an uninterrupted night of sleep, sleep deprivation (sleep restricted to 03:00–07:00), and recovery. Measurement and Results: Blood samples were obtained each morning to measure toll-like receptor-4 activation of monocyte intracellular production of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Partial sleep deprivation induced a significant increase in the production of IL-6 and/or TNF-α that persisted after a night of recovery sleep (F(2,121.2) = 3.8, P < 0.05). Age moderated the effects of sleep loss, such that younger adults had an increase in inflammatory cytokine production that was not present in older adults (F(2,121.2) = 4.0, P < 0.05). Conclusion: Older adults exhibit reduced toll-like receptor 4 stimulated cellular inflammation that, unlike in younger adults, is not activated after a night of partial sleep loss. Whereas sleep loss increases cellular inflammation in younger adults and may contribute to inflammatory disorders, blunted toll-like receptor activation in older adults may increase the risk of infectious disease seen with aging. Citation: Carroll JE, Carrillo C, Olmstead R, Witarama T, Breen EC, Yokomizo M, Seeman TE, Irwin MR. Sleep deprivation and divergent toll-like receptor-4 activation of cellular inflammation in aging. SLEEP

  12. Parallel Activation in Bilingual Phonological Processing

    ERIC Educational Resources Information Center

    Lee, Su-Yeon

    2011-01-01

    In bilingual language processing, the parallel activation hypothesis suggests that bilinguals activate their two languages simultaneously during language processing. Support for the parallel activation mainly comes from studies of lexical (word-form) processing, with relatively less attention to phonological (sound) processing. According to…

  13. [Measurement of cellular activity with the MTT test. Optimization of the method].

    PubMed

    Bank, U; Reinhold, D; Ansorge, S

    1991-01-01

    Since a few years activity of mitochondrial dehydrogenases has been determined by MTT-test as alternative method to measurement of the cellular activity and proliferation by incorporation of tritiated thymidine. In this test the tetrazolium salt MTT is converted into blue, insoluble formazan dye crystals, which have to be dissolved by a suitable extraction mixture. The present paper describes a modified extraction method using an isopropanol-dimethylformamide mixture acidified to pH 5.5. The modified method enables an optimal, easy to handle, less time and work consuming MTT-assay. A MTT concentration of 1 mg/ml was found to be optimal. The extinction maximum was identified at 566 nm.

  14. Blood biochemical and cellular changes during decompression and simulated extravehicular activity

    NASA Technical Reports Server (NTRS)

    Jauchem, J. R.; Waligora, J. M.; Johnson, P. C. Jr

    1990-01-01

    Blood biochemical and cellular parameters were measured in human subjects before and after exposure to a decompression schedule involving 6 h of oxygen prebreathing. The exposure was designed to simulate extravehicular activity for 6 h (subjects performed exercise while exposed to 29.6 kPa). There were no significant differences between blood samples from subjects who were susceptible (n = 11) versus those who were resistant (n = 27) to formation of venous gas emboli. Although several statistically significant (P less than 0.05) changes in blood parameters were observed following the exposure (increases in white blood cell count, prothrombin time, and total bilirubin, and decreases in triglycerides, very-low-density lipoprotein cholesterol, and blood urea nitrogen), the changes were small in magnitude and blood factor levels remained within normal clinical ranges. Thus, the decompression schedule used in this study is not likely to result in blood changes that would pose a threat to astronauts during extravehicular activity.

  15. Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits.

    PubMed

    Neufer, P Darrell; Bamman, Marcas M; Muoio, Deborah M; Bouchard, Claude; Cooper, Dan M; Goodpaster, Bret H; Booth, Frank W; Kohrt, Wendy M; Gerszten, Robert E; Mattson, Mark P; Hepple, Russell T; Kraus, William E; Reid, Michael B; Bodine, Sue C; Jakicic, John M; Fleg, Jerome L; Williams, John P; Joseph, Lyndon; Evans, Mary; Maruvada, Padma; Rodgers, Mary; Roary, Mary; Boyce, Amanda T; Drugan, Jonelle K; Koenig, James I; Ingraham, Richard H; Krotoski, Danuta; Garcia-Cazarin, Mary; McGowan, Joan A; Laughlin, Maren R

    2015-07-01

    The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine. PMID:26073496

  16. Dietary essential fatty acids, prostaglandins (PG) production and cellular cytotoxic activity in Balb/c mice

    SciTech Connect

    Fritsche, K.L.; Johnston, P.V.

    1986-03-05

    Several investigators have implicated PG as natural feedback inhibitors of cellular cytotoxic activity. It was of interest to determine if dietarily modulated PG synthesis alters cytotoxic response to a varial challenge. Weanling Balb/c mice were fed ad libitum semi-purified diets with 10% by weight corn oil or linseed oil, providing a 1:42 or 2:1 ratio of 18:3n-3 to 18:2n-6, respectively for 4-5 weeks. Mice were then immunochallenged with an intraperitoneal injection of 10/sup 4/ or 10/sup 5/ vaccinia virus. Peritoneal exudate cells and splenocytes were collected on days 3,6 and 9 post-challenge and assessed for cytotoxic activities using the /sup 51/Cr release assay. Peritoneal macrophage (M0), splenic natural killer and cytotoxic T lymphocyte cytotoxic activities were tested against the SV3T3, YAC-1, and vaccinia virus-infected 3T3 cell lines, respectively. PGE/sub 2/ and PGF/sub 2..cap alpha../ production by M0 and splenocytes from dietarily treated, non-challenged mice was determined by radioimmunoassay of cell supernatants following a 60 minute exposure to the calcium ionophore A23187. Peritoneal M0 and splenocyte populations from the linseed oil fed mice produced approximately 50% of the PGE/sub 2/ and PGF/sub 2..cap alpha../ as compared to the corn oil fed mice. However, no significant differences in cellular cytotoxic activity between the linseed oil and corn oil fed mice were observed.

  17. PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes.

    PubMed

    Anta, B; Pérez-Rodríguez, A; Castro, J; García-Domínguez, C A; Ibiza, S; Martínez, N; Durá, L M; Hernández, S; Gragera, T; Peña-Jiménez, D; Yunta, M; Zarich, N; Crespo, P; Serrador, J M; Santos, E; Muñoz, A; Oliva, J L; Rojas-Cabañeros, J M

    2016-01-01

    The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment. PMID:27468687

  18. Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.

    PubMed

    Hallett, Ryan A; Zimmerman, Seth P; Yumerefendi, Hayretin; Bear, James E; Kuhlman, Brian

    2016-01-15

    Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies. PMID:26474029

  19. PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes

    PubMed Central

    Anta, B; Pérez-Rodríguez, A; Castro, J; García- Domínguez, C A; Ibiza, S; Martínez, N; Durá, L M; Hernández, S; Gragera, T; Peña-Jiménez, D; Yunta, M; Zarich, N; Crespo, P; Serrador, J M; Santos, E; Muñoz, A; Oliva, J L; Rojas-Cabañeros, J M

    2016-01-01

    The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment. PMID:27468687

  20. Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri.

    PubMed

    Kurn, N; Sela, B A

    1981-12-01

    Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes. PMID:6459931

  1. PGA1-induced apoptosis involves specific activation of H-Ras and N-Ras in cellular endomembranes.

    PubMed

    Anta, B; Pérez-Rodríguez, A; Castro, J; García-Domínguez, C A; Ibiza, S; Martínez, N; Durá, L M; Hernández, S; Gragera, T; Peña-Jiménez, D; Yunta, M; Zarich, N; Crespo, P; Serrador, J M; Santos, E; Muñoz, A; Oliva, J L; Rojas-Cabañeros, J M

    2016-07-28

    The cyclopentenone prostaglandin A1 (PGA1) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA1, did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.

  2. Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.

    PubMed

    Hallett, Ryan A; Zimmerman, Seth P; Yumerefendi, Hayretin; Bear, James E; Kuhlman, Brian

    2016-01-15

    Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

  3. Cellular aging and cancer

    PubMed Central

    Hornsby, Peter J.

    2010-01-01

    Aging is manifest in a variety of changes over time, including changes at the cellular level. Cellular aging acts primarily as a tumor suppressor mechanism, but also may enhance cancer development under certain circumstances. One important process of cellular aging is oncogene-induced senescence, which acts as an important anti-cancer mechanism. Cellular senescence resulting from damage caused by activated oncogenes prevents the growth or potentially neoplastic cells. Moreover, cells that have entered senescence appear to be targets for elimination by the innnate immune system. In another aspect of cellular aging, the absence of telomerase activity in normal tissues results in such cells lacking a telomere maintenance mechanism. One consequence is that in aging there is an increase in cells with shortened telomeres. In the presence of active oncogenes that cause expansion of a neoplastic clone, shortening of telomeres leading to telomere dysfunction prevents the indefinite expansion of the clone because the cells enter crisis. Crisis results from fusions and other defects caused by dysfunctional telomeres and is a terminal state of the neoplastic clone. In this way the absence of telomerase in human cells, while one cause of cellular aging, also acts as an anti-cancer mechanism. PMID:20705476

  4. QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

    EPA Science Inventory

    New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

  5. Theoretical aspects and modelling of cellular decision making, cell killing and information-processing in photodynamic therapy of cancer

    PubMed Central

    2013-01-01

    Background The aim of this report is to provide a mathematical model of the mechanism for making binary fate decisions about cell death or survival, during and after Photodynamic Therapy (PDT) treatment, and to supply the logical design for this decision mechanism as an application of rate distortion theory to the biochemical processing of information by the physical system of a cell. Methods Based on system biology models of the molecular interactions involved in the PDT processes previously established, and regarding a cellular decision-making system as a noisy communication channel, we use rate distortion theory to design a time dependent Blahut-Arimoto algorithm where the input is a stimulus vector composed of the time dependent concentrations of three PDT related cell death signaling molecules and the output is a cell fate decision. The molecular concentrations are determined by a group of rate equations. The basic steps are: initialize the probability of the cell fate decision, compute the conditional probability distribution that minimizes the mutual information between input and output, compute the cell probability of cell fate decision that minimizes the mutual information and repeat the last two steps until the probabilities converge. Advance to the next discrete time point and repeat the process. Results Based on the model from communication theory described in this work, and assuming that the activation of the death signal processing occurs when any of the molecular stimulants increases higher than a predefined threshold (50% of the maximum concentrations), for 1800s of treatment, the cell undergoes necrosis within the first 30 minutes with probability range 90.0%-99.99% and in the case of repair/survival, it goes through apoptosis within 3-4 hours with probability range 90.00%-99.00%. Although, there is no experimental validation of the model at this moment, it reproduces some patterns of survival ratios of predicted experimental data. Conclusions

  6. Benzothiophene inhibitors of MK2. Part 1: structure-activity relationships, assessments of selectivity and cellular potency.

    PubMed

    Anderson, David R; Meyers, Marvin J; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Long, Scott A; Pierce, Betsy S; Mahoney, Matthew W; Mourey, Robert J

    2009-08-15

    Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.

  7. Cellular antioxidant activity of phenylaminoethyl selenides as monitored by chemiluminescence of peroxalate nanoparticles and by reduction of lipopolysaccharide-induced oxidative stress.

    PubMed

    Cowan, Elizabeth A; Taylor, Jennifer L; Oldham, Charlie D; Dasari, Madhuri; Doyle, Donald; Murthy, Niren; May, Sheldon W

    2013-12-10

    Hydrogen peroxide (H2O2), produced in living cells by oxidases and by other biochemical reactions, plays an important role in cellular processes such as signaling and cell cycle progression. Nevertheless, H2O2 and other reactive oxygen species are capable of inducing damage to DNA and other cellular components, and oxidative stress caused by overproduction of cellular oxidants has been linked to pathologies such as inflammatory diseases and cancer. Therefore, new approaches for reducing the accumulation of cellular oxidants are of considerable interest from both a biotechnological and a therapeutic perspective. Recognizing that selenium is an essential component of the active sites of several antioxidant enzymes, we have developed a family of novel phenylaminoethyl selenide compounds that are readily taken up into cells and have low toxicity in vivo. We now report chemiluminescent imaging of hydrogen peroxide consumption by phenylaminoethyl selenides, via the use of peroxalate nanoparticle methodology. Further, we demonstrate the ability of phenylaminoethyl selenides to decrease lipopolysaccharide-induced oxidative stress in human embryonic kidney cells. We also report the successful encapsulation of a phenylaminoethyl selenide within poly(lactide-co-glycolide) nanoparticles, and we show that these selenide-loaded nanoparticles exhibit antioxidant activity in cells. Taken together, these results significantly enhance the attractiveness of phenylaminoethyl selenides as potential agents for supplementing cellular defenses against reactive oxygen species. PMID:24315639

  8. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity

    NASA Astrophysics Data System (ADS)

    Shen, Duanwen; Bai, Mingfeng; Tang, Rui; Xu, Baogang; Ju, Xiaoming; Pestell, Richard G.; Achilefu, Samuel

    2013-04-01

    Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

  13. Graphene Enhances Cellular Proliferation through Activating the Epidermal Growth Factor Receptor.

    PubMed

    Liu, Wei; Sun, Cheng; Liao, Chunyang; Cui, Lin; Li, Haishan; Qu, Guangbo; Yu, Wenlian; Song, Naining; Cui, Yuan; Wang, Zheng; Xie, Wenping; Chen, Huiming; Zhou, Qunfang

    2016-07-27

    Graphene has promising applications in food packaging, water purification, and detective sensors for contamination monitoring. However, the biological effects of graphene are not fully understood. It is necessary to clarify the potential risks of graphene exposure to humans through diverse routes, such as foods. In the present study, graphene, as the model nanomaterial, was used to test its potential effects on the cell proliferation based on multiple representative cell lines, including HepG2, A549, MCF-7, and HeLa cells. Graphene was characterized by Raman spectroscopy, particle size analysis, atomic force microscopy, and transmission electron microscopy. The cellular responses to graphene exposure were evaluated using flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and alamarBlue assays. Rat cerebral astrocyte cultures, as the non-cancer cells, were used to assess the potential cytotoxicity of graphene as well. The results showed that graphene stimulation enhanced cell proliferation in all tested cell cultures and the highest elevation in cell growth was up to 60%. A western blot assay showed that the expression of epidermal growth factor (EGF) was upregulated upon graphene treatment. The phosphorylation of EGF receptor (EGFR) and the downstream proteins, ShC and extracellular regulating kinase (ERK), were remarkably induced, indicating that the activation of the mitogen-activated protein kinase (MAPK)/ERK signaling pathway was triggered. The activation of PI3 kinase p85 and AKT showed that the PI3K/AKT signaling pathway was also involved in graphene-induced cell proliferation, causing the increase of cell ratios in the G2/M phase. No influences on cell apoptosis were observed in graphene-treated cells when compared to the negative controls, proving the low cytotoxicity of this emerging nanomaterial. The findings in this study revealed the potential cellular biological effect of graphene, which may give useful hints on its biosafety

  14. In silico, in vitro and cellular analysis with a kinome-wide inhibitor panel correlates cellular LRRK2 dephosphorylation to inhibitor activity on LRRK2.

    PubMed

    Vancraenenbroeck, Renée; De Raeymaecker, Joren; Lobbestael, Evy; Gao, Fangye; De Maeyer, Marc; Voet, Arnout; Baekelandt, Veerle; Taymans, Jean-Marc

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase

  15. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  16. Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

    SciTech Connect

    Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

    2012-06-10

    Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

  17. Characteristics of Middle School Students Learning Actions in Outdoor Mathematical Activities with the Cellular Phone

    ERIC Educational Resources Information Center

    Daher, Wajeeh; Baya'a, Nimer

    2012-01-01

    Learning in the cellular phone environment enables utilizing the multiple functions of the cellular phone, such as mobility, availability, interactivity, verbal and voice communication, taking pictures or recording audio and video, measuring time and transferring information. These functions together with mathematics-designated cellular phone…

  18. 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

  19. 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.

  20. The relationship between redox enzyme activity and electrochemical potential-cellular and mechanistic implications from protein film electrochemistry.

    PubMed

    Gates, Andrew J; Kemp, Gemma L; To, Chun Yip; Mann, James; Marritt, Sophie J; Mayes, Andrew G; Richardson, David J; Butt, Julea N

    2011-05-01

    In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies.

  1. The relationship between redox enzyme activity and electrochemical potential-cellular and mechanistic implications from protein film electrochemistry.

    PubMed

    Gates, Andrew J; Kemp, Gemma L; To, Chun Yip; Mann, James; Marritt, Sophie J; Mayes, Andrew G; Richardson, David J; Butt, Julea N

    2011-05-01

    In protein film electrochemistry a redox protein of interest is studied as an electroactive film adsorbed on an electrode surface. For redox enzymes this configuration allows quantification of the relationship between catalytic activity and electrochemical potential. Considered as a function of enzyme environment, i.e., pH, substrate concentration etc., the activity-potential relationship provides a fingerprint of activity unique to a given enzyme. Here we consider the nature of the activity-potential relationship in terms of both its cellular impact and its origin in the structure and catalytic mechanism of the enzyme. We propose that the activity-potential relationship of a redox enzyme is tuned to facilitate cellular function and highlight opportunities to test this hypothesis through computational, structural, biochemical and cellular studies. PMID:21423952

  2. Insight into the impact of dietary saturated fat on tissue-specific cellular processes underlying obesity-related diseases.

    PubMed

    Enos, Reilly T; Velázquez, Kandy T; Murphy, E Angela

    2014-06-01

    This study investigated the influence of three high-fat diets (HFDs), differing in the percentage of total calories from saturated fat (SF) (6%, 12%, 24%) but identical in total fat (40%), for a 16-week period in mice on a variety of tissue-specific cellular processes believed to be at the root of obesity-related diseases. Specifically, we examined ectopic lipid accumulation, oxidative capacity [peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA and protein; mtDNA; Cox IV and cytochrome C protein; citrate synthase activity; and gene expression of fission 1, mitofusin (Mfn) 1 and Mfn2], oxidative stress (4-hydroxy-2-nonenal), endoplasmic reticulum (ER) stress (binding immunoglobulin protein, activating transcription factor 6-p50, p-eukaryotic initiation factor 2 alpha and x-box binding protein 1 spliced protein), inflammatory [p-c-Jun N-terminal kinase (JNK), p-nuclear factor kappa-B, p-p38 mitogen-activated protein kinase) and insulin signaling (p-Akt), and inflammation [tumor necrosis factor-alpha, monocyte chemotactic protein-1, interleukin-6, F4/80, toll-like receptor (TLR)2 and TLR4 gene expression] in various tissues, including the adipose tissue, liver, skeletal muscle and heart. In general, adipose and hepatic tissues were the only tissues which displayed evidence of dysfunction. All HFDs down-regulated adipose, cardiac and hepatic PGC-1α mRNA and hepatic citrate synthase activity, and induced adipose tissue oxidative stress, whereas only the 6%-SF and 12%-SF diet produced hepatic steatosis. However, compared to the 6%-SF and 24%-SF diets, consumption of the 12%-SF diet resulted in the greatest degree of dysregulation (hepatic ER and oxidative stress, JNK activation, increased F4/80 gene expression and down-regulation of adipose tissue Akt signaling). These findings suggest that the saturated fatty acid composition of an HFD can greatly influence the processes responsible for obesity-related diseases - nonalcoholic fatty

  3. Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

    SciTech Connect

    Herr, Michael J.; Longhurst, Celia M.; Baker, Benjamin; Homayouni, Ramin; Speich, Henry E.; Kotha, Jayaprakash; Jennings, Lisa K.

    2014-05-16

    Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

  4. Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions.

    PubMed

    Agarinis, C; Orsini, V; Megel, P; Abraham, Y; Yang, H; Mickanin, C; Myer, V; Bouwmeester, T; Tchorz, J S; Parker, C N

    2016-03-01

    The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic-nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.

  5. The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

    PubMed

    Salari, Vahid; Scholkmann, Felix; Bokkon, Istvan; Shahbazi, Farhad; Tuszynski, Jack

    2016-01-01

    For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to 'internal photons' inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350-700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation.

  6. The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

    PubMed Central

    Salari, Vahid; Scholkmann, Felix; Bokkon, Istvan; Shahbazi, Farhad; Tuszynski, Jack

    2016-01-01

    For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to ‘internal photons’ inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350–700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation. PMID:26950936

  7. The Physical Mechanism for Retinal Discrete Dark Noise: Thermal Activation or Cellular Ultraweak Photon Emission?

    PubMed

    Salari, Vahid; Scholkmann, Felix; Bokkon, Istvan; Shahbazi, Farhad; Tuszynski, Jack

    2016-01-01

    For several decades the physical mechanism underlying discrete dark noise of photoreceptors in the eye has remained highly controversial and poorly understood. It is known that the Arrhenius equation, which is based on the Boltzmann distribution for thermal activation, can model only a part (e.g. half of the activation energy) of the retinal dark noise experimentally observed for vertebrate rod and cone pigments. Using the Hinshelwood distribution instead of the Boltzmann distribution in the Arrhenius equation has been proposed as a solution to the problem. Here, we show that the using the Hinshelwood distribution does not solve the problem completely. As the discrete components of noise are indistinguishable in shape and duration from those produced by real photon induced photo-isomerization, the retinal discrete dark noise is most likely due to 'internal photons' inside cells and not due to thermal activation of visual pigments. Indeed, all living cells exhibit spontaneous ultraweak photon emission (UPE), mainly in the optical wavelength range, i.e., 350-700 nm. We show here that the retinal discrete dark noise has a similar rate as UPE and therefore dark noise is most likely due to spontaneous cellular UPE and not due to thermal activation. PMID:26950936

  8. SEROLOGICAL ANALYSES OF CELLULAR SLIME-MOLD DEVELOPMENT. I. CHANGES IN ANTIGENIC ACTIVITY DURING CELL AGGREATION.

    PubMed

    SONNEBORN, D R; SUSSMAN, M; LEVINE, L

    1964-06-01

    Sonneborn, D. R. (Brandeis University, Waltham, Mass.), M. Sussman, and L. Levine. Serological analysis of cellular slime-mold development. I. Changes in antigenic activity during cell aggregation. J. Bacteriol. 87:1321-1329. 1964.-During aggregation in Dictyostelium discoideum, the concentration of a single antigenic determinant increased markedly, starting from very low or undetectable levels. Subsequently, the determinant appeared to segregate preferentially into the stalks of terminal fruiting bodies. Sera containing the antibody specific for this determinant inhibited the aggregation of D. discoideum without disturbing cell viability. The properties of the antigen during fractionation are consistent with the supposition that it may be a protein associated with the cell membrane. The ability or inability of three species to coaggregate with D. discoideum was correlated with the presence or absence of the antigenic determinant in aggregates of these species.

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

    PubMed Central

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

    2014-01-01

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

  10. Inhibition of cellular Shp2 activity by a methyl ester analog of SPI-112.

    PubMed

    Chen, Liwei; Pernazza, Daniele; Scott, Latanya M; Lawrence, Harshani R; Ren, Yuan; Luo, Yunting; Wu, Xin; Sung, Shen-Shu; Guida, Wayne C; Sebti, Said M; Lawrence, Nicholas J; Wu, Jie

    2010-09-15

    The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2(E76K) mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2(E76K)-dependent cell survival, which is associated with inhibition of Shp2(E76K) PTP activity, Shp2(E76K)-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-gamma (IFN-gamma)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

  11. Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

    NASA Technical Reports Server (NTRS)

    Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

    1999-01-01

    The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

  12. Cellular Redistribution of Protein Tyrosine Phosphatases LAR and PTPσ by Inducible Proteolytic Processing

    PubMed Central

    Aicher, Babette; Lerch, Markus M.; Müller, Thomas; Schilling, James; Ullrich, Axel

    1997-01-01

    Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPσ. PTPσ biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPσ underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCα. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPσ cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and β-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPσ in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell–cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPσ is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of

  13. TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions.

    PubMed

    Brandao, Katherine; Deason-Towne, Francina; Zhao, Xiaoyun; Perraud, Anne-Laure; Schmitz, Carsten

    2014-12-01

    The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg(2+) homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other's cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg(2+)-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg(2+), but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect-a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular

  14. Characterization of the cellular activity of PDE 4 inhibitors using two novel PDE 4 reporter cell lines.

    PubMed

    Wunder, Frank; Quednau, Ramona; Geerts, Andreas; Barg, Martina; Tersteegen, Adrian

    2013-10-01

    We report here the generation and pharmacological characterization of two novel PDE 4B1 and PDE 4D3 reporter cell lines. Intracellular cAMP levels are monitored in these cells by a cAMP-sensitive biosensor. We used the recombinant PDE 4B1 and PDE 4D3 reporter cell lines to characterize the cellular effects of various competitive and allosteric PDE 4 inhibitors. In addition, we compared the cellular activity of these PDE 4 inhibitors with the in vitro inhibition of full-length PDE 4D3 and a truncated enzyme comprising the PDE 4D3 catalytic domain. Two different groups of PDE 4 inhibitors could be identified. The first group, including competitive inhibitors like roflumilast, cilomilast and piclamilast, shows similar in vitro activity on full-length and truncated PDE 4D3 and comparably low cellular activity. The second group, including the allosteric inhibitors PMNPQ, D159153, and D159404, shows much better inhibition of full-length versus truncated PDE 4D3. In addition, these compounds show high cellular activity. Our data obtained with the prototype PDE 4 inhibitor rolipram show that rolipram has properties intermediate between the two groups. The results imply that these novel PDE 4 reporter cell lines are well-suited for the characterization of the cellular activity of PDE 4 inhibitors and may also support a better understanding of the complex PDE 4 pharmacology.

  15. Cellular uptake and activity of heparin functionalised cerium oxide nanoparticles in monocytes.

    PubMed

    Ting, S R Simon; Whitelock, John M; Tomic, Romana; Gunawan, Cindy; Teoh, Wey Yang; Amal, Rose; Lord, Megan S

    2013-06-01

    Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution. PMID:23478040

  16. Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo

    PubMed Central

    Packer, Adam M.; Russell, Lloyd E.; Dalgleish, Henry W.P.; Häusser, Michael

    2016-01-01

    We describe an all-optical strategy for simultaneously manipulating and recording the activity of multiple neurons with cellular resolution in vivo. Concurrent two-photon optogenetic activation and calcium imaging is enabled by coexpression of a red-shifted opsin and a genetically encoded calcium indicator. A spatial light modulator allows tens of user-selected neurons to be targeted for spatiotemporally precise optogenetic activation, while simultaneous fast calcium imaging provides high-resolution network-wide readout of the manipulation with negligible optical crosstalk. Proof-of-principle experiments in mouse barrel cortex demonstrate interrogation of the same neuronal population during different behavioral states, and targeting of neuronal ensembles based on their functional signature. This approach extends the optogenetic toolkit beyond the specificity obtained with genetic or viral approaches, enabling high-throughput, flexible and long-term optical interrogation of functionally defined neural circuits with single-cell and single-spike resolution in the mammalian brain in vivo. PMID:25532138

  17. Evidence of cellular immune activation in children with opsoclonus-myoclonus: cerebrospinal fluid neopterin.

    PubMed

    Pranzatelli, Michael R; Hyland, Keith; Tate, Elizabeth D; Arnold, Lauren A; Allison, Tyler J; Soori, Gamini S

    2004-12-01

    To evaluate cellular immune activation in opsoclonus-myoclonus syndrome, we measured the inflammatory marker neopterin in the cerebrospinal fluid of 16 children with opsoclonus-myoclonus and neuroblastoma, 24 children with opsoclonus-myoclonus but no tumor, and 19 age-matched controls. The mean concentration in opsoclonus-myoclonus was 2.3-fold higher than in controls (P = .008). Neopterin was greatly elevated in four of the most neurologically severe cases, up to 8.3-fold above the highest control level. Thirteen of the 40 children with opsoclonus-myoclonus but no controls had a neopterin concentration >2 SD above the control mean (P = .005). In this high neopterin subgroup, neurologic severity was significantly greater and the duration of neurologic symptoms was less. In 16 children re-examined on immunotherapy, including adrenocorticotropic hormone (ACTH) combination therapy, treatment was associated with a significant reduction in both neopterin and neurologic severity. Neopterin did not differ significantly between the tumor and non-tumor opsoclonus-myoclonus etiologies. No abnormalities of tetrahydrobiopterin were found. Although cerebrospinal fluid neopterin lacked the sensitivity to be a biomarker of disease activity in opsoclonus-myoclonus, elevated concentrations do support a role for T-cell activation and cell-mediated immunity in its pathophysiology.

  18. Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease.

    PubMed

    Rosenbusch, Katharina E; Kortholt, Arjan

    2016-01-01

    Human LRRK2 (Leucine-Rich Repeat Kinase 2) has been associated with both familial and idiopathic Parkinson's disease (PD). Although several LRRK2 mediated pathways and interaction partners have been identified, the cellular functions of LRRK2 and LRRK2 mediated progression of PD are still only partially understood. LRRK2 belongs to the group of Roco proteins which are characterized by the presence of a Ras-like G-domain (Roc), a C-terminal of Roc domain (COR), a kinase, and several protein-protein interaction domains. Roco proteins exhibit a complex activation mechanism involving intramolecular signaling, dimerization, and substrate/effector binding. Importantly, PD mutations in LRRK2 have been linked to a decreased GTPase and impaired kinase activity, thus providing putative therapeutic targets. To fully explore these potential targets it will be crucial to understand the function and identify the pathways responsible for LRRK2-linked PD. Here, we review the recent progress in elucidating the complex LRRK2 activation mechanism, describe the accumulating evidence that link LRRK2-mediated PD to mitochondrial dysfunction and aberrant autophagy, and discuss possible ways for therapeutically targeting LRRK2.

  19. Antibody-dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells.

    PubMed

    Mise, Naoko; Takami, Mariko; Suzuki, Akane; Kamata, Toshiko; Harada, Kazuaki; Hishiki, Tomoro; Saito, Takeshi; Terui, Keita; Mitsunaga, Tetsuya; Nakata, Mitsuyuki; Ikeuchi, Takayuki; Nakayama, Toshinori; Yoshida, Hideo; Motohashi, Shinichiro

    2016-03-01

    Anti-ganglioside GD2 antibodies mainly work through antibody-dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high-risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti-GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti-tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti-GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co-cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti-GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK-NKT cell contact or NK cell-dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell-based immunotherapy could be an appropriate candidate for anti-GD2 antibody therapy for neuroblastoma.

  20. Redox mechanism as alternative to ligand binding for receptor activation delivering disregulated cellular signals.

    PubMed

    Nakashima, I; Pu, M Y; Nishizaki, A; Rosila, I; Ma, L; Katano, Y; Ohkusu, K; Rahman, S M; Isobe, K; Hamaguchi, M

    1994-02-01

    Cross-linking with specific ligand is a general requirement for ordered activation of cell surface receptors. In this study we demonstrated a novel pathway for disregulated receptor activation through a redox mechanism. Treatment of murine thymocytes or spleen cells with thiol-reactive HgCl2, a known inducer of autoimmune proliferative lymphocyte disorders in rodents, was found to induce tyrosine phosphorylation of several cellular proteins, which was up to 100 times as extensive as that triggered by stimulation with antireceptor antibody or mitogen. Through the cross-linkage by thiol-reactive bivalent mercury, transmembrane CD4, CD3, and CD45 and glycosylphosphatidylinositol-anchored Thy-1 were aggregated together on thymocytes or T lymphocytes. Along with the aggregation of Thy-1 and CD4, nonreceptor protein tyrosine kinase p56lck was aggregated and activated. These events were linked to extensive protein tyrosine phosphorylation, which was visualized as a well localized spot beneath the membrane. Under appropriate conditions, this novel pathway of multiple receptor aggregation delivered a disregulated signal into T lymphocytes, which cross-talked to the antireceptor antibody-induced signal, for prolonged cell proliferation and IL-2 production. These results suggest a novel mechanism of disregulation of the ligand-dependent receptor function.

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

    PubMed Central

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

    2016-01-01

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

  2. Cellular Activation of the Self-Quenched Fluorescent Reporter Probe in Tumor Microenvironment

    PubMed Central

    Bogdanov, Alexei A; Lin, Charles P; Simonova, Maria; Matuszewski, Lars; Weissleder, Ralph

    2002-01-01

    Abstract The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 µm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5–3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes. PMID:11988842

  3. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

  4. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

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

    PubMed

    Jacob, Claus

    2014-01-01

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

  6. Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson's disease.

    PubMed

    Wu, Yuncheng; Li, Xinqun; Zhu, Julie Xiaohong; Xie, Wenjie; Le, Weidong; Fan, Zhen; Jankovic, Joseph; Pan, Tianhong

    2011-01-01

    Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson's disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models.

  7. New water-soluble ruthenium(II) cytotoxic complex: biological activity and cellular distribution.

    PubMed

    Morais, Tânia S; Santos, Filipa C; Jorge, Tiago F; Côrte-Real, Leonor; Madeira, Paulo J Amorim; Marques, Fernanda; Robalo, M Paula; Matos, António; Santos, Isabel; Garcia, M Helena

    2014-01-01

    A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=η(5)-cyclopentadienyl, mTPPMS=diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy=2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. PMID:24145065

  8. A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

    PubMed Central

    Massignan, Tania; Cimini, Sara; Stincardini, Claudia; Cerovic, Milica; Vanni, Ilaria; Elezgarai, Saioa R.; Moreno, Jorge; Stravalaci, Matteo; Negro, Alessandro; Sangiovanni, Valeria; Restelli, Elena; Riccardi, Geraldina; Gobbi, Marco; Castilla, Joaquín; Borsello, Tiziana; Nonno, Romolo; Biasini, Emiliano

    2016-01-01

    Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrPC) into PrPSc, a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrPSc is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrPC may provide an opportunity to overcome these problems. PrPC ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrPC, and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrPC-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrPC-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer’s Disease. These results demonstrate that molecules binding to PrPC may produce a dual effect of blocking prion replication and inhibiting PrPC-mediated toxicity. PMID:26976106

  9. Dysfunction of nucleus accumbens-1 activates cellular senescence and inhibits tumor cell proliferation and oncogenesis.

    PubMed

    Zhang, Yi; Cheng, Yan; Ren, Xingcong; Hori, Tsukasa; Huber-Keener, Kathryn J; Zhang, Li; Yap, Kai Lee; Liu, David; Shantz, Lisa; Qin, Zheng-Hong; Zhang, Suping; Wang, Jianrong; Wang, Hong-Gang; Shih, Ie-Ming; Yang, Jin-Ming

    2012-08-15

    Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, has emerging roles in cancer. We report here that NAC1 acts as a negative regulator of cellular senescence in transformed and nontransformed cells, and dysfunction of NAC1 induces senescence and inhibits its oncogenic potential. We show that NAC1 deficiency markedly activates senescence and inhibits proliferation in tumor cells treated with sublethal doses of γ-irradiation. In mouse embryonic fibroblasts from NAC1 knockout mice, following infection with a Ras virus, NAC1-/- cells undergo significantly more senescence and are either nontransformed or less transformed in vitro and less tumorigenic in vivo when compared with NAC1+/+ cells. Furthermore, we show that the NAC1-caused senescence blunting is mediated by ΔNp63, which exerts its effect on senescence through p21, and that NAC1 activates transcription of ΔNp63 under stressful conditions. Our results not only reveal a previously unrecognized function of NAC1, the molecular pathway involved and its impact on pathogenesis of tumor initiation and development, but also identify a novel senescence regulator that may be exploited as a potential target for cancer prevention and treatment.

  10. Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming

    PubMed Central

    Marthaler, Adele G.; Adachi, Kenjiro; Tiemann, Ulf; Wu, Guangming; Sabour, Davood; Velychko, Sergiy; Kleiter, Ingo; Schöler, Hans R.; Tapia, Natalia

    2016-01-01

    Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming. PMID:26762895

  11. PCL/alginate composite scaffolds for hard tissue engineering: fabrication, characterization, and cellular activities.

    PubMed

    Kim, Yong Bok; Kim, Geun Hyung

    2015-02-01

    Alginates have been used widely in biomedical applications because of good biocompatibility, low cost, and rapid gelation in the presence of calcium ions. However, poor mechanical properties and fabrication-ability for three-dimensional shapes have been obstacles in hard-tissue engineering applications. To overcome these shortcomings of alginates, we suggest a new composite system, consisting of a synthetic polymer, poly(ε-caprolactone), and various weight fractions (10-40 wt %) of alginate. The fabricated composite scaffolds displayed a multilayered 3D structure, consisting of microsized composite struts, and they provided a 100% offset for each layer. To show the feasibility of the scaffold for hard tissue regeneration, the composite scaffolds fabricated were assessed not only for physical properties, including surface roughness, tensile strength, and water absorption and wetting, but also in vitro osteoblastic cellular responses (cell-seeding efficiency, cell viability, fluorescence analyses, alkaline phosphatase (ALP) activity, and mineralization) by culturing with preosteoblasts (MC3T3-E1). Due to the alginate components in the composites, the scaffolds showed significantly enhanced wetting behavior, water-absorption (∼12-fold), and meaningful biological activities (∼2.1-fold for cell-seeding efficiency, ∼2.5-fold for cell-viability at 7 days, ∼3.4-fold for calcium deposition), compared with a pure PCL scaffold.

  12. The bright side of plasmonic gold nanoparticles; activation of Nrf2, the cellular protective pathway

    NASA Astrophysics Data System (ADS)

    Goldstein, Alona; Soroka, Yoram; Frušić-Zlotkin, Marina; Lewis, Aaron; Kohen, Ron

    2016-06-01

    Plasmonic gold nanoparticles (AuNPs) are widely investigated for cancer therapy, due to their ability to strongly absorb light and convert it to heat and thus selectively destroy tumor cells. In this study we shed light on a new aspect of AuNPs and their plasmonic excitation, wherein they can provide anti-oxidant and anti-inflammatory protection by stimulating the cellular protective Nrf2 pathway. Our study was carried out on cells of the immune system, macrophages, and on skin cells, keratinocytes. A different response to AuNPs was noted in the two types of cells, explained by their distinct uptake profiles. In keratinocytes, the exposure to AuNPs, even at low concentrations, was sufficient to activate the Nrf2 pathway, without any irradiation, due to the presence of free AuNPs inside the cytosol. In contrast, in macrophages, the plasmonic excitation of the AuNPs by a low, non-lethal irradiation dose was required for their release from the constraining vesicles. The mechanism by which AuNPs activate the Nrf2 pathway was studied. Direct and indirect activation were suggested, based on the inherent ability of the AuNPs to react with thiol groups and to generate reactive oxygen species, in particular, under plasmonic excitation. The ability of AuNPs to directly activate the Nrf2 pathway renders them good candidates for treatment of disorders in which the up-regulation of Nrf2 is beneficial, specifically for topical treatment of inflammatory skin diseases.

  13. Lunar Dust and Lunar Simulant Activation, Monitoring, Solution and Cellular Toxicity Properties

    NASA Technical Reports Server (NTRS)

    Jeevarajan, A.S.; Wallace, W.T.

    2009-01-01

    During the Apollo missions, many undesirable situations were encountered that must be mitigated prior to returning humans to the moon. Lunar dust (that part of the lunar regolith less than 20 m in diameter) was found to produce several problems with astronaut s suits and helmets, mechanical seals and equipment, and could have conceivably produced harmful physiological effects for the astronauts. For instance, the abrasive nature of the dust was found to cause malfunctions of various joints and seals of the spacecraft and suits. Additionally, though efforts were made to exclude lunar dust from the cabin of the lunar module, a significant amount of material nonetheless found its way inside. With the loss of gravity correlated with ascent of the lunar module from the lunar surface to rendezvous with the command module, much of the major portions of the contaminating soil and dust began to float, irritating the astronaut s eyes and being inhaled into their lungs. Our goal has been to understand some of the properties of lunar dust that could lead to possible hazards for humans. Due to the lack of an atmosphere, there is nothing to protect the lunar soil from ultraviolet radiation, solar wind, and meteorite impacts. These processes could all serve to activate the soil, or produce reactive surface species. In order to understand the possible toxic effects of the reactive dust, it is necessary to reactivate the dust, as samples returned during the Apollo missions were exposed to the atmosphere of the Earth. We have used grinding and UV exposure to mimic some of the processes occurring on the Moon. The level of activation has been monitored using two methods: fluorescence spectroscopy and electron paramagnetic resonance spectroscopy (EPR). These techniques allow the monitoring of hydroxyl radical production in solution. We have found that grinding of lunar dust produces 2-3 times the concentration of hydroxyl radicals as lunar simulant and 10 times that of quartz. Exposure

  14. Efficient process development for bulk silicon etching using cellular automata simulation techniques

    NASA Astrophysics Data System (ADS)

    Marchetti, James; He, Yie; Than, Olaf; Akkaraju, Sandeep

    1998-09-01

    This paper describes cellular automata simulation techniques used to predict the anisotropic etching of single-crystal silicon. In particular, this paper will focus on the application of wet etching of silicon wafers using typical anisotropic etchants such as KOH, TMAH, and EDP. Achieving a desired final 3D geometry of etch silicon wafers often is difficult without requiring a number of fabrication design iterations. The result is wasted time and resources. AnisE, a tool to simulate anisotropic etching of silicon wafers using cellular automata simulation, was developed in order to efficiently prototype and manufacture MEMS devices. AnisE has been shown to effectively decrease device development time and costs by up to 50% and 60%, respectively.

  15. Using Cultured Mammalian Neurons to Study Cellular Processes and Neurodegeneration: A Suite of Undergraduate Lab Exercises

    PubMed Central

    Catlin, Rachel; Taylor, Abigail; Ditchek, Lisa; Burnett, Samantha; Khan, Showkhin; Todd, Olivia; Adams, Marguerite; Touhey, Eva; Wynkoop, Andrew; Ryan, James

    2016-01-01

    Cell culture is a powerful tool for exploring cellular function. Culturing primary neurons has revealed how neurons communicate in learning and memory (Kandel, 2006) and provided insights into the mechanisms of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (Alberio et al., 2012; Trinchese, et al., 2004). Here we describe a series of four modular laboratory exercises to integrate this neuroscience technique in undergraduate teaching laboratories. First, we describe the modular approach. Then we provide educators with simple techniques for culturing rat primary neurons, performing immunohistochemistry to label cellular components, and illustrating neurodegeneration caused by reactive oxygen species. We describe teaching exercises that culminate in student-generated research projects. Finally, we describe potential barriers students may face when integrating modern cell culture experiments into teaching laboratories. PMID:27385922

  16. Using Cultured Mammalian Neurons to Study Cellular Processes and Neurodegeneration: A Suite of Undergraduate Lab Exercises.

    PubMed

    Catlin, Rachel; Taylor, Abigail; Ditchek, Lisa; Burnett, Samantha; Khan, Showkhin; Todd, Olivia; Adams, Marguerite; Touhey, Eva; Wynkoop, Andrew; Ryan, James

    2016-01-01

    Cell culture is a powerful tool for exploring cellular function. Culturing primary neurons has revealed how neurons communicate in learning and memory (Kandel, 2006) and provided insights into the mechanisms of neurodegenerative diseases such as Parkinson's and Alzheimer's disease (Alberio et al., 2012; Trinchese, et al., 2004). Here we describe a series of four modular laboratory exercises to integrate this neuroscience technique in undergraduate teaching laboratories. First, we describe the modular approach. Then we provide educators with simple techniques for culturing rat primary neurons, performing immunohistochemistry to label cellular components, and illustrating neurodegeneration caused by reactive oxygen species. We describe teaching exercises that culminate in student-generated research projects. Finally, we describe potential barriers students may face when integrating modern cell culture experiments into teaching laboratories. PMID:27385922

  17. Antibacterial and Anti-inflammatory Activities of Ppc-1, Active Principle of the Cellular Slime Mold Polysphondylium pseudo-candidum.

    PubMed

    Azelmat, Jabrane; Fiorito, Serena; Genovese, Salvatore; Epifano, Francesco; Grenier, Daniel

    2015-01-01

    The diisopentenyloxy quinolobactin derivative 3-methylbut-2-enyl-4-methoxy-8-[(3-methylbut-2-enyl)oxy] quinoline-2-carboxylate, also named as Ppc-1, has been initially isolated from the fruiting bodies of the cellular slime mold Polysphondylium pseudo-candidum. Given that few data are available in the literature concerning the biological properties of this compound, this study was undertaken to evaluate its antibacterial and anti-inflammatory properties. Ppc-1 exerted antibacterial activity on the Gram negative periodontopathogen Porphyromonas gingivalis, while it had no such effect on the other bacterial species tested. The antibacterial activity of Ppc-1 appeared to result from its ability to permeate the cell membrane. Using the U937-3xκB-LUC human monocytic cell line, Ppc-1 was found to dose-dependently inhibit the lipopolysaccharide-induced NF-κB activation, a signaling pathway that has been associated with inflammatory mediator secretion. In conclusion, Ppc-1, by exhibiting a dual mode of action including antibacterial and anti-inflammatory activities, may represent a promising targeted therapeutic agent for periodontal diseases.

  18. Antibacterial and Anti-inflammatory Activities of Ppc-1, Active Principle of the Cellular Slime Mold Polysphondylium pseudo-candidum.

    PubMed

    Azelmat, Jabrane; Fiorito, Serena; Genovese, Salvatore; Epifano, Francesco; Grenier, Daniel

    2015-01-01

    The diisopentenyloxy quinolobactin derivative 3-methylbut-2-enyl-4-methoxy-8-[(3-methylbut-2-enyl)oxy] quinoline-2-carboxylate, also named as Ppc-1, has been initially isolated from the fruiting bodies of the cellular slime mold Polysphondylium pseudo-candidum. Given that few data are available in the literature concerning the biological properties of this compound, this study was undertaken to evaluate its antibacterial and anti-inflammatory properties. Ppc-1 exerted antibacterial activity on the Gram negative periodontopathogen Porphyromonas gingivalis, while it had no such effect on the other bacterial species tested. The antibacterial activity of Ppc-1 appeared to result from its ability to permeate the cell membrane. Using the U937-3xκB-LUC human monocytic cell line, Ppc-1 was found to dose-dependently inhibit the lipopolysaccharide-induced NF-κB activation, a signaling pathway that has been associated with inflammatory mediator secretion. In conclusion, Ppc-1, by exhibiting a dual mode of action including antibacterial and anti-inflammatory activities, may represent a promising targeted therapeutic agent for periodontal diseases. PMID:25925558

  19. 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

  20. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.

    PubMed

    Shinkai, Yasuhiro; Sumi, Daigo; Fukami, Ikuo; Ishii, Tetsuro; Kumagai, Yoshito

    2006-03-20

    Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity. PMID:16516206

  1. Sulforaphane, an activator of Nrf2, suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.

    PubMed

    Shinkai, Yasuhiro; Sumi, Daigo; Fukami, Ikuo; Ishii, Tetsuro; Kumagai, Yoshito

    2006-03-20

    Sulforaphane (SFN) is an activator of the transcription factor Nrf2, which plays a critical role in metabolism and excretion of xenobiotics. Exposure of primary mouse hepatocytes to SFN resulted in activation of Nrf2 and significant elevation of protein expressions responsible for excretion of arsenic into extracellular space. Pretreatment with SFN 24 h prior to arsenite exposure reduced not only arsenic accumulation in the cells but also cellular toxicity of this metalloid. Therefore, our findings indicate a potential function of SFN in reducing cellular arsenic levels, thereby diminishing arsenic toxicity.

  2. Macro-cellular silica foams: synthesis during the natural creaming process of an oil-in-water emulsion.

    PubMed

    Sen, T; Tiddy, G J T; Casci, J L; Anderson, M W

    2003-09-01

    The room-temperature synthesis of a macro-mesoporous silica material during the natural creaming process of an oil-in-water emulsion is reported. The material has 3-dimensional interconnected macropores with a strut-like structure similar to meso-cellular silica foams with mesoporous walls of worm-hole structure. The material has very high surface area (approximately 800 m2 g(-1)) with narrow mesopore size distribution.

  3. Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function

    PubMed Central

    Nagayach, Aarti; Patro, Nisha; Patro, Ishan

    2014-01-01

    Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes. PMID:25400546

  4. Abnormalities in the cellular phase of blood fibrinolytic activity in systemic lupus erythematosus and in venous thromboembolism

    SciTech Connect

    Moroz, L.A.; MacLean, L.D.; Langleben, D.

    1986-09-15

    Fibrinolytic activities of whole blood and plasma were determined by /sup 125/I-fibrin radiometric assay in 16 normal subjects, and in 11 patients with systemic lupus erythematosus (SLE), 14 with progressive systemic sclerosis (PSS), 23 with venous thromboembolic disease, and 20 patients awaiting elective surgery. Mean whole blood and plasma activities for patients with PSS, and for those awaiting elective surgery, were similar to normal values, as was the mean plasma activity in patients with SLE. However, mean whole blood activity in SLE was significantly decreased compared with normals (p less than 0.05), with mean plasma activity accounting for 44% of mean whole blood activity (compared with 17% in normal subjects), representing a 67% decrease in mean calculated cellular phase activity in SLE, when compared with normals. Since the numbers of cells (neutrophils, monocytes) possibly involved in cellular activity were not decreased, the findings suggest a functional defect in fibrinolytic activity of one or more blood cell types in SLE. An additional finding was the participation of the cellular phase as well as the well-known plasma phase of blood in the fibrinolytic response to thromboembolism.

  5. Papillomavirus E2 Proteins and the Host Brd4 Protein Associate with Transcriptionally Active Cellular Chromatin▿ †

    PubMed Central

    Jang, Moon Kyoo; Kwon, Deukwoo; McBride, Alison A.

    2009-01-01

    The interaction of papillomavirus E2 proteins with cellular Brd4 protein is important for transcriptional regulation of viral genes and partitioning of viral genomes. Bovine papillomavirus type 1 (BPV-1) E2 binds cellular chromatin in complex with Brd4 in both mitotic and interphase cells. To identify specific sites of E2 interaction on cellular chromatin, a genome-wide chromatin immunoprecipitation-on-chip analysis was carried out using human promoter sequences. Both E2 and Brd4 were found bound to most transcriptionally active promoters in C33A cells. These promoters were also bound by RNA polymerase II and were modified by histone H3 acetylation and K4 trimethylation, all indicators of active transcription. E2 binding strongly correlated with Brd4 and RNA polymerase II occupancy and H3K4me3 modification at all human promoters, indicating that E2 bound to active promoters. E2 binding did not correlate with the presence of consensus E2 binding sites in the promoters. Furthermore, the mRNA levels of E2-bound cellular genes were not significantly changed by E2 expression. Thus, the papillomavirus E2 proteins bind to transcriptionally active cellular genes but do not change their activity. We propose that this may be a way for the virus to ensure that the viral genome is retained in transcriptionally active regions of the nucleus to escape silencing. Therefore, E2-mediated tethering of viral genomes to host chromatin has multiple roles: to partition the viral genome to daughter cells, to ensure that the genomes are retained in the nucleus, and to make certain that the genomes are retained in functionally active nuclear domains. PMID:19129460

  6. Chemical Composition and, Cellular Evaluation of the Antioxidant Activity of Desmodium adscendens Leaves

    PubMed Central

    Muanda, François Nsemi; Bouayed, Jaouad; Djilani, Abdelouaheb; Yao, Chunyan; Soulimani, Rachid; Dicko, Amadou

    2011-01-01

    Desmodium adscendens plant is widely used as juice or tea in various parts of the world against a wide range of diseases. This study determines the quality and the quantity of polyphenols, flavonoids, anthocyanins, and tannins in D. adscendens leaves by UV-spectrophotometry and RP-HPLC methods. In addition, the antioxidant capacity of these phenolic compounds is evaluated by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic)), DPPH (2,2-diphenyl-1 picrylhydrazyl), and Cellular tests. D. adscendens leaves are mainly composite of flavonoid compounds with 12.8 mg of catechin equivalent (CE)/g dw. The amounts of total polyphenol compounds are 11.1 mg of gallic acid equivalent (GAE)/g dw. The quantity of total anthocyanin and total tannin compounds is not considerable 0.0182 mg CgE/g dw and 0.39 mg CE/g dw, respectively. A direct correlation between phenolic compounds and antioxidant activity is observed (R2 = 0.96). The RP-HPLC analyses reveal that the main phenolic compound identified in the methanol-water extract is quercetrin dihydrat (2.11 mg/mL). According to the results, it is observed that D. adscendens leaves possess a considerable scavenging antioxidant and antiradical capacity, therefore these antioxidant properties might increase the therapeutic value of this medicinal plant. PMID:20976084

  7. TWO-DIMENSIONAL CELLULAR AUTOMATON MODEL FOR THE EVOLUTION OF ACTIVE REGION CORONAL PLASMAS

    SciTech Connect

    López Fuentes, Marcelo; Klimchuk, James A.

    2015-02-01

    We study a two-dimensional cellular automaton (CA) model for the evolution of coronal loop plasmas. The model is based on the idea that coronal loops are made of elementary magnetic strands that are tangled and stressed by the displacement of their footpoints by photospheric motions. The magnetic stress accumulated between neighbor strands is released in sudden reconnection events or nanoflares that heat the plasma. We combine the CA model with the Enthalpy Based Thermal Evolution of Loops model to compute the response of the plasma to the heating events. Using the known response of the X-Ray Telescope on board Hinode, we also obtain synthetic data. The model obeys easy-to-understand scaling laws relating the output (nanoflare energy, temperature, density, intensity) to the input parameters (field strength, strand length, critical misalignment angle). The nanoflares have a power-law distribution with a universal slope of –2.5, independent of the input parameters. The repetition frequency of nanoflares, expressed in terms of the plasma cooling time, increases with strand length. We discuss the implications of our results for the problem of heating and evolution of active region coronal plasmas.

  8. Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia

    PubMed Central

    Carlucci, Annalisa; Adornetto, Annagrazia; Scorziello, Antonella; Viggiano, Davide; Foca, Mariapaola; Cuomo, Ornella; Annunziato, Lucio; Gottesman, Max; Feliciello, Antonio

    2008-01-01

    A-kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post-translationally by the ubiquitin/proteasome pathway. Seven In-Absentia Homolog 2 (Siah2), an E3–ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2-mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2-dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121-signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability. PMID:18323779

  9. A beacon of hope in stroke therapy-Blockade of pathologically activated cellular events in excitotoxic neuronal death as potential neuroprotective strategies.

    PubMed

    Hoque, Ashfaqul; Hossain, M Iqbal; Ameen, S Sadia; Ang, Ching-Seng; Williamson, Nicholas; Ng, Dominic C H; Chueh, Anderly C; Roulston, Carli; Cheng, Heung-Chin

    2016-04-01

    Excitotoxicity, a pathological process caused by over-stimulation of ionotropic glutamate receptors, is a major cause of neuronal loss in acute and chronic neurological conditions such as ischaemic stroke, Alzheimer's and Huntington's diseases. Effective neuroprotective drugs to reduce excitotoxic neuronal loss in patients suffering from these neurological conditions are urgently needed. One avenue to achieve this goal is to clearly define the intracellular events mediating the neurotoxic signals originating from the over-stimulated glutamate receptors in neurons. In this review, we first focus on the key cellular events directing neuronal death but not involved in normal physiological processes in the neurotoxic signalling pathways. These events, referred to as pathologically activated events, are potential targets for the development of neuroprotectant therapeutics. Inhibitors blocking some of the known pathologically activated cellular events have been proven to be effective in reducing stroke-induced brain damage in animal models. Notable examples are inhibitors suppressing the ion channel activity of neurotoxic glutamate receptors and those disrupting interactions of specific cellular proteins occurring only in neurons undergoing excitotoxic cell death. Among them, Tat-NR2B9c and memantine are clinically effective in reducing brain damage caused by some acute and chronic neurological conditions. Our second focus is evaluation of the suitability of the other inhibitors for use as neuroprotective therapeutics. We also discuss the experimental approaches suitable for bridging our knowledge gap in our current understanding of the excitotoxic signalling mechanism in neurons and discovery of new pathologically activated cellular events as potential targets for neuroprotection. PMID:26899498

  10. Gravity-induced cellular and molecular processes in plants studied under altered gravity conditions

    NASA Astrophysics Data System (ADS)

    Vagt, Nicole; Braun, Markus

    With the ability to sense gravity plants possess a powerful tool to adapt to a great variety of environmental conditions and to respond to environmental changes in a most beneficial way. Gravity is the only constant factor that provides organisms with reliable information for their orientation since billions of years. Any deviation of the genetically determined set-point angle of the plants organs from the vector of gravity is sensed by specialized cells, the statocytes of roots and shoots in higher plants. Dense particles, so-called statoliths, sediment in the direction of gravity and activate membrane-bound gravireceptors. A physiological signalling-cascade is initiated that eventually results in the gravitropic curvature response, namely, the readjust-ment of the growth direction. Experiments under microgravity conditions have significantly contributed to our understanding of plant gravity-sensing and gravitropic reorientation. For a gravity-sensing lower plant cell type, the rhizoid of the green alga Chara, and for statocytes of higher plant roots, it was shown that the interactions between statoliths and the actomyosin system consisting of the actin cytoskeleton and motor proteins (myosins) are the basis for highly efficient gravity-sensing processes. In Chara rhizoids, the actomyosin represents a guid-ing system that directs sedimenting statoliths to a specific graviperception site. Parabolic flight experiments aboard the airbus A300 Zero-G have provided evidence that lower and higher plant cells use principally the same statolith-mediated gravireceptor-activation mechanism. Graviper-ception is not dependent on mechanical pressure mediated through the weight of the sedimented statoliths, but on direct interactions between the statoliths's surface and yet unknown gravire-ceptor molecules. In contrast to Chara rhizoids, in the gravity-sensing cells of higher plants, the actin cytoskeleton is not essentially involved in the early phases of gravity sensing. Dis

  11. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage.

    PubMed

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound "natural Aβ", sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson's disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  12. Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

    PubMed Central

    West, Ewan; Osborne, Craig; Nolan, William; Bate, Clive

    2015-01-01

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ) and the loss of synapses. Aggregation of the cellular prion protein (PrPC) by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI) anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2) and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage. PMID:26043272

  13. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.

    PubMed

    Aragón, T; de la Luna, S; Novoa, I; Carrasco, L; Ortín, J; Nieto, A

    2000-09-01

    Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

  14. Pregnancy affects cellular activity, but not tissue mechanical properties, in the healing rabbit medial collateral ligament.

    PubMed

    Hart, D A; Reno, C; Frank, C B; Shrive, N G

    2000-05-01

    Recently, evidence has been accumulating that ligament and joint laxity is altered in women and rabbits during pregnancy. Furthermore, many female adolescents injure ligaments through participation in athletics and other activities. Therefore, to determine whether pregnancy has different effects on the injured and uninjured medial collateral ligament of the rabbit knee, we investigated cellular changes (mRNA levels) and alterations in tissue properties (biomechanics) accompanying pregnancy in animals with the medial collateral ligament injured during adolescence and bred for their primigravid pregnancy as young adults. Assessment of mRNA levels for matrix molecules, matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, growth factors and sex hormone receptors, inflammatory cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 by semiquantitative reverse transcription-polymerase chain reaction revealed that pregnancy had different impacts on scar and uninjured tissue for six of 15 genes assessed. A pregnancy-associated increase in laxity of the medial collateral ligament was observed for rabbits in the uninjured primigravida group; however, no increase was observed for injured rabbits during pregnancy. The injured ligament was already significantly more lax than the normal counterpart, and pregnancy did not lead to additional laxity or prevent the normal decline in laxity as the scar matured in nonpregnant animals. These results indicate that the impact of pregnancy on laxity and cell activity of the medial collateral ligament is dependent on whether the ligament is uninjured or injured. Pregnancy had no significant effect on structural (stiffness and failure load), material (stress at failure and Young's modulus), or viscoelastic (cyclic and static relaxation) properties of tissue from uninjured or injured medial collateral ligament. Therefore, the properties of the healing ligament were not adversely affected during pregnancy in this

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

    SciTech Connect

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

    2015-08-21

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

  16. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  17. Characterization of a cellular denitrase activity that reverses nitration of cyclooxygenase

    PubMed Central

    Nuriel, Tal; Cheung, Cynthia; Summers, Barbara; Lamon, Brian D.; Gross, Steven S.; Hajjar, David P.

    2013-01-01

    Protein 3-nitrotyrosine (3-NT) formation is frequently regarded as a simple biomarker of disease, an irreversible posttranslational modification that can disrupt protein structure and function. Nevertheless, evidence that protein 3-NT modifications may be site selective and reversible, thus allowing for physiological regulation of protein activity, has begun to emerge. We have previously reported that cyclooxygenase (COX)-1 undergoes heme-dependent nitration of Tyr385, an internal and catalytically essential residue. In the present study, we demonstrate that nitrated COX-1 undergoes a rapid reversal of nitration by substrate-selective and biologically regulated denitrase activity. Using nitrated COX-1 as a substrate, denitrase activity was validated and quantified by analytic HPLC with electrochemical detection and determined to be constitutively active in murine and human endothelial cells, macrophages, and a variety of tissue samples. Smooth muscle cells, however, contained little denitrase activity. Further characterizing this denitrase activity, we found that it was inhibited by free 3-NT and may be enhanced by endogenous nitric oxide and exogenously administered carbon monoxide. Finally, we describe a purification protocol that results in significant enrichment of a discrete denitrase-containing fraction, which maintains activity throughout the purification process. These findings reveal that nitrated COX-1 is a substrate for a denitrase in cells and tissues, implying that the reciprocal processes of nitration and denitration may modulate bioactive lipid synthesis in the setting of inflammation. In addition, our data reveal that denitration is a controlled process that may have broad importance for regulating cell signaling events in nitric oxide-generating systems during oxidative/nitrosative stress. PMID:23792683

  18. Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment*

    PubMed Central

    Geng, Jie; Sivaramakrishnan, Sivaraj; Raghavan, Malini

    2013-01-01

    The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function. PMID:24196954

  19. Identification of Yeast Mutants Exhibiting Altered Sensitivity to Valinomycin and Nigericin Demonstrate Pleiotropic Effects of Ionophores on Cellular Processes

    PubMed Central

    Bhatia-Kissova, Ingrid; Valachovic, Martin; Klobucnikova, Vlasta; Zeiselova, Lucia; Griac, Peter; Nosek, Jozef

    2016-01-01

    Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the Saccharomyces cerevisiae nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments. PMID:27711131

  20. Mapping whole-brain activity with cellular resolution by light-sheet microscopy and high-throughput image analysis (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Silvestri, Ludovico; Rudinskiy, Nikita; Paciscopi, Marco; Müllenbroich, Marie Caroline; Costantini, Irene; Sacconi, Leonardo; Frasconi, Paolo; Hyman, Bradley T.; Pavone, Francesco S.

    2016-03-01

    Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information. Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution. The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.

  1. Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

    PubMed Central

    Ito, T; Sasaki, Y; Wands, J R

    1996-01-01

    The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation. PMID:8622697

  2. Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A.

    PubMed

    Kim, Jeong-Keun; Park, Keun-Tae; Lee, Hyun-Sun; Kim, Mijin; Lim, Young-Hee

    2012-08-01

    The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.

  3. Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation

    PubMed Central

    Stellavato, Antonietta; Corsuto, Luisana; D’Agostino, Antonella; La Gatta, Annalisa; Diana, Paola; Bernini, Patrizia; De Rosa, Mario

    2016-01-01

    Hyaluronic Acid (HA)-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM), acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA) provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity) and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH) digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR) and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at longer time (up

  4. Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

    PubMed Central

    Dayaram, Tajhal; Lemoine, Francene J.; Donehower, Lawrence A.; Marriott, Susan J.

    2013-01-01

    Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome. PMID:23405243

  5. Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

    PubMed

    Indrawattana, Nitaya; Sookrung, Nitat; Kulkeaw, Kasem; Seesuay, Watee; Kongngoen, Thida; Chongsa-nguan, Manas; Tungtrongchitr, Anchalee; Chaicumpa, Wanpen

    2010-03-01

    Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called tetanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant

  6. Tuberculosis in HIV-positive patients: cellular response and immune activation in the lung.

    PubMed

    Law, K F; Jagirdar, J; Weiden, M D; Bodkin, M; Rom, W N

    1996-04-01

    The host response to Mycobacterium tuberculosis is dependent on the accumulation and activation of cytotoxic and memory CD4+ T cells, resulting in granuloma formation and delayed type hypersensitivity. We characterized the cellular response of radiographically involved lung segments from 17 HIV-positive and 11 HIV-negative patients with acute tuberculosis (TB) using bronchoalveolar lavage (BAL) and compared the response to uninvolved segments, normal control subjects and peripheral blood. In both HIV-positive and HIV-negative patients, radiographically involved segments had significantly increased numbers of total cells per milliliter, percent of neutrophils recovered, and percent of lymphocytes recovered compared with uninvolved segments or normal control subjects, but HIV-positive patients had a lower proportion of lymphocytes in the involved segments than HIV-negative patients with tuberculosis (19 +/- 5% versus 33 +/- 5%; p < 0.05). Lymphocyte subset analysis demonstrated that HIV-positive patients had markedly reduced percentages of CD4+ lymphocytes (CD4+ lymphocytes in HIV-positive TB involved site 25 +/- 6%; HIV-negative TB involved site 73 +/- 2%; p < 0.01) and an increase in the percentage of CD8+ lymphocytes (HIV positive involved site 61 +/- 6% versus HIV negative involved site 19 +/- 3%; p < 0.01). Immunohistochemistry of lung biopsy tissue in five HIV-negative patients showed similar lymphocyte subset profiles as BAL, indicating that BAL reflects cell populations in tissue granulomas. BAL lymphocytes from four HIV-positive and four HIV-negative tuberculosis patients demonstrated immune activation by staining with a murine antibody to TIA-1, a cytoplasmic protein associated with cytotoxicity and apoptosis (HIV positive 48 +/- 6%, HIV negative 31 +/- 7%, normals 11 +/- 5%). Steady state mRNA for gamma-interferon was decreased in four HIV-positive patients when compared with four HIV-negative patients. IL-8 production was comparable in HIV-negative and

  7. Lunar Dust and Lunar Simulant Activation, Monitoring, Solution and Cellular Toxicity Properties

    NASA Technical Reports Server (NTRS)

    Wallace, William; Jeevarajan, A. S.

    2009-01-01

    During the Apollo missions, many undesirable situations were encountered that must be mitigated prior to returning humans to the moon. Lunar dust (that part of the lunar regolith less than 20 microns in diameter) was found to produce several problems with mechanical equipment and could have conceivably produced harmful physiological effects for the astronauts. For instance, the abrasive nature of the dust was found to cause malfunctions of various joints and seals of the spacecraft and suits. Additionally, though efforts were made to exclude lunar dust from the cabin of the lunar module, a significant amount of material nonetheless found its way inside. With the loss of gravity correlated with ascent from the lunar surface, much of the finer fraction of this dust began to float and was inhaled by the astronauts. The short visits tothe Moon during Apollo lessened exposure to the dust, but the plan for future lunar stays of up to six months demands that methods be developed to minimize the risk of dust inhalation. The guidelines for what constitutes "safe" exposure will guide the development of engineering controls aimed at preventing the presence of dust in the lunar habitat. This work has shown the effects of grinding on the activation level of lunar dust, the changes in dissolution properties of lunar simulant, and the production of cytokines by cellular systems. Grinding of lunar dust leads to the production of radicals in solution and increased dissolution of lunar simulant in buffers of different pH. Additionally, ground lunar simulant has been shown to promote the production of IL-6 and IL-8, pro-inflammatory cytokines, by alveolar epithelial cells. These results provide evidence of the need for further studies on these materials prior to returning to the lunar surface.

  8. Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

    PubMed

    Habermeyer, Michael; Fritz, Jessica; Barthelmes, Hans U; Christensen, Morten O; Larsen, Morten K; Boege, Fritz; Marko, Doris

    2005-09-01

    In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).

  9. Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

    PubMed Central

    Buri, Marcus V.; Domingues, Tatiana M.; Paredes-Gamero, Edgar J.; Casaes-Rodrigues, Rafael L.; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

  10. Array of amorphous calcium phosphate particles improves cellular activity on a hydrophobic surface.

    PubMed

    Kim, InAe; Kim, Hyun Jung; Kim, Hyun-Man

    2010-04-01

    Poor interaction between cells and surfaces, especially hydrophobic surfaces, results in delayed proliferation and increased apoptosis due to low cell adhesion signaling. To improve cell adhesion, hydrophilic array of amorphous calcium phosphate (ACP) was fabricated on a surface. A phosphate-buffered solution containing calcium ions was prepared at low temperature to prevent spontaneous precipitation. Then, the ion solution was heated to generate nuclei of ACP nanoparticles. The ACP nanoparticles adhered to the hydrophobic polystyrene surface forming an array composed of ACP particles. Multiple treatments of these nuclei with fresh CaP ion solutions increased the diameter and decreased the solubility of ACP particles enough to mediate cellular adhesion. The particle density in the array was dependent on the ion concentration of the CaP ion solutions. The ACP array improved a wide variety of activities when osteoblastic MC3T3-E1 cells were cultured on the ACP array fabricated on a hydrophobic bacteriological dish surface, compared to those cultured without the ACP array in vitro. The use of ACP array resulted in a lower apoptosis and also increased the spreading of cells to form stress fibers and focal contacts. Cells cultured on the ACP array proliferated more than cells cultured on a hydrophobic surface without the ACP array. The ACP array increased the expression of markers of differentiation in osteoblast. These results indicate that an array of ACP can be used as a coating material for enhancing biocompatibility in tissue engineering or biomaterials rather than modifying the surface with organic molecules. PMID:20119940

  11. Cellular localization of type 1 plasminogen activator inhibitor messenger RNA and protein in murine renal tissue.

    PubMed Central

    Keeton, M.; Eguchi, Y.; Sawdey, M.; Ahn, C.; Loskutoff, D. J.

    1993-01-01

    Type 1 plasminogen activator inhibitor (PAI-1) may be markedly increased in the plasma of patients with endotoxemia and/or renal disease. To investigate renal PAI-1 production during acute endotoxemia, a murine model system was used. Mice were injected with either saline alone or saline containing 50 micrograms endotoxin, and sacrificed 3 hours later and their tissues analyzed for PAI-1 messenger RNA (mRNA) and antigen. Northern blot analysis confirmed that the level of renal PAI-1 mRNA was greatly increased in the endotoxemic mice relative to the saline controls. In situ hybridization was then performed to determine the cellular localization of PAI-1 mRNA within the renal tissues. In the control kidneys, low levels of PAI-1 mRNA were detected in the renal papilla and in the muscular walls of renal arteries. However, in the endotoxemic mice, an intense hybridization signal for PAI-1 mRNA was observed in glomerular and peritubular cells. These cells also stained positively for von Willebrand factor antigen, an endothelial cell-specific marker. The PAI-1 mRNA hybridization signal could further be observed in peritubular endothelial cells in the medulla and in endothelial cells of veins and arteries throughout the kidney. Immunochemical analysis revealed that PAI-1 antigen co-localized to the cytoplasm of cells expressing PAI-1 mRNA. This study provides the first direct evidence that PAI-1 is induced in endothelial cells of the kidney during endotoxemia in vivo and suggests a role for PAI-1 in the pathogenesis of renal disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8424466

  12. Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium

    PubMed Central

    Vinh, Nguyen X.; Viswanathan, Ganesh A.; Chetty, Madhu; Wangikar, Pramod P.

    2015-01-01

    Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell’s metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production. PMID:25973856

  13. Coupling of Cellular Processes and Their Coordinated Oscillations under Continuous Light in Cyanothece sp. ATCC 51142, a Diazotrophic Unicellular Cyanobacterium.

    PubMed

    Krishnakumar, S; Gaudana, Sandeep B; Vinh, Nguyen X; Viswanathan, Ganesh A; Chetty, Madhu; Wangikar, Pramod P

    2015-01-01

    Unicellular diazotrophic cyanobacteria such as Cyanothece sp. ATCC 51142 (henceforth Cyanothece), temporally separate the oxygen sensitive nitrogen fixation from oxygen evolving photosynthesis not only under diurnal cycles (LD) but also in continuous light (LL). However, recent reports demonstrate that the oscillations in LL occur with a shorter cycle time of ~11 h. We find that indeed, majority of the genes oscillate in LL with this cycle time. Genes that are upregulated at a particular time of day under diurnal cycle also get upregulated at an equivalent metabolic phase under LL suggesting tight coupling of various cellular events with each other and with the cell's metabolic status. A number of metabolic processes get upregulated in a coordinated fashion during the respiratory phase under LL including glycogen degradation, glycolysis, oxidative pentose phosphate pathway, and tricarboxylic acid cycle. These precede nitrogen fixation apparently to ensure sufficient energy and anoxic environment needed for the nitrogenase enzyme. Photosynthetic phase sees upregulation of photosystem II, carbonate transport, carbon concentrating mechanism, RuBisCO, glycogen synthesis and light harvesting antenna pigment biosynthesis. In Synechococcus elongates PCC 7942, a non-nitrogen fixing cyanobacteria, expression of a relatively smaller fraction of genes oscillates under LL condition with the major periodicity being 24 h. In contrast, the entire cellular machinery of Cyanothece orchestrates coordinated oscillation in anticipation of the ensuing metabolic phase in both LD and LL. These results may have important implications in understanding the timing of various cellular events and in engineering cyanobacteria for biofuel production.

  14. Cellular signalling and free-radical modulating activities of the novel peptidomimetic L-glutamyl-histamine.

    PubMed

    Babizhayev, M A; Semiletov, Y A; Lul'kin, Y A; Sakina, N L; Savel'yeva, E L; Deyev, A I; Alimbarova, L I; Barinskii, I F; Nicolay, J; Paillet, C; Langrand, G; Seguin, F

    1999-05-01

    A novel histamine-containing peptidomimetic, L-glutamyl-histamine (L-Glu-Hist), has been synthesized and characterized as a possible cytokine mimic which might lead to cellular responses of improved specificity. The energy-minimized 3-D conformations of L-Glu-Hist derived from its chemical structure stabilize Fe2+-chelating complexes. L-Glu-Hist concentration-dependently accelerates a decrease in ferrous iron in ferrous sulfate solution and shows ferroxidase-like activity at concentrations less than 3 mM in the phenanthroline assay, whereas in the concentration range 3-20 mM it restricts the availability of Fe2+ to phenanthroline by chelation of iron ions. At low concentrations (less than or about 1 mM), L-Glu-Hist stimulates peroxidation of phosphatidylcholine in liposomes catalyzed by a superoxide anion radical (O2)-generating system (Fe2+ + ascorbate) and, at high concentrations (*10 mM), it suppresses lipid peroxidation (LPO) in liposomes. The stimulation of LPO by L-Glu-Hist is related to its ability at low concentrations (*0.05 mM) to release O2 free radicals as determined by the superoxide dismutase-inhibitable reduction of cytochrome c. The release of O2 by L-Glu-Hist might result from its ferroxidase-like activity, while its inhibition of LPO is due to chelation of Fe2+, prevention of the formation of free radicals, and degradation of lipid hydroperoxides at 5-20 mM L-Glu-Hist concentrations. L-Glu-Hist releases O2 at concentrations which stimulate [3H]thymidine incorporation into DNA and proliferation of mouse spleen lymphocytes and also of mononuclear cells from human blood. The induction of lymphocyte proliferation by L-Glu-Hist is dose-dependent in the 0.01-0.05 mM concentration range, although the maximal stimulation of LPO in the O2-dependent system is observed at higher L-Glu-Hist concentrations (*1 mM). Thus, low concentrations of oxygen free radicals released by L-Glu-Hist may provide a very fast, specific, and sensitive trigger for lymphocyte

  15. The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets.

    PubMed

    Arena, Ellen T; Auweter, Sigrid D; Antunes, L Caetano M; Vogl, A Wayne; Han, Jun; Guttman, Julian A; Croxen, Matthew A; Menendez, Alfredo; Covey, Scott D; Borchers, Christoph H; Finlay, B Brett

    2011-11-01

    To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

  16. Cellular differentiation in the process of generation of the eukaryotic cell

    NASA Astrophysics Data System (ADS)

    Nakamura, Hakobu; Hase, Atsushi

    1990-11-01

    Primitive atmosphere of the earth did not contain oxygen gas (O2) when the proto-cells were generated successfully as the resut of chemical evolution and then evolved. Therefore, they first had acquired anaerobic energy metabolism, fermentation. The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions. Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle. On the other hand, O2-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem. These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacteriumChlorogloea fritschii and aerobic photosynthetic bacteriaRhodospirillum rubrum andErythrobacter sp. Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium. Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O2 -respiratory enzymes show similarities to corresponding prokaryotic enzymes. However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules. In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only

  17. Cellular and transcriptomic analysis of human mesenchymal stem cell response to plasma-activated hydroxyapatite coating.

    PubMed

    Tan, Fei; O'Neill, Feidhlim; Naciri, Mariam; Dowling, Denis; Al-Rubeai, Mohamed

    2012-04-01

    Atmospheric pressure plasma has recently emerged as a technique with a promising future in the medical field. In this work we used the technique as a post-deposition modification process as a means to activate hydroxyapatite (HA) coatings. Contact angle goniometry, optical profilometry, scanning electron microscopy morphology imaging and X-ray photoelectron spectroscopy analysis demonstrate that surface wettability is improved after treatment, without inducing any concomitant damage to the coating. The protein adsorption pattern has been found to be preferable for MSC, and this may result in greater cell attachment and adhesion to plasma-activated HA than to untreated samples. Cell cycle distribution analysis using flow cytometry reveals a faster transition from G(1) to S phase, thus leading to a faster cell proliferation rate on plasma-activated HA. This indicates that the improvement in surface wettability independently enhances cell attachment and cell proliferation, which is possibly mediated by FAK phosphorylation. Pathway-specific polymerase chain reaction arrays revealed that wettability has a substantial influence on gene expression during osteogenic differentiation of human MSC. Plasma-activated HA tends to enhance this process by systemically deregulating multiple genes. In addition, the majority of these deregulated genes had been appropriately translated, as confirmed by ELISA protein quantification. Lastly, alizarin red staining showed that plasma-activated HA is capable of improving mineralization for up to 3 weeks of in vitro culture. It was concluded from this study that atmospheric pressure plasma is a potent tool for modifying the biological function of a material without causing thermal damage, such that adhesion molecules and drugs might be deposited on the original coating to improve performance.

  18. The first cellular bioenergetic process: primitive generation of a proton-motive force.

    PubMed

    Koch, A L; Schmidt, T M

    1991-10-01

    It is proposed that the energy-transducing system of the first cellular organism and its precursor was fueled by the oxidation of hydrogen sulfide and ferric sulfide to iron pyrites and two [H+] on the outside surface of a vesicle (the cell membrane), with the concomitant reduction of CO or CO2 on the interior. The resulting proton gradient across the cell membrane provides a proton-motive force, so that a variety of kinds of work can be done. It is envisioned as providing a selective advantage for cells capable of harvesting this potential. The proposed reactants for these reactions are consistent with the predicted composition of the Earth's early environment. Modern-day homologs of the ancestral components of the energy-transducing system are thought to be membrane-associated ferredoxins for the extracellular redox reaction, carbon monoxide dehydrogenase for the carbon fixation reaction, and ATPase for the harvesting of the proton gradient. With a source of consumable energy, the cell could drive chemical reactions and transport events in such a way as to be exploited by Darwinian evolution. PMID:1663558

  19. The first cellular bioenergetic process: primitive generation of a proton-motive force.

    PubMed

    Koch, A L; Schmidt, T M

    1991-10-01

    It is proposed that the energy-transducing system of the first cellular organism and its precursor was fueled by the oxidation of hydrogen sulfide and ferric sulfide to iron pyrites and two [H+] on the outside surface of a vesicle (the cell membrane), with the concomitant reduction of CO or CO2 on the interior. The resulting proton gradient across the cell membrane provides a proton-motive force, so that a variety of kinds of work can be done. It is envisioned as providing a selective advantage for cells capable of harvesting this potential. The proposed reactants for these reactions are consistent with the predicted composition of the Earth's early environment. Modern-day homologs of the ancestral components of the energy-transducing system are thought to be membrane-associated ferredoxins for the extracellular redox reaction, carbon monoxide dehydrogenase for the carbon fixation reaction, and ATPase for the harvesting of the proton gradient. With a source of consumable energy, the cell could drive chemical reactions and transport events in such a way as to be exploited by Darwinian evolution.

  20. TissueMiner: A multiscale analysis toolkit to quantify how cellular processes create tissue dynamics

    PubMed Central

    Etournay, Raphaël; Merkel, Matthias; Popović, Marko; Brandl, Holger; Dye, Natalie A; Aigouy, Benoît; Salbreux, Guillaume; Eaton, Suzanne; Jülicher, Frank

    2016-01-01

    Segmentation and tracking of cells in long-term time-lapse experiments has emerged as a powerful method to understand how tissue shape changes emerge from the complex choreography of constituent cells. However, methods to store and interrogate the large datasets produced by these experiments are not widely available. Furthermore, recently developed methods for relating tissue shape changes to cell dynamics have not yet been widely applied by biologists because of their technical complexity. We therefore developed a database format that stores cellular connectivity and geometry information of deforming epithelial tissues, and computational tools to interrogate it and perform multi-scale analysis of morphogenesis. We provide tutorials for this computational framework, called TissueMiner, and demonstrate its capabilities by comparing cell and tissue dynamics in vein and inter-vein subregions of the Drosophila pupal wing. These analyses reveal an unexpected role for convergent extension in shaping wing veins. DOI: http://dx.doi.org/10.7554/eLife.14334.001 PMID:27228153

  1. Bubble nucleation process in a metal tritide modeled using a cellular automaton

    NASA Astrophysics Data System (ADS)

    Segard, M.; Fabre, A.; Thiébaut, S.; Montheillet, F.

    2012-01-01

    The modeling of the 3He bubble nucleation phase that occurs during the aging of metal tritides such as palladium tritide is undertaken using a cellular automaton describing the material at the atomic scale. In that model, using simple rules of cell state change, the physical phenomena involved in the bubble nucleation, namely tritium diffusion, formation of 3He by radioactive tritium decay, 3He diffusion, 3He self-trapping, bubble growth, influence of pre-existing trapping sites such as vacancies, are taken into consideration. Calculations steps are related to physical time through the decay of tritium atoms into 3He atoms which is characterized by a half-life of 12.32 years. That work has shown that the bubble density and distribution are almost stable within a few days of aging - typically 5-10 -, whatever the input parameters, the system making created bubbles to grow thereafter instead of creating new ones. The reached bubble density is very dependent on the 3He mobility - related to temperature for instance - during the first days of aging. The smaller it is, the higher the density. With a larger density of trapping sites, bubbles appear earlier and their density is higher. These results are discussed and compared to available experimental and theoretical works on palladium tritide.

  2. Long-Term Calorie Restriction Enhances Cellular Quality-Control Processes in Human Skeletal Muscle.

    PubMed

    Yang, Ling; Licastro, Danilo; Cava, Edda; Veronese, Nicola; Spelta, Francesco; Rizza, Wanda; Bertozzi, Beatrice; Villareal, Dennis T; Hotamisligil, Gökhan S; Holloszy, John O; Fontana, Luigi

    2016-01-26

    Calorie restriction (CR) retards aging, acts as a hormetic intervention, and increases serum corticosterone and HSP70 expression in rodents. However, less is known regarding the effects of CR on these factors in humans. Serum cortisol and molecular chaperones and autophagic proteins were measured in the skeletal muscle of subjects on CR diets for 3-15 years and in control volunteers. Serum cortisol was higher in the CR group than in age-matched sedentary and endurance athlete groups (15.6 ± 4.6 ng/dl versus 12.3 ± 3.9 ng/dl and 11.2 ± 2.7 ng/dl, respectively; p ≤ 0.001). HSP70, Grp78, beclin-1, and LC3 mRNA and/or protein levels were higher in the skeletal muscle of the CR group compared to controls. Our data indicate that CR in humans is associated with sustained rises in serum cortisol, reduced inflammation, and increases in key molecular chaperones and autophagic mediators involved in cellular protein quality control and removal of dysfunctional proteins and organelles. PMID:26774472

  3. Long-Term Calorie Restriction Enhances Cellular Quality-Control Processes in Human Skeletal Muscle.

    PubMed

    Yang, Ling; Licastro, Danilo; Cava, Edda; Veronese, Nicola; Spelta, Francesco; Rizza, Wanda; Bertozzi, Beatrice; Villareal, Dennis T; Hotamisligil, Gökhan S; Holloszy, John O; Fontana, Luigi

    2016-01-26

    Calorie restriction (CR) retards aging, acts as a hormetic intervention, and increases serum corticosterone and HSP70 expression in rodents. However, less is known regarding the effects of CR on these factors in humans. Serum cortisol and molecular chaperones and autophagic proteins were measured in the skeletal muscle of subjects on CR diets for 3-15 years and in control volunteers. Serum cortisol was higher in the CR group than in age-matched sedentary and endurance athlete groups (15.6 ± 4.6 ng/dl versus 12.3 ± 3.9 ng/dl and 11.2 ± 2.7 ng/dl, respectively; p ≤ 0.001). HSP70, Grp78, beclin-1, and LC3 mRNA and/or protein levels were higher in the skeletal muscle of the CR group compared to controls. Our data indicate that CR in humans is associated with sustained rises in serum cortisol, reduced inflammation, and increases in key molecular chaperones and autophagic mediators involved in cellular protein quality control and removal of dysfunctional proteins and organelles.

  4. Processing and properties of multiscale cellular thermoplastic fiber reinforced composite (CellFRC)

    NASA Astrophysics Data System (ADS)

    Sorrentino, L.; Cafiero, L.; D'Auria, M.; Iannace, S.

    2015-12-01

    High performance fiber reinforced polymer composites are made by embedding high strength/modulus fibers in a polymeric matrix. They are a class of materials that owe its success to the impressive specific mechanical properties with respect to metals. In many weight-sensitive applications, where high mechanical properties and low mass are required, properties per unit of mass are more important than absolute properties and further weight reduction is desirable. A route to reach this goal could be the controlled induction of porosity into the polymeric matrix, while still ensuring load transfer to the reinforcing fibers and fiber protection from the environment. Cellular lightweight fiber reinforced composites (CellFRC) were prepared embedding gas bubbles of controlled size within a high performance thermoplastic matrix reinforced with continuous fibers. Pores were induced after the composite was first saturated with CO2 and then foamed by using an in situ foaming/shaping technology based on compression moulding with adjustable mould cavities. The presence of micro- or submicro-sized cells in the new CellFRC reduced the apparent density of the structure and led to significant improvements of its impact properties. Both structural and functional performances were further improved through the use of a platelet-like nanofiller (Expanded Graphite) dispersed into the matrix.

  5. SPT genes: key players in the regulation of transcription, chromatin structure and other cellular processes.

    PubMed

    Yamaguchi, Y; Narita, T; Inukai, N; Wada, T; Handa, H

    2001-02-01

    Suppressor of Ty (SPT) genes were originally identified through a genetic screen for mutations in the yeast Saccharomyces cerevisiae that restore gene expression disrupted by the insertion of the transposon Ty. Classic members of the SPT gene family, SPT11, SPT12, and SPT15, encode for the histones H2A and H2B, and for TATA-binding protein (TBP), respectively. Over the past few years, molecular complexes and cellular functions in which other SPT gene products involve have been discovered through genetic and biochemical studies in yeast and several other organisms: Key regulators of transcription and chromatin structure, such as DSIF, SAGA, and FACT, all contain SPT gene products as essential subunits. In addition, accumulating evidence suggests that SPT gene products play more diverse roles, including roles in DNA replication, DNA recombination and developmental regulation. Here we review the current understanding of the functions and roles of the SPT genes, with special emphasis on the role of SPT5 in transcript elongation and in neuronal development in vertebrates.

  6. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

    PubMed

    Atamna, Hani; Atamna, Wafa; Al-Eyd, Ghaith; Shanower, Gregory; Dhahbi, Joseph M

    2015-12-01

    Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear. Since MB is redox-active, we investigated its effect on the NAD/NADH ratio in IMR90 cells. The transient increase in NAD/NADH observed in MB-treated cells triggered an investigation of the energy regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex-IV biogenesis. Subsequently MB-treated cells exhibited >100% increase in complex-IV activity and a 28% decline in cellular oxidants. The telomeres erosion rate was also significantly lower in MB-treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence. We identified that the anti-senescence activity of MB (transient activator) was 8-times higher than that of AICAR (chronic activator). Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense pathways as a possible key factor in MB's potent anti-senescence activity.

  7. Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue

    PubMed Central

    Atamna, Hani; Atamna, Wafa; Al-Eyd, Ghaith; Shanower, Gregory; Dhahbi, Joseph M.

    2015-01-01

    Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear. Since MB is redox-active, we investigated its effect on the NAD/NADH ratio in IMR90 cells. The transient increase in NAD/NADH observed in MB-treated cells triggered an investigation of the energy regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex-IV biogenesis. Subsequently MB-treated cells exhibited >100% increase in complex-IV activity and a 28% decline in cellular oxidants. The telomeres erosion rate was also significantly lower in MB-treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence. We identified that the anti-senescence activity of MB (transient activator) was 8-times higher than that of AICAR (chronic activator). Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense pathways as a possible key factor in MB's potent anti-senescence activity. PMID:26386875

  8. Autoinducer AI-2 is involved in regulating a variety of cellular processes in Salmonella Typhimurium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LuxS/AI-2 mediated cell signaling is a known strategy that modulates a variety of bacterial processes in prokaryotes. Salmonella Typhimurium is known to possess LuxS/AI-2 mediated cell signaling. Until now, the Lsr- ABC transporter system (LuxS- regulated) is the only known process controlled by t...

  9. Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses

    PubMed Central

    Saha, Rajib; Liu, Deng; Hoynes-O’Connor, Allison; Liberton, Michelle; Yu, Jingjie; Bhattacharyya-Pakrasi, Maitrayee; Balassy, Andrea; Zhang, Fuzhong; Maranas, Costas D.

    2016-01-01

    ABSTRACT Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP+ showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium. PMID:27143387

  10. Tempo-spatially resolved cellular dynamics of human immunodeficiency virus transacting activator of transcription (Tat) peptide-modified nanocargos in living cells

    NASA Astrophysics Data System (ADS)

    Wei, Lin; Yang, Qiaoyu; Xiao, Lehui

    2014-08-01

    Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs could not actively target the cell nuclei, which is contrary to previous observations based on fixed cell results. More importantly, the inheritance of TGNPs to the daughter cells through mitosis was found to be the major route to metabolize TGNPs by HeLa cells. These understandings on the cellular uptake mechanism and intracellular fate of nanocargos in living cells would provide deep insight on how to improve and controllably manipulate their translocation efficiency for targeted drug delivery.Understanding the cellular uptake mechanism and intracellular fate of nanocarriers in living cells is of great importance for the rational design of efficient drug delivery cargos as well as the development of robust biomedical diagnostic probes. In present study, with a dual wavelength view darkfield microscope (DWVD), the tempo-spatially resolved dynamics of Tat peptide-functionalized gold nanoparticles (TGNPs, with size similar to viruses) in living HeLa cells were extensively explored. It was found that energy-dependent endocytosis (both clathrin- and caveolae-mediated processes were involved) was the prevailing pathway for the cellular uptake of TGNPs. The time-correlated dynamic spatial distribution information revealed that TGNPs

  11. Cellular Interaction and Toxicity Depends on Physiochemical Properties and Surface Modification of Redox Active Nanomaterials

    PubMed Central

    Dowding, Janet M.; Das, Soumen; Kumar, Amit; Dosani, Talib; McCormack, Rameech; Gupta, Ankur; Sayle, Thi X. T.; Sayle, Dean C.; von Kalm, Laurence; Seal, Sudipta; Self, William T.

    2013-01-01

    The study of the chemical and biological properties of CeO2 NPs (CNPs) has expanded recently due to its therapeutic potential, and the methods used to synthesize these materials are diverse. Moreover, conflicting reports exists regarding the toxicity of CNP. To help resolve these discrepancies, we must first determine whether CeO2 NPs made by different methods are similar or different in their physiochemical and catalytic properties. In this paper, we have synthesized several forms of CNPs using identical precursors through a wet chemical process but using different oxidizer/reducer H2O2 (CNP1), NH4OH (CNP2) or hexamethylenetetramine (HMT-CNP1). Physiochemical properties of these CeO2 NPs were extensively studied and found to be different depending on the preparation methods. Unlike CNP1 and CNP2, HMT-CNP1 were readily taken into endothelial cells and their aggregation can be visualized using light microscopy. Exposure to HMT-CNP1 also reduced cell viability (MTT) at a 10-fold lower concentration than CNP1 or CNP2. Surprisingly, exposure to HMT-CNP1 led to substantial decreases in the ATP levels. Mechanistic studies revealed that HMT-CNP1 exhibited substantial ATPase (phosphatase) activity. Though CNP2 also exhibits ATPase activity, CNP1 lacked ATPase activity. The difference in catalytic (ATPase) activity of different CeO2 NPs preparation may be due to differences in their morphology and oxygen extraction energy. These results suggest the combination of increased uptake and ATPase activity of HMT-CNP1 may underlie the biomechanism of the toxicity of this preparation of CNPs, and may suggest ATPase activity should be considered when synthesizing CNPs for use in biomedical applications. PMID:23668322

  12. Early Inflammatory Responses Following Cell Grafting in the CNS Trigger Activation of the Subventricular Zone: A Proposed Model of Sequential Cellular Events.

    PubMed

    Praet, Jelle; Santermans, Eva; Daans, Jasmijn; Le Blon, Debbie; Hoornaert, Chloé; Goossens, Herman; Hens, Niel; Van der Linden, Annemie; Berneman, Zwi; Ponsaerts, Peter

    2015-01-01

    While multiple rodent preclinical studies, and to a lesser extent human clinical trials, claim the feasibility, safety, and potential clinical benefit of cell grafting in the central nervous system (CNS), currently only little convincing knowledge exists regarding the actual fate of the grafted cells and their effect on the surrounding environment (or vice versa). Our preceding studies already indicated that only a minor fraction of the initially grafted cell population survives the grafting process, while the surviving cell population becomes invaded by highly activated microglia/macrophages and surrounded by reactive astrogliosis. In the current study, we further elaborate on early cellular and inflammatory events following syngeneic grafting of eGFP(+) mouse embryonic fibroblasts (mEFs) in the CNS of immunocompetent mice. Based on obtained quantitative histological data, we here propose a detailed mathematically derived working model that sequentially comprises hypoxia-induced apoptosis of grafted mEFs, neutrophil invasion, neoangiogenesis, microglia/macrophage recruitment, astrogliosis, and eventually survival of a limited number of grafted mEFs. Simultaneously, we observed that the cellular events following mEF grafting activates the subventricular zone neural stem and progenitor cell compartment. This proposed model therefore further contributes to our understanding of cell graft-induced cellular responses and will eventually allow for successful manipulation of this intervention.

  13. Control of ADAM17 activity by regulation of its cellular localisation

    PubMed Central

    Lorenzen, Inken; Lokau, Juliane; Korpys, Yvonne; Oldefest, Mirja; Flynn, Charlotte M.; Künzel, Ulrike; Garbers, Christoph; Freeman, Matthew; Grötzinger, Joachim; Düsterhöft, Stefan

    2016-01-01

    An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17. PMID:27731361

  14. Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

    PubMed

    Novak-Hofer, I; Amstutz, H P; Mäcke, H R; Schwarzbach, R; Zimmermann, K; Morgenthaler, J J; Schubiger, P A

    1995-01-01

    Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

  15. Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

    PubMed

    Novak-Hofer, I; Amstutz, H P; Mäcke, H R; Schwarzbach, R; Zimmermann, K; Morgenthaler, J J; Schubiger, P A

    1995-01-01

    Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite. PMID:7805039

  16. Impacts of silver nanoparticles on cellular and transcriptional activity of nitrogen-cycling bacteria.

    PubMed

    Yang, Yu; Wang, Jing; Xiu, Zongming; Alvarez, Pedro J J

    2013-07-01

    The widespread use of silver nanoparticles (AgNPs) raises the potential for environmental releases that could impact microbial ecosystem services. In the present study, the authors address how the AgNPs and Ag(+) that they release may impact nitrogen-cycling bacteria. The authors studied the cellular and transcriptional response of the denitrifier Pseudomonas stutzeri, the nitrogen fixer Azotobacter vinelandii, and the nitrifier Nitrosomonas europaea exposed to 35 nm (carbon-coated) AgNPs or to Ag(+) (added as AgNO3 ). Based on minimum inhibitory concentrations (MICs), Ag(+) was 20 times to 48 times more toxic to the tested strains than AgNPs (including Ag(+) released during exposure). Exposure to sublethal concentrations of AgNPs or Ag(+) (representing 10% of the respective MIC for AgNO3 ) resulted in no significant effect on the expression of the denitrifying genes narG, napB, nirH, and norB in P. stutzeri or the nitrogen-fixing genes nifD, nifH, vnfD, and anfD in A. vinelandii, whereas nitrifying genes (amoA1 and amoC2) in N. europaea were upregulated (2.1- to 3.3-fold). This stimulatory effect disappeared at higher silver concentrations (60% of the Ag(+) MIC), and toxicity was exerted at concentrations higher than 60% of the Ag(+) MIC. The MIC for N. europaea was 8 times to 24 times lower than for the other strains, indicating higher susceptibility to AgNPs. This was corroborated by the lower half-lethal concentration for N. europaea (87 µg/L) compared with P. stutzeri (124 µg/L) and A. vinelandii (>250 µg/L) when cells were exposed with Ag(+) for 24 h in 1 mM bicarbonate buffer. This suggests that ammonia oxidation would be the most vulnerable nitrogen-cycling process in wastewater treatment plants receiving AgNPs and in agricultural soils amended with biosolids that concentrate them. PMID:23554086

  17. Impacts of silver nanoparticles on cellular and transcriptional activity of nitrogen-cycling bacteria.

    PubMed

    Yang, Yu; Wang, Jing; Xiu, Zongming; Alvarez, Pedro J J

    2013-07-01

    The widespread use of silver nanoparticles (AgNPs) raises the potential for environmental releases that could impact microbial ecosystem services. In the present study, the authors address how the AgNPs and Ag(+) that they release may impact nitrogen-cycling bacteria. The authors studied the cellular and transcriptional response of the denitrifier Pseudomonas stutzeri, the nitrogen fixer Azotobacter vinelandii, and the nitrifier Nitrosomonas europaea exposed to 35 nm (carbon-coated) AgNPs or to Ag(+) (added as AgNO3 ). Based on minimum inhibitory concentrations (MICs), Ag(+) was 20 times to 48 times more toxic to the tested strains than AgNPs (including Ag(+) released during exposure). Exposure to sublethal concentrations of AgNPs or Ag(+) (representing 10% of the respective MIC for AgNO3 ) resulted in no significant effect on the expression of the denitrifying genes narG, napB, nirH, and norB in P. stutzeri or the nitrogen-fixing genes nifD, nifH, vnfD, and anfD in A. vinelandii, whereas nitrifying genes (amoA1 and amoC2) in N. europaea were upregulated (2.1- to 3.3-fold). This stimulatory effect disappeared at higher silver concentrations (60% of the Ag(+) MIC), and toxicity was exerted at concentrations higher than 60% of the Ag(+) MIC. The MIC for N. europaea was 8 times to 24 times lower than for the other strains, indicating higher susceptibility to AgNPs. This was corroborated by the lower half-lethal concentration for N. europaea (87 µg/L) compared with P. stutzeri (124 µg/L) and A. vinelandii (>250 µg/L) when cells were exposed with Ag(+) for 24 h in 1 mM bicarbonate buffer. This suggests that ammonia oxidation would be the most vulnerable nitrogen-cycling process in wastewater treatment plants receiving AgNPs and in agricultural soils amended with biosolids that concentrate them.

  18. Processed sweet corn has higher antioxidant activity.

    PubMed

    Dewanto, Veronica; Wu, Xianzhong; Liu, Rui Hai

    2002-08-14

    Processed fruits and vegetables have been long considered to have lower nutritional value than the fresh produce due to the loss of vitamin C during processing. Vitamin C in apples has been found to contribute <0.4% of total antioxidant activity, indicating most of the activity comes from the natural combination of phytochemicals. This suggests that processed fruits and vegetables may retain their antioxidant activity despite the loss of vitamin C. Here it is shown that thermal processing at 115 degrees C for 25 min significantly elevated the total antioxidant activity of sweet corn by 44% and increased phytochemical content such as ferulic acid by 550% and total phenolics by 54%, although 25% vitamin C loss was observed. Processed sweet corn has increased antioxidant activity equivalent to 210 mg of vitamin C/100 g of corn compared to the remaining 3.2 mg of vitamin C in the sample that contributed only 1.5% of its total antioxidant activity. These findings do not support the notion that processed fruits and vegetables have lower nutritional value than fresh produce. This information may have a significant impact on consumers' food selection by increasing their consumption of fruits and vegetables to reduce the risk of chronic diseases. PMID:12166989

  19. Identification of the molecular mechanisms in cellular processes that elicit a surface plasmon resonance (SPR) response using simultaneous surface plasmon-enhanced fluorescence (SPEF) microscopy.

    PubMed

    Chabot, Vincent; Miron, Yannick; Charette, Paul G; Grandbois, Michel

    2013-12-15

    Surface plasmon resonance (SPR) has developed into a powerful approach for label-free monitoring of cellular behavior. Most cellular responses, however, involve a complex cascade of molecular events which makes identifying the specific components of cellular behavior dynamics contributing to the aggregate SPR signal problematic. Recently, a number of groups have used surface plasmon-enhanced fluorescence (SPEF) microscopy on living cells. In this work, we show that SPEF microscopy can be used to identify the molecular mechanisms responsible for SPR detection of cellular processes. By specifically labeling the actin cytoskeleton in human epithelial kidney cells (HEK 293) and rat vascular smooth muscle cells (A7r5), we correlate cell reorganization observed in SPEF with SPR signal variations reflecting aggregate cellular changes. HEK 293 cells stimulated with angiotensin-II exhibited transient contraction, appearing as an SPR signal decrease with a subsequent increase above the initial baseline. SPEF micrographs showed a decrease in cellular area followed by actin densification and cell spreading. A7r5 stimulated with Latrunculin A showed actin cytoskeleton depolymerization, generating a steady SPR signal decrease, with SPEF micrographs showing extensive collapse of cell actin structures. We observed that SPR monitoring of cellular response is strongly dependent on minute variations in cellular footprint on the substrate as well as changes in the molecular density in the basal portions of the cells. Therefore, combining SPR with imaging of selective fluorescent markers by SPEF allows a more comprehensive deconvolution of the cellular signal in relation to molecular events within the cells.

  20. Ancient cellular structures and modern humans: change of survival strategies before prolonged low solar activity period

    NASA Astrophysics Data System (ADS)

    Ragulskaya, Mariya; Rudenchik, Evgeniy; Gromozova, Elena; Voychuk, Sergei; Kachur, Tatiana

    The study of biotropic effects of modern space weather carries the information about the rhythms and features of adaptation of early biological systems to the outer space influence. The influence of cosmic rays, ultraviolet waves and geomagnetic field on early life has its signs in modern biosphere processes. These phenomena could be experimentally studied on present-day biological objects. Particularly inorganic polyphosphates, so-called "fossil molecules", attracts special attention as the most ancient molecules which arose in inanimate nature and have been accompanying biological objects at all stages of evolution. Polyphosphates-containing graves of yeast's cells of Saccharomyces cerevisiae strain Y-517, , from the Ukrainian Collection of Microorganisms was studied by daily measurements during 2000-2013 years. The IZMIRAN daily data base of physiological parameters dynamics during 2000-2013 years were analyzed simultaneously (25 people). The analysis showed significant simultaneous changes of the statistical parameters of the studied biological systems in 2004 -2006. The similarity of simultaneous changes of adaptation strategies of human organism and the cell structures of Saccharomyces cerevisiae during the 23-24 cycles of solar activity are discussed. This phenomenon could be due to a replacement of bio-effective parameters of space weather during the change from 23rd to 24th solar activity cycle and nonstandard geophysical peculiarities of the 24th solar activity cycle. It could be suggested that the observed similarity arose as the optimization of evolution selection of the living systems in expectation of probable prolonged period of low solar activity (4-6 cycles of solar activity).

  1. The pathogenic activation of calpain: a marker and mediator of cellular toxicity and disease states

    PubMed Central

    Vanderklish, Peter W; Bahr, Ben A

    2000-01-01

    Over-activation of calpain, a ubiquitous calcium-sensitive protease, has been linked to a variety of degenerative conditions in the brain and several other tissues. Dozens of substrates for calpain have been identified and several of these have been used to measure activation of the protease in the context of experimentally induced and naturally occurring pathologies. Calpain-mediated cleavage of the cytoskeletal protein spectrin, in particular, results in a set of large breakdown products (BDPs) that are unique in that they are unusually stable. Over the last 15 years, measurements of BDPs in experimental models of stroke-type excitotoxicity, hypoxia/ischemia, vasospasm, epilepsy, toxin exposure, brain injury, kidney malfunction, and genetic defects, have established that calpain activation is an early and causal event in the degeneration that ensues from acute, definable insults. The BDPs also have been found to increase with normal ageing and in patients with Alzheimer's disease, and the calpain activity may be involved in related apoptotic processes in conjunction with the caspase family of proteases. Thus, it has become increasingly clear that regardless of the mode of disturbance in calcium homeostasis or the cell type involved, calpain is critical to the development of pathology and therefore a distinct and powerful therapeutic target. The recent development of antibodies that recognize the site at which spectrin is cleaved has greatly facilitated the temporal and spatial resolution of calpain activation in situ. Accordingly, sensitive spectrin breakdown assays now are utilized to identify potential toxic side-effects of compounds and to develop calpain inhibitors for a wide range of indications including stroke, cerebral vasospasm, and kidney failure. PMID:11168679

  2. Investigation of mechanical properties for open cellular structure CoCrMo alloy fabricated by selective laser melting process

    NASA Astrophysics Data System (ADS)

    Azidin, A.; Taib, Z. A. M.; Harun, W. S. W.; Che Ghani, S. A.; Faisae, M. F.; Omar, M. A.; Ramli, H.

    2015-12-01

    Orthodontic implants have been a major focus through mechanical and biological performance in advance to fabricate shape of complex anatomical. Designing the part with a complex mechanism is one of the challenging process and addition to achieve the balance and desired mechanical performance brought to the right manufacture technique to fabricate. Metal additive manufacturing (MAM) is brought forward to the newest fabrication technology in this field. In this study, selective laser melting (SLM) process was utilized on a medical grade cobalt-chrome molybdenum (CoCrMo) alloy. The work has focused on mechanical properties of the CoCrMo open cellular structures samples with 60%, 70%, and 80% designed volume porosity that could potentially emulate the properties of human bone. It was observed that hardness values decreased as the soaking time increases except for bottom face. For compression test, 60% designed volume porosity demonstrated highest ultimate compressive strength compared to 70% and 80%.

  3. A cardiac electrical activity model based on a cellular automata system in comparison with neural network model.

    PubMed

    Khan, Muhammad Sadiq Ali; Yousuf, Sidrah

    2016-03-01

    Cardiac Electrical Activity is commonly distributed into three dimensions of Cardiac Tissue (Myocardium) and evolves with duration of time. The indicator of heart diseases can occur randomly at any time of a day. Heart rate, conduction and each electrical activity during cardiac cycle should be monitor non-invasively for the assessment of "Action Potential" (regular) and "Arrhythmia" (irregular) rhythms. Many heart diseases can easily be examined through Automata model like Cellular Automata concepts. This paper deals with the different states of cardiac rhythms using cellular automata with the comparison of neural network also provides fast and highly effective stimulation for the contraction of cardiac muscles on the Atria in the result of genesis of electrical spark or wave. The specific formulated model named as "States of automaton Proposed Model for CEA (Cardiac Electrical Activity)" by using Cellular Automata Methodology is commonly shows the three states of cardiac tissues conduction phenomena (i) Resting (Relax and Excitable state), (ii) ARP (Excited but Absolutely refractory Phase i.e. Excited but not able to excite neighboring cells) (iii) RRP (Excited but Relatively Refractory Phase i.e. Excited and able to excite neighboring cells). The result indicates most efficient modeling with few burden of computation and it is Action Potential during the pumping of blood in cardiac cycle.

  4. A cardiac electrical activity model based on a cellular automata system in comparison with neural network model.

    PubMed

    Khan, Muhammad Sadiq Ali; Yousuf, Sidrah

    2016-03-01

    Cardiac Electrical Activity is commonly distributed into three dimensions of Cardiac Tissue (Myocardium) and evolves with duration of time. The indicator of heart diseases can occur randomly at any time of a day. Heart rate, conduction and each electrical activity during cardiac cycle should be monitor non-invasively for the assessment of "Action Potential" (regular) and "Arrhythmia" (irregular) rhythms. Many heart diseases can easily be examined through Automata model like Cellular Automata concepts. This paper deals with the different states of cardiac rhythms using cellular automata with the comparison of neural network also provides fast and highly effective stimulation for the contraction of cardiac muscles on the Atria in the result of genesis of electrical spark or wave. The specific formulated model named as "States of automaton Proposed Model for CEA (Cardiac Electrical Activity)" by using Cellular Automata Methodology is commonly shows the three states of cardiac tissues conduction phenomena (i) Resting (Relax and Excitable state), (ii) ARP (Excited but Absolutely refractory Phase i.e. Excited but not able to excite neighboring cells) (iii) RRP (Excited but Relatively Refractory Phase i.e. Excited and able to excite neighboring cells). The result indicates most efficient modeling with few burden of computation and it is Action Potential during the pumping of blood in cardiac cycle. PMID:27087101

  5. An emerging molecular and cellular framework for memory processing by the hippocampus.

    PubMed

    Wittenberg, Gayle M; Tsien, Joe Z

    2002-10-01

    The hippocampus plays a central role in memory consolidation, a process for converting short-term memory into cortically stored, long-lasting memory in the mammalian brain. Here, we review recent data and discuss the 'synaptic re-entry reinforcement' (SRR) hypothesis, which can account for the role of the hippocampus in memory consolidation at both the molecular and systems levels. The central idea of the SRR hypothesis is that reactivation of neural ensembles in the hippocampus during the consolidation period results in multiple rounds of NMDA-receptor-dependent synaptic reinforcement of the hippocampal memory traces created during initial learning. In addition, such reactivation and reinforcement processes permit the hippocampus to act as a 'coincidence regenerator', providing coordinated input that drives the coherent reactivation of cortical neurons, resulting in the progressive strengthening of cortical memory traces through reactivation of cortical NMDA receptors. PMID:12220877

  6. Discovery of plant extracts that greatly delay yeast chronological aging and have different effects on longevity-defining cellular processes.

    PubMed

    Lutchman, Vicky; Medkour, Younes; Samson, Eugenie; Arlia-Ciommo, Anthony; Dakik, Pamela; Cortes, Berly; Feldman, Rachel; Mohtashami, Sadaf; McAuley, Mélissa; Chancharoen, Marisa; Rukundo, Belise; Simard, Éric; Titorenko, Vladimir I

    2016-03-29

    We discovered six plant extracts that increase yeast chronological lifespan to a significantly greater extent than any of the presently known longevity-extending chemical compounds. One of these extracts is the most potent longevity-extending pharmacological intervention yet described. We show that each of the six plant extracts is a geroprotector which delays the onset and decreases the rate of yeast chronological aging by eliciting a hormetic stress response. We also show that each of these extracts has different effects on cellular processes that define longevity in organisms across phyla. These effects include the following: 1) increased mitochondrial respiration and membrane potential; 2) augmented or reduced concentrations of reactive oxygen species; 3) decreased oxidative damage to cellular proteins, membrane lipids, and mitochondrial and nuclear genomes; 4) enhanced cell resistance to oxidative and thermal stresses; and 5) accelerated degradation of neutral lipids deposited in lipid droplets. Our findings provide new insights into mechanisms through which chemicals extracted from certain plants can slow biological aging. PMID:26918729

  7. Discovery of plant extracts that greatly delay yeast chronological aging and have different effects on longevity-defining cellular processes

    PubMed Central

    Samson, Eugenie; Arlia-Ciommo, Anthony; Dakik, Pamela; Cortes, Berly; Feldman, Rachel; Mohtashami, Sadaf; McAuley, Mélissa; Chancharoen, Marisa; Rukundo, Belise; Simard, Éric; Titorenko, Vladimir I.

    2016-01-01

    We discovered six plant extracts that increase yeast chronological lifespan to a significantly greater extent than any of the presently known longevity-extending chemical compounds. One of these extracts is the most potent longevity-extending pharmacological intervention yet described. We show that each of the six plant extracts is a geroprotector which delays the onset and decreases the rate of yeast chronological aging by eliciting a hormetic stress response. We also show that each of these extracts has different effects on cellular processes that define longevity in organisms across phyla. These effects include the following: 1) increased mitochondrial respiration and membrane potential; 2) augmented or reduced concentrations of reactive oxygen species; 3) decreased oxidative damage to cellular proteins, membrane lipids, and mitochondrial and nuclear genomes; 4) enhanced cell resistance to oxidative and thermal stresses; and 5) accelerated degradation of neutral lipids deposited in lipid droplets. Our findings provide new insights into mechanisms through which chemicals extracted from certain plants can slow biological aging. PMID:26918729

  8. Increased cellular activity in rat insular cortex after water and salt ingestion induced by fluid depletion.

    PubMed

    Pastuskovas, Cinthia V; Cassell, Martin D; Johnson, Alan Kim; Thunhorst, Robert L

    2003-04-01

    Insular cortex (IC) receives inputs from multiple sensory systems, including taste, and from receptors that monitor body electrolyte and fluid balance and blood pressure. This work analyzed metabolic activity of IC cells after water and sodium ingestion induced by sodium depletion. Rats were injected with the diuretic furosemide (10 mg/kg body wt), followed 5 min later by injections of the angiotensin-converting enzyme inhibitor captopril (5 mg/kg body wt). After 90 min, some rats received water and 0.3 M NaCl to drink for 2 h while others did not. A third group had access to water and saline but was not depleted of fluids. All rats were killed for processing of brain tissue for Fos-immunoreactivity (Fos-ir). Nondepleted animals had weak-to-moderate levels of Fos-ir within subregions of IC. Fluid-depleted rats without fluid access had significantly increased Fos-ir in all areas of IC. Levels of Fos-ir were highest in fluid-depleted rats that drank water and sodium. Fos-ir levels were highest in anterior regions of IC and lowest in posterior regions of IC. These results implicate visceral, taste, and/or postingestional factors in the increased metabolic activity of cells in IC.

  9. Activation of cellular oncogenes by chemical carcinogens in Syrian hamster embryo fibroblasts

    SciTech Connect

    Ebert, R.; Reiss, E.; Roellich, G.; Schiffmann, D. ); Barrett, J.C.; Wiseman, R.W. ); Pechan, R.

    1990-08-01

    Carcinogen-induced point mutations resulting in activation of ras oncogenes have been demonstrated in various experimental systems such as skin carcinogenesis, mammary, and liver carcinogenesis. In many cases, the data support the conclusion that these point mutations are critical changes in the initiation of these tumors. The Syrian hamster embryo (SHE) cell transformation model system has been widely used to study the multistep process of chemically induced neoplastic transformation. Recent data suggest that activation of the Ha-ras gene via point mutation is one of the crucial events in the transformation of these cells. The authors have now cloned the c-Ha-ras proto-oncogene from SHE cDNA-libraries, and we have performed polymerase chain reaction and direct sequencing to analyze tumor cell lines induced by different chemical carcinogens for the presence of point mutations. No changes were detectable at codons 12, 13, 59, 61, and 117 or adjacent regions in tumor cell lines induced by diethylstilbestrol, asbestos, benzo(a)pyrene, trenbolone, or aflatoxin B{sub 1}. Thus, it is not known whether point mutations in the Ha-ras proto-oncogene are essential for the acquisition of the neoplastic phenotype of SHE cells. Activation of other oncogenes or inactivation of tumor suppressor genes may be responsible for the neoplastic progression of these cells. However, in SHE cells neoplastically transformed by diethylstilbestrol or trenbolone, a significant elevation of the c-Ha-ras expression was observed. Enhanced expression of c-myc was detected in SHE cells transformed by benzo(a)pyrene or trenbolone.

  10. Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

    SciTech Connect

    Lee, Jun Han; Kim, Sung-Hak; Pascua, Philippe Noriel Q.; Song, Min-Suk; Baek, Yun Hee; Jin, Xun; Choi, Joong-Kook; Kim, Chul-Joong; Kim, Hyunggee; Choi, Young Ki

    2010-02-05

    To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.

  11. Morphology of Filamentous Fungi: Linking Cellular Biology to Process Engineering Using Aspergillus niger

    NASA Astrophysics Data System (ADS)

    Krull, Rainer; Cordes, Christiana; Horn, Harald; Kampen, Ingo; Kwade, Arno; Neu, Thomas R.; Nörtemann, Bernd

    In various biotechnological processes, filamentous fungi, e.g. Aspergillus niger, are widely applied for the production of high value-added products due to their secretion efficiency. There is, however, a tangled relationship between the morphology of these microorganisms, the transport phenomena and the related productivity. The morphological characteristics vary between freely dispersed mycelia and distinct pellets of aggregated biomass. Hence, advantages and disadvantages for mycel or pellet cultivation have to be balanced out carefully. Due to this inadequate understanding of morphogenesis of filamentous microorganisms, fungal morphology, along with reproducibility of inocula of the same quality, is often a bottleneck of productivity in industrial production. To obtain an optimisation of the production process it is of great importance to gain a better understanding of the molecular and cell biology of these microorganisms as well as the approaches in biochemical engineering and particle technique, in particular to characterise the interactions between the growth conditions, cell morphology, spore-hyphae-interactions and product formation. Advances in particle and image analysis techniques as well as micromechanical devices and their applications to fungal cultivations have made available quantitative morphological data on filamentous cells. This chapter provides the ambitious aspects of this line of action, focussing on the control and characterisation of the morphology, the transport gradients and the approaches to understand the metabolism of filamentous fungi. Based on these data, bottlenecks in the morphogenesis of A. niger within the complex production pathways from gene to product should be identified and this may improve the production yield.

  12. Sampangine (a Copyrine Alkaloid) Exerts Biological Activities through Cellular Redox Cycling of Its Quinone and Semiquinone Intermediates.

    PubMed

    Mahdi, Fakhri; Morgan, J Brian; Liu, Wenlong; Agarwal, Ameeta K; Jekabsons, Mika B; Liu, Yang; Zhou, Yu-Dong; Nagle, Dale G

    2015-12-24

    The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity. PMID:26637046

  13. A cellular automata model for social-learning processes in a classroom context

    NASA Astrophysics Data System (ADS)

    Bordogna, C. M.; Albano, E. V.

    2002-02-01

    A model for teaching-learning processes that take place in the classroom is proposed and simulated numerically. Recent ideas taken from the fields of sociology, educational psychology, statistical physics and computational science are key ingredients of the model. Results of simulations are consistent with well-established empirical results obtained in classrooms by means of different evaluation tools. It is shown that students engaged in collaborative groupwork reach higher achievements than those attending traditional lectures only. However, in many cases, this difference is subtle and consequently very difficult to be detected using tests. The influence of the number of students forming the collaborative groups on the average knowledge achieved is also studied and discussed.

  14. Cellular Aspects of Shigella Pathogenesis: Focus on the Manipulation of Host Cell Processes

    PubMed Central

    Killackey, Samuel A.; Sorbara, Matthew T.; Girardin, Stephen E.

    2016-01-01

    Shigella is a Gram-negative bacterium that is responsible for shigellosis. Over the years, the study of Shigella has provided a greater understanding of how the host responds to bacterial infection, and how bacteria have evolved to effectively counter the host defenses. In this review, we provide an update on some of the most recent advances in our understanding of pivotal processes associated with Shigella infection, including the invasion into host cells, the metabolic changes that occur within the bacterium and the infected cell, cell-to-cell spread mechanisms, autophagy and membrane trafficking, inflammatory signaling and cell death. This recent progress sheds a new light into the mechanisms underlying Shigella pathogenesis, and also more generally provides deeper understanding of the complex interplay between host cells and bacterial pathogens in general. PMID:27066460

  15. Cellular mechanisms of deep brain stimulation: activity-dependent focal circuit reprogramming?

    PubMed Central

    Veerakumar, Avin; Berton, Olivier

    2015-01-01

    Deep brain stimulation (DBS) is a well-established treatment modality for movement disorders. As more behavioral disorders are becoming understood as specific disruptions in neural circuitry, the therapeutic realm of DBS is broadening to encompass a wider range of domains, including disorders of compulsion, affect, and memory, but current understanding of the cellular mechanisms of DBS remains limited. We review progress made during the last decade focusing in particular on how recent methods for targeted circuit manipulations, imaging and reconstruction are fostering preclinical and translational advances that improve our neurobiological understanding of DBS’s action in psychiatric disorders. PMID:26719852

  16. Cardiac hypertrophy, arrhythmogenicity and the new myocardial phenotype. II. The cellular adaptational process.

    PubMed

    Swynghedauw, B; Chevalier, B; Charlemagne, D; Mansier, P; Carré, F

    1997-07-01

    Ventricular fibrosis is not the only structural determinant of arrhythmias in left ventricular hypertrophy. In an experimental model of compensatory cardiac hypertrophy (CCH) the degree of cardiac hypertrophy is also independently linked to ventricular arrhythmias. Cardiac hypertrophy reflects the level of adaptation, and matches the adaptational modifications of the myocardial phenotype. We suggest that these modifications have detrimental aspects. The increased action potential (AP) and QT duration and the prolonged calcium transient both favour spontaneous calcium oscillations, and both are potentially arrhythmogenic and linked to phenotypic changes in membrane proteins. To date, only two ionic currents have been studied in detail: Ito is depressed (likely the main determinant in AP durations), and If, the pacemaker current, is induced in the overloaded ventricular myocytes. In rat CCH, the two components of the sarcoplasmic reticulum, namely Ca(2+)-ATPase and ryanodine receptors, are down-regulated in parallel. Nevertheless, while the inward calcium current is unchanged, the functionally linked duo composed of the Na+/Ca2+ exchanged and (Na+, K+)-ATPase, is less active. Such an imbalance may explain the prolonged calcium transient. The changes in heart rate variability provide information about the state of the autonomic nervous system and has prognostic value even in CCH. Transgenic studies have demonstrated that the myocardial adrenergic and muscarinic receptor content is also a determining factor. During CCH, several phenotypic membrane changes participate in the slowing of contraction velocity and are thus adaptational. They also have a detrimental counterpart and, together with fibrosis, favour arrhythmias. PMID:9302342

  17. Processing and characterization of multi-cellular monolithic bioceramics for bone regenerative scaffolds

    NASA Astrophysics Data System (ADS)

    Ari-Wahjoedi, Bambang; Ginta, Turnad Lenggo; Parman, Setyamartana; Abustaman, Mohd Zikri Ahmad

    2014-10-01

    Multicellular monolithic ceramic body is a ceramic material which has many gas or liquid passages partitioned by thin walls throughout the bulk material. There are many currently known advanced industrial applications of multicellular ceramics structures i.e. as supports for various catalysts, electrode support structure for solid oxide fuel cells, refractories, electric/electronic materials, aerospace vehicle re-entry heat shields and biomaterials for dental as well as orthopaedic implants by naming only a few. Multicellular ceramic bodies are usually made of ceramic phases such as mullite, cordierite, aluminum titanate or pure oxides such as silica, zirconia and alumina. What make alumina ceramics is excellent for the above functions are the intrinsic properties of alumina which are hard, wear resistant, excellent dielectric properties, resists strong acid and alkali attacks at elevated temperatures, good thermal conductivities, high strength and stiffness as well as biocompatible. In this work the processing technology leading to truly multicellular monolithic alumina ceramic bodies and their characterization are reported. Ceramic slip with 66 wt.% solid loading was found to be optimum as impregnant to the polyurethane foam template. Mullitic ceramic composite of alumina-sodium alumino disilicate-Leucite-like phases with bulk and true densities of 0.852 and 1.241 g cm-3 respectively, pore linear density of ±35 cm-1, linear and bulk volume shrinkages of 7-16% and 32 vol.% were obtained. The compressive strength and elastic modulus of the bioceramics are ≈0.5-1.0 and ≈20 MPa respectively.

  18. Processing and characterization of multi-cellular monolithic bioceramics for bone regenerative scaffolds

    SciTech Connect

    Ari-Wahjoedi, Bambang; Ginta, Turnad Lenggo; Parman, Setyamartana; Abustaman, Mohd Zikri Ahmad

    2014-10-24

    Multicellular monolithic ceramic body is a ceramic material which has many gas or liquid passages partitioned by thin walls throughout the bulk material. There are many currently known advanced industrial applications of multicellular ceramics structures i.e. as supports for various catalysts, electrode support structure for solid oxide fuel cells, refractories, electric/electronic materials, aerospace vehicle re-entry heat shields and biomaterials for dental as well as orthopaedic implants by naming only a few. Multicellular ceramic bodies are usually made of ceramic phases such as mullite, cordierite, aluminum titanate or pure oxides such as silica, zirconia and alumina. What make alumina ceramics is excellent for the above functions are the intrinsic properties of alumina which are hard, wear resistant, excellent dielectric properties, resists strong acid and alkali attacks at elevated temperatures, good thermal conductivities, high strength and stiffness as well as biocompatible. In this work the processing technology leading to truly multicellular monolithic alumina ceramic bodies and their characterization are reported. Ceramic slip with 66 wt.% solid loading was found to be optimum as impregnant to the polyurethane foam template. Mullitic ceramic composite of alumina-sodium alumino disilicate-Leucite-like phases with bulk and true densities of 0.852 and 1.241 g cm{sup −3} respectively, pore linear density of ±35 cm{sup −1}, linear and bulk volume shrinkages of 7-16% and 32 vol.% were obtained. The compressive strength and elastic modulus of the bioceramics are ≈0.5-1.0 and ≈20 MPa respectively.

  19. The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers

    NASA Astrophysics Data System (ADS)

    Lin, Jiang-Jen; Lin, Wen-Chun; Dong, Rui-Xuan; Hsu, Shan-hui

    2012-02-01

    Silver nanoparticles (AgNPs) are known for their excellent antibacterial activities. The possible toxicity, however, is a major concern for their applications. Three types of AgNPs were prepared in this study by chemical processes. Each was stabilized by a polymer surfactant, which was expected to reduce the exposure of cells to AgNPs and therefore their cytotoxicity. The polymer stabilizers included poly(oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol) (PVA). The cytotoxicity of these chemically produced AgNPs to mouse skin fibroblasts (L929), human hepatocarcinoma cells (HepG2), and mouse monocyte macrophages (J774A1) was compared to that of physically produced AgNPs and gold nanoparticles (AuNPs) as well as the standard reference material RM8011 AuNPs. Results showed that SMA-AgNPs were the least cytotoxic among all materials, but cytotoxicity was still observed at higher silver concentrations (>30 ppm). Macrophages demonstrated the inflammatory response with cell size increase and viability decrease upon exposure to 10 ppm of the chemically produced AgNPs. SMA-AgNPs did not induce hemolysis at a silver concentration below 1.5 ppm. Regarding the antibacterial activity, POEM-AgNPs and SMA-AgNPs at 1 ppm silver content showed 99.9% and 99.3% growth inhibition against E. coli, while PVA-AgNPs at the same silver concentration displayed 79.1% inhibition. Overall, SMA-AgNPs demonstrated better safety in vitro and greater antibacterial effects than POEM-AgNPs and PVA-AgNPs. This study suggested that polymer stabilizers may play an important role in determining the toxicity of AgNPs.

  20. Activation of HSPA1A promoter by environmental pollutants: An early and rapid response to cellular damage.

    PubMed

    Xin, Lili; Wang, Jianshu; Fan, Guoqiang; Wu, Yanhu; Guo, Sifan

    2015-05-01

    We established the HepG2-luciferase cells containing a luciferase reporter gene regulated by human HSPA1A promoter. The screening of heat shock and three typical environmental toxicants revealed differences in their capacities to activate HSPA1A promoter in HepG2-luciferase cells. After heat shock, a progressive time-dependent increase in relative luciferase activity was detected peaking at 8h of recovery. Benzo[a]pyrene, formaldehyde and sodium bisulfite induced significant time-dependent elevation of relative luciferase activity, which were positively correlated with MDA concentration, Olive tail moment and micronuclei frequency. The significant increase in relative luciferase activity was already evident after 4h of benzo[a]pyrene, 1h of formaldehyde and sodium bisulfite exposure, when no increases in cellular damage were detected by other toxicity tests. Therefore, the HepG2-luciferase cells are useful model for examining the overall cellular responses to oxidative stress and genotoxic damage, and provide a reporter system for rapid and sensitive screening of environmental pollutants. PMID:25863329

  1. Processing of Color Words Activates Color Representations

    ERIC Educational Resources Information Center

    Richter, Tobias; Zwaan, Rolf A.

    2009-01-01

    Two experiments were conducted to investigate whether color representations are routinely activated when color words are processed. Congruency effects of colors and color words were observed in both directions. Lexical decisions on color words were faster when preceding colors matched the color named by the word. Color-discrimination responses…

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

    PubMed

    Yao, Cui-Luan; Somero, George N

    2013-06-01

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

  3. Hemin activation of innate cellular response blocks human immunodeficiency virus type-1-induced osteoclastogenesis

    SciTech Connect

    Takeda, Kazuyo; Adhikari, Rewati; Yamada, Kenneth M.; Dhawan, Subhash

    2015-08-14

    The normal skeletal developmental and homeostatic process termed osteoclastogenesis is exacerbated in numerous pathological conditions and causes excess bone loss. In cancer and HIV-1-infected patients, this disruption of homeostasis results in osteopenia and eventual osteoporesis. Counteracting the factors responsible for these metabolic disorders remains a challenge for preventing or minimizing this co-morbidity associated with these diseases. In this report, we demonstrate that a hemin-induced host protection mechanism not only suppresses HIV-1 associated osteoclastogenesis, but it also exhibits anti-osteoclastogenic activity for non-infected cells. Since the mode of action of hemin is both physiological and pharmacological through induction of heme oxygenase-1 (HO-1), an endogenous host protective response to an FDA-licensed therapeutic used to treat another disease, our study suggests an approach to developing novel, safe and effective therapeutic strategies for treating bone disorders, because hemin administration in humans has previously met required FDA safety standards. - Highlights: • HIV-1 infection induced osteoclastogenesis in primary human macrophages. • Heme oxygenase-1 (HO-1) induction inhibited HIV-1-induced osteoclastogenesis in macrophages. • HO-1 induction suppressed RANKL-enhanced osteoclastogenesis in HIV-1-infected macrophages. • This inverse relationship between HO-1 and HIV-1 pathogenesis may define a novel host defense response against HIV-1 infection.

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

    SciTech Connect

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

    2015-04-15

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

  5. Measurement of whole body cellular and collagen nitrogen, potassium, and other elements by neutron activation and whole body counting

    SciTech Connect

    James, H.M.; Fabricius, P.J.; Dykes, P.W.

    1987-09-01

    Whole body nitrogen can be measured by neutron activation analysis with an acceptable radiation dose; it is an index of body protein which, in normal subjects, is 65% cellular protein and 35% extracellular connective collagen. Whole body potassium can be measured by whole body counting without irradiating the subject; it is an index of body cell mass. We measured whole body nitrogen, potassium, extracellular water, intracellular water, and fat-folds. The differences between 37 malnourished patients and five normal subjects suggested that the patients had 9 kg less cell mass than normal, but no difference in extracellular mass. Measurements were made on eight patients before and after 14 days of total parenteral nutrition; balance of nitrogen intake and excretion also was measured. The changes were consistent with mean increases of 3 kg of cellular mass and 3 kg of fat with no change of extracellular mass. The accuracy and sensitivity of the whole body measurements need further confirmation for use in patients with changing body composition. Where tissue wasting is largely from the cellular compartment, potassium could be a more sensitive index of wasting than nitrogen. Multielement analysis of nitrogen, potassium, chlorine, and carbon will probably be valuable in elucidating body composition in malnutrition.

  6. Esterification of Ginsenoside Rh2 Enhanced Its Cellular Uptake and Antitumor Activity in Human HepG2 Cells.

    PubMed

    Chen, Fang; Deng, Ze-Yuan; Zhang, Bing; Xiong, Zeng-Xing; Zheng, Shi-Lian; Tan, Chao-Li; Hu, Jiang-Ning

    2016-01-13

    Our previous research had indicated that the octyl ester derivative of ginsenoside Rh2 (Rh2-O) might have a higher bioavailability than Rh2 in the Caco-2 cell line. The aim of this study was to investigate the cellular uptake and antitumor effects of Rh2-O in human HepG2 cells as well as its underlying mechanism compared with Rh2. Results showed that Rh2-O exhibited a higher cellular uptake (63.24%) than Rh2 (36.76%) when incubated with HepG2 cells for 24 h. Rh2-O possessed a dose- and time-dependent inhibitory effect against the proliferation of HepG2 cells. The IC50 value of Rh2-O for inhibition of HepG2 cell proliferation was 20.15 μM, which was roughly half the value of Rh2. Rh2-O induced apoptosis of HepG2 cells through a mitochondrial-mediated intrinsic pathway. In addition, the accumulation of ROS was detected in Rh2-O-treated HepG2 cells, which participated in the apoptosis of HepG2 cells. Conclusively, the findings above all suggested that Rh2-O as well as Rh2 inducing HepG2 cells apoptosis might involve similar mechanisms; however, Rh2-O had better antitumor activities than Rh2, probably due to its higher cellular uptake. PMID:26672619

  7. Effect of GO-Fe3O4 and rotating magnetic field on cellular metabolic activity of mammalian cells.

    PubMed

    Urbas, Karolina; Jedrzejczak-Silicka, Magdalena; Rakoczy, Rafal; Zaborski, Daniel; Mijowska, Ewa

    2016-04-01

    The effect of hybrid material-graphene flakes with Fe3O4 nanospheres (GO-Fe3O4), graphene oxide (GO) and magnetite nanospheres (Fe3O4) in rotating magnetic field on mammalian cells metabolism has been studied. Several reports shown that exposure to magnetic field may have influence on cellular membrane permeability. Thus, the aim of presented study was to determine the cellular response of L929 fibroblast cells to nanomaterials and rotating magnetic field for 8-h exposure experiment. The GO had tendency to adsorb proteins, thus cell metabolism was decreased and the effect of that mechanism was enhanced by impact of nanospheres and rotating magnetic field. The highest reduction of cellular metabolism was recorded for WST-1 and NR assays at concentration 100 µg/mL of all tested nanomaterials and magnetic induction value 10.06 mT. The lactate dehydrogenase leakage assay has shown significant changes in membrane permeability. Further studies need to be carried out to precisely determine the mechanism of that process. PMID:26809700

  8. The activated sludge process: Fundamentals of operation

    SciTech Connect

    Junkins, R.; Deeny, K.J.; Eckhoff, T.H.

    1983-01-01

    The procedures given here - based on extensive and intensive experience. Background information on process mechanics is followed by detailed consideration of control and troubleshooting practices. Contents: PREFACE AND INTRODUCTION; PROCESS MECHANICS; Basic Mechanism of Activated Sludge Systems; Formation of Activated Sludge; Growth of Microorganisms; Classifications of Microorganisms: Type, Environment, Age; Solids Separation and Return; FACTORS AFFECTING OPERATION; Raw Wastewater Strength; Dissolved Oxygen; pH; Temperature; Nutrients; Toxicity; Mixing; Detention Time; Hydraulics; PROCESS MODIFICATIONS; Conventional; Complete Mix; Contact-Stabilization; Extended Aeration; Others; PROCESS MONITORING; Visual; Analytical Indicators; OPERATIONAL CONTROL; Sludge Volume Index; Sludge Age; Mean Cell Residence Time; Food/Microorganism Ratio; Organic Loading Rate; Solids Loading Rate; Clarifier Overflow Rate; Weir Overflow Rate; Sludge Recycle Rate, Sludge Wastage Rate; Chemical Feed Rate; TROUBLESHOOTING; Low BOD Removal; Low D.O. in Aeration Baisn; Poor Settling; PLANT START-UP; Introduction; Pre Start-up Checkup; Wastewater Analysis; Seed Screening; Process Checklist; Mechanical Checklist; Familiarization and Training; Start-up; Seeding; Process Monitoring; Transition; Typical Start-up Problems; Foaming; Settling Problems; Low BOD Removal; INDEX.

  9. Speech perception as an active cognitive process

    PubMed Central

    Heald, Shannon L. M.; Nusbaum, Howard C.

    2014-01-01

    One view of speech perception is that acoustic signals are transformed into representations for pattern matching to determine linguistic structure. This process can be taken as a statistical pattern-matching problem, assuming realtively stable linguistic categories are characterized by neural representations related to auditory properties of speech that can be compared to speech input. This kind of pattern matching can be termed a passive process which implies rigidity of processing with few demands on cognitive processing. An alternative view is that speech recognition, even in early stages, is an active process in which speech analysis is attentionally guided. Note that this does not mean consciously guided but that information-contingent changes in early auditory encoding can occur as a function of context and experience. Active processing assumes that attention, plasticity, and listening goals are important in considering how listeners cope with adverse circumstances that impair hearing by masking noise in the environment or hearing loss. Although theories of speech perception have begun to incorporate some active processing, they seldom treat early speech encoding as plastic and attentionally guided. Recent research has suggested that speech perception is the product of both feedforward and feedback interactions between a number of brain regions that include descending projections perhaps as far downstream as the cochlea. It is important to understand how the ambiguity of the speech signal and constraints of context dynamically determine cognitive resources recruited during perception including focused attention, learning, and working memory. Theories of speech perception need to go beyond the current corticocentric approach in order to account for the intrinsic dynamics of the auditory encoding of speech. In doing so, this may provide new insights into ways in which hearing disorders and loss may be treated either through augementation or therapy. PMID

  10. Multifunctional Hyaluronic Acid and Chondroitin Sulfate Nanoparticles: Impact of Glycosaminoglycan Presentation on Receptor Mediated Cellular Uptake and Immune Activation.

    PubMed

    Oommen, Oommen P; Duehrkop, Claudia; Nilsson, Bo; Hilborn, Jöns; Varghese, Oommen P

    2016-08-17

    Hyaluronic acid (HA) and chondroitin sulfate (CS) polymers are extensively used for various biomedical applications, such as for tissue engineering, drug delivery, and gene delivery. Although both these biopolymers are known to target cell surface CD44 receptors, their relative cellular targeting properties and immune activation potential have never been evaluated. In this article, we present the synthesis and characterization of novel self-assembled supramolecular HA and CS nanoparticles (NPs). These NPs were developed using fluorescein as a hydrophobic component that induced amphiphilicity in biopolymers and also efficiently stabilized anticancer drug doxorubicin (DOX) promoting a near zero-order drug release. The cellular uptake and cytotoxicity studies of these NPs in different human cancer lines, namely, human colorectal carcinoma cell line HCT116 and human breast cancer cell line MCF-7 demonstrated dose dependent cytotoxicity. Interestingly, both NPs showed CD44 dependent cellular uptake with the CS-DOX NP displaying higher dose-dependent cytotoxicity than the HA-DOX NP in different mammalian cells tested. Immunological evaluation of these nanocarriers in an ex vivo human whole blood model revealed that unlike unmodified polymers, the HA NP and CS NP surprisingly showed platelet aggregation and thrombin-antithrombin complex formation at high concentrations (0.8 mg/mL). We also observed a clear difference in early- and late-stage complement activation (C3a and sC5b-9) with CS and CS NP triggering significant complement activation at high concentrations (0.08-0.8 mg/mL), unlike HA and HA NP. These results offer new insight into designing glycosaminoglycan-based NPs and understanding their hematological responses and targeting ability. PMID:27468113

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

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

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

  12. Imaging large-scale neural activity with cellular resolution in awake, mobile mice.

    PubMed

    Dombeck, Daniel A; Khabbaz, Anton N; Collman, Forrest; Adelman, Thomas L; Tank, David W

    2007-10-01

    We report a technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice with minimal motion artifact. The apparatus combines an upright, table-mounted two-photon microscope with a spherical treadmill consisting of a large, air-supported Styrofoam ball. Mice, with implanted cranial windows, are head restrained under the objective while their limbs rest on the ball's upper surface. Following adaptation to head restraint, mice maneuver on the spherical treadmill as their heads remain motionless. Image sequences demonstrate that running-associated brain motion is limited to approximately 2-5 microm. In addition, motion is predominantly in the focal plane, with little out-of-plane motion, making the application of a custom-designed Hidden-Markov-Model-based motion correction algorithm useful for postprocessing. Behaviorally correlated calcium transients from large neuronal and astrocytic populations were routinely measured, with an estimated motion-induced false positive error rate of <5%.

  13. Oxidation of DNA bases by tumor promoter-activated processes.

    PubMed Central

    Frenkel, K

    1989-01-01

    Evidence has accumulated showing that active oxygen species participate in at least one stage of tumor promotion. Tumor promoters can induce various types of cells to undergo processes that result in formation of active oxygen species. They stimulate polymorphonuclear leukocytes (PMNs) to undergo an oxidative burst that is characterized by rapid formation of .O2- and H2O2. We find that in vitro formation of H2O2 by tumor promoter-activated PMNs correlates with their in vivo first-stage promoting activity. Moreover, two thymidine derivatives are formed in DNA coincubated with tumor promoter-stimulated PMNs: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG). The amounts of HMdU and dTG formed correlate with the first-stage tumor-promoting potencies of the agents used for PMN stimulation and with the amount of H2O2 generated. We find that HMdU is also formed in the DNA of HeLa cells coincubated with 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated PMNs, with the amount of HMdU being proportional to that of TPA used. Even in the absence of PMNs, HMdU is increasingly formed in cellular DNA with increased TPA concentration, although at much lower levels than in the presence of PMNs. When rat liver microsomes are incubated with benzo[a]pyrene (BaP), a complete carcinogen, H2O2 is also generated. Production of H2O2 increases linearly with increasing concentrations of BaP. Furthermore, HMdU is formed in DNA exposed to BaP-treated microsomes, and its formation is inhibited by catalase. These results suggest that carcinogen-induced processes generating H2O2 are associated with the first-stage promoting activity of complete carcinogens. PMID:2667984

  14. PLAGL2 translocation and SP-C promoter activity-A cellular response of lung cells to hypoxia

    SciTech Connect

    Guo, Yuhong; Yang, Meng-Chun; Weissler, Jonathan C.; Yang, Yih-Sheng . E-mail: Yih-Sheng.Yang@UTSouthwestern.edu

    2007-08-31

    Cobalt is a transition metal which can substitute for iron in the oxygen-sensitive protein and mimic hypoxia. Cobalt was known to be associated with the development of lung disease. In this study, when lung cells were exposed to hypoxia-induced by CoCl{sub 2} at a sub-lethal concentration (100 {mu}M), their thyroid transcription factor-1 (TTF-1) expression was greatly reduced. Under this condition, SP-B promoter activity was down-regulated, but SP-C promoter remained active. Therefore, we hypothesized that other factor(s) besides TTF-1 might contribute to the modulation of SP-C promoter in hypoxic lung cells. Pleomorphic adenoma gene like-2 (PLAGL2), a previously identified TTF-1-independent activator of the SP-C promoter, was not down-regulated, nor increased, within those cells. Its cellular location was redistributed from the cytoplasm to the nucleus. Chromatin immunoprecipitation (ChIP) and quantitative RT-PCR analyses demonstrated that nuclear PLAGL2 occupied and transactivated the endogenous SP-C promoter in lung cells. Thereby, through relocating and accumulating of PLAGL2 inside the nucleus, PLAGL2 interacted with its target genes for various cellular functions. These results further suggest that PLAGL2 is an oxidative stress responding regulator in lung cells.

  15. Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

    PubMed Central

    Hirner, Heidrun; Günes, Cagatay; Bischof, Joachim; Wolff, Sonja; Grothey, Arnhild; Kühl, Marion; Oswald, Franz; Wegwitz, Florian; Bösl, Michael R.; Trauzold, Anna; Henne-Bruns, Doris; Peifer, Christian; Leithäuser, Frank; Deppert, Wolfgang; Knippschild, Uwe

    2012-01-01

    Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data

  16. The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

    PubMed Central

    2012-01-01

    Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important

  17. Process for preparing active oxide powders

    DOEpatents

    Berard, Michael F.; Hunter, Jr., Orville; Shiers, Loren E.; Dole, Stephen L.; Scheidecker, Ralph W.

    1979-02-20

    An improved process for preparing active oxide powders in which cation hydroxide gels, prepared in the conventional manner are chemically dried by alternately washing the gels with a liquid organic compound having polar characteristics and a liquid organic compound having nonpolar characteristics until the mechanical water is removed from the gel. The water-free cation hydroxide is then contacted with a final liquid organic wash to remove the previous organic wash and speed drying. The dried hydroxide treated in the conventional manner will form a highly sinterable active oxide powder.

  18. RhoD activated by fibroblast growth factor induces cytoneme-like cellular protrusions through mDia3C

    PubMed Central

    Koizumi, Kazuhisa; Takano, Kazunori; Kaneyasu, Akiko; Watanabe-Takano, Haruko; Tokuda, Emi; Abe, Tomoyuki; Watanabe, Naoki; Takenawa, Tadaomi; Endo, Takeshi

    2012-01-01

    The small GTPase RhoD regulates actin cytoskeleton to collapse actin stress fibers and focal adhesions, resulting in suppression of cell migration and cytokinesis. It also induces alignment of early endosomes along actin filaments and reduces their motility. We show here that a constitutively activated RhoD generated two types of actin-containing thin peripheral cellular protrusions distinct from Cdc42-induced filopodia. One was longer, almost straight, immotile, and sensitive to fixation, whereas the other was shorter, undulating, motile, and resistant to fixation. Moreover, cells expressing wild-type RhoD extended protrusions toward fibroblast growth factor (FGF) 2/4/8–coated beads. Stimulation of wild-type RhoD-expressing cells with these FGFs also caused formation of cellular protrusions. Nodules moved through the RhoD-induced longer protrusions, mainly toward the cell body. Exogenously expressed FGF receptor was associated with these moving nodules containing endosome-like vesicles. These results suggest that the protrusions are responsible for intercellular communication mediated by FGF and its receptor. Accordingly, the protrusions are morphologically and functionally equivalent to cytonemes. RhoD was activated by FGF2/4/8. Knockdown of RhoD interfered with FGF-induced protrusion formation. Activated RhoD specifically bound to mDia3C and facilitated actin polymerization together with mDia3C. mDia3C was localized to the tips or stems of the protrusions. In addition, constitutively activated mDia3C formed protrusions without RhoD or FGF stimulation. Knockdown of mDia3 obstructed RhoD-induced protrusion formation. These results imply that RhoD activated by FGF signaling forms cytoneme-like protrusions through activation of mDia3C, which induces actin filament formation. PMID:23034183

  19. Profile-QSAR: a novel meta-QSAR method that combines activities across the kinase family to accurately predict affinity, selectivity, and cellular activity.

    PubMed

    Martin, Eric; Mukherjee, Prasenjit; Sullivan, David; Jansen, Johanna

    2011-08-22

    Profile-QSAR is a novel 2D predictive model building method for kinases. This "meta-QSAR" method models the activity of each compound against a new kinase target as a linear combination of its predicted activities against a large panel of 92 previously studied kinases comprised from 115 assays. Profile-QSAR starts with a sparse incomplete kinase by compound (KxC) activity matrix, used to generate Bayesian QSAR models for the 92 "basis-set" kinases. These Bayesian QSARs generate a complete "synthetic" KxC activity matrix of predictions. These synthetic activities are used as "chemical descriptors" to train partial-least squares (PLS) models, from modest amounts of medium-throughput screening data, for predicting activity against new kinases. The Profile-QSAR predictions for the 92 kinases (115 assays) gave a median external R²(ext) = 0.59 on 25% held-out test sets. The method has proven accurate enough to predict pairwise kinase selectivities with a median correlation of R²(ext) = 0.61 for 958 kinase pairs with at least 600 common compounds. It has been further expanded by adding a "C(k)XC" cellular activity matrix to the KxC matrix to predict cellular activity for 42 kinase driven cellular assays with median R²(ext) = 0.58 for 24 target modulation assays and R²(ext) = 0.41 for 18 cell proliferation assays. The 2D Profile-QSAR, along with the 3D Surrogate AutoShim, are the foundations of an internally developed iterative medium-throughput screening (IMTS) methodology for virtual screening (VS) of compound archives as an alternative to experimental high-throughput screening (HTS). The method has been applied to 20 actual prospective kinase projects. Biological results have so far been obtained in eight of them. Q² values ranged from 0.3 to 0.7. Hit-rates at 10 uM for experimentally tested compounds varied from 25% to 80%, except in K5, which was a special case aimed specifically at finding "type II" binders, where none of the compounds were predicted to be

  20. The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

    PubMed Central

    Freitas, Fernanda Zanolli; Virgilio, Stela; Cupertino, Fernanda Barbosa; Kowbel, David John; Fioramonte, Mariana; Gozzo, Fabio Cesar; Glass, N. Louise; Bertolini, Maria Célia

    2016-01-01

    When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. PMID:26994287

  1. Chiral Ruthenium(II) Polypyridyl Complexes: Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Cellular Uptake

    PubMed Central

    Yu, Qianqian; Liu, Yanan; Wang, Chuan; Sun, Dongdong; Yang, Xingcheng; Liu, Yanyu; Liu, Jie

    2012-01-01

    Two ruthenium(II) complexes, Λ-[Ru(phen)2(p-HPIP)]2+ and Δ-[Ru(phen)2(p-HPIP)]2+, were synthesized and characterized via proton nuclear magnetic resonance spectroscopy, electrospray ionization-mass spectrometry, and circular dichroism spectroscopy. This study aims to clarify the anticancer effect of metal complexes as novel and potent telomerase inhibitors and cellular nucleus target drug. First, the chiral selectivity of the compounds and their ability to stabilize quadruplex DNA were studied via absorption and emission analyses, circular dichroism spectroscopy, fluorescence-resonance energy transfer melting assay, electrophoretic mobility shift assay, and polymerase chain reaction stop assay. The two chiral compounds selectively induced and stabilized the G-quadruplex of telomeric DNA with or without metal cations. These results provide new insights into the development of chiral anticancer agents for G-quadruplex DNA targeting. Telomerase repeat amplification protocol reveals the higher inhibitory activity of Λ-[Ru(phen)2(p-HPIP)]2+ against telomerase, suggesting that Λ-[Ru(phen)2(p-HPIP)]2+ may be a potential telomerase inhibitor for cancer chemotherapy. MTT assay results show that these chiral complexes have significant antitumor activities in HepG2 cells. More interestingly, cellular uptake and laser-scanning confocal microscopic studies reveal the efficient uptake of Λ-[Ru(phen)2(p-HPIP)]2+ by HepG2 cells. This complex then enters the cytoplasm and tends to accumulate in the nucleus. This nuclear penetration of the ruthenium complexes and their subsequent accumulation are associated with the chirality of the isomers as well as with the subtle environment of the ruthenium complexes. Therefore, the nucleus can be the cellular target of chiral ruthenium complexes for anticancer therapy. PMID:23236402

  2. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    PubMed Central

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  3. Enhanced multistatic active sonar signal processing.

    PubMed

    Zhao, Kexin; Liang, Junli; Karlsson, Johan; Li, Jian

    2013-07-01

    Multistatic active sonar systems involve the transmission and reception of multiple probing sequences and can achieve significantly enhanced performance of target detection and localization through exploiting spatial diversity. This paper mainly focuses on two signal processing aspects of such systems, namely, enhanced range-Doppler imaging and improved target parameter estimation. The main contributions of this paper are (1) a hybrid dense-sparse method is proposed to generate range-Doppler images with both low sidelobe levels and high accuracy; (2) a generalized K-Means clustering (GKC) method for target association is developed to associate the range measurements from different transmitter-receiver pairs, which is actually a range fitting procedure; (3) the extended invariance principle-based weighted least-squares method is developed for accurate target position and velocity estimation. The effectiveness of the proposed multistatic active sonar signal processing techniques is verified using numerical examples.

  4. Formin’ cellular structures

    PubMed Central

    Bogdan, Sven; Schultz, Jörg; Grosshans, Jörg

    2014-01-01

    Members of the Diaphanous (Dia) protein family are key regulators of fundamental actin driven cellular processes, which are conserved from yeast to humans. Researchers have uncovered diverse physiological roles in cell morphology, cell motility, cell polarity, and cell division, which are involved in shaping cells into tissues and organs. The identification of numerous binding partners led to substantial progress in our understanding of the differential functions of Dia proteins. Genetic approaches and new microscopy techniques allow important new insights into their localization, activity, and molecular principles of regulation. PMID:24719676

  5. Active voltammetric microsensors with neural signal processing.

    SciTech Connect

    Vogt, M. C.

    1998-12-11

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

  6. Assays to measure the activation of membrane tyrosine kinase receptors: focus on cellular methods.

    PubMed

    Minor, Lisa K

    2003-09-01

    Many methods have been explored as means to measure the activation and inhibition of tyrosine kinase receptors, in vitro using the isolated kinase domain, and in living cells. Kinase activity has been measured in enzyme assays using a peptide substrate, but with different detection systems. These include the radioactive FlashPlate assay, the fluorescent resonance energy transfer (FRET) assay, the dissociation-enhance lanthanide fluorescence immunoassay (DELFIA) and other formats. These methods have successfully identified inhibitors of receptor activity. Cell-based assays have recently emerged to measure receptor activation and inhibition. When membrane tyrosine kinase receptors become activated, they increase their state of phosphorylation. This phosphorylation may lead to an increase in tyrosine kinase-specific activity. Methods have been developed that take advantage of these properties. These include measuring the ligand-stimulated total tyrosine phosphorylation of the receptor using a DELFIA or an ELISA assay, measuring ligand-stimulated enzyme activation of the receptor by quantifying enzyme activity, and dimerization of the activated receptor using bioluminescence resonance energy transfer (BRET). Although cell-based assays are still in their infancy, these techniques may prove a valuable addition to the receptor screening strategy.

  7. Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells.

    PubMed

    Haage, Amanda; Schneider, Ian C

    2014-08-01

    The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

  8. Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity.

    PubMed

    He, Bing; Martin, Adam; Wieschaus, Eric

    2016-07-01

    Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows. PMID:27226317

  9. Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity

    PubMed Central

    Martin, Adam; Wieschaus, Eric

    2016-01-01

    Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows. PMID:27226317

  10. Modified Vaccinia virus Ankara: innate immune activation and induction of cellular signalling.

    PubMed

    Price, Philip J R; Torres-Domínguez, Lino E; Brandmüller, Christine; Sutter, Gerd; Lehmann, Michael H

    2013-09-01

    Attenuated poxviruses are currently under development as vaccine vectors against a number of diseases including, influenza, HIV, malaria and tuberculosis. Modified Vaccinia virus Ankara (MVA) is an attenuated, replication deficient vaccinia virus (VACV) strain which, similar to replication competent VACV, is highly immunogenic. The lack of productive viral replication further improves the safety profile of MVA as a vector, minimizing the potential for reversion to virulent forms particularly if used in immunocompromised individuals. Despite its inability to replicate in most mammalian cells, MVA still efficiently expresses viral and recombinant genes making it a potent antigen delivery platform. Moreover, due to the loss of various immunomodulatory factors MVA infection leads to rapid local immune responses, fulfilling a requirement of an adjuvant. In this review we take a look at the immunostimulatory properties of MVA, paying particular attention to the signalling of the innate immune system in response to MVA and VACV infection. Understanding the cellular and molecular mechanisms modulated by VACV will help in the future design and engineering of new vaccines and may provide insight into previously unknown mechanisms of dominant virus-host interactions.

  11. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    PubMed

    Xing, Jiali; Wang, Gang; Zhang, Qiuxiang; Liu, Xiaoming; Gu, Zhennan; Zhang, Hao; Chen, Yong Q; Chen, Wei

    2015-01-01

    Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA) assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs) of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS), reducing power (RP), and inhibition of linoleic acid peroxidation (ILAP). Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs. PMID:25789875

  12. Activation of consolidation processes of alumina ceramics

    NASA Astrophysics Data System (ADS)

    Matrenin, S. V.; Zenin, B. S.; Tayukin, R. V.

    2016-02-01

    The methods for activating sintering ceramics based on Al2O3 by mechanical activation in the planetary mill, by adding in the mixture of nanopowders (NP) Al, Al2O3, and submicron powder TiO2, and by applying the technology of spark plasma sintering (SPS) are developed. It has been shown that adding the nanopowder up to 20 wt. % Al2O3 in a coarse powder α-Al2O3 activates the sintering process resulting in increased density and hardness of the sintered alumina ceramics. Substantial effect of increasing density of alumina ceramics due to adding the submicron powder TiO2 in the compound of initial powder mixtures has been established.

  13. Precise quantification of cellular uptake of cell-penetrating peptides using fluorescence-activated cell sorting and fluorescence correlation spectroscopy.

    PubMed

    Rezgui, Rachid; Blumer, Katy; Yeoh-Tan, Gilbert; Trexler, Adam J; Magzoub, Mazin

    2016-07-01

    Cell-penetrating peptides (CPPs) have emerged as a potentially powerful tool for drug delivery due to their ability to efficiently transport a whole host of biologically active cargoes into cells. Although concerted efforts have shed some light on the cellular internalization pathways of CPPs, quantification of CPP uptake has proved problematic. Here we describe an experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules. This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane. PMID:27033412

  14. Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

    PubMed

    Guzman-Villanueva, Diana; El-Sherbiny, Ibrahim M; Vlassov, Alexander V; Herrera-Ruiz, Dea; Smyth, Hugh D C

    2014-10-01

    The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA. PMID:25063077

  15. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

    PubMed Central

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song

    2015-01-01

    ABSTRACT Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCE Influenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we

  16. Mapping social behavior-induced brain activation at cellular resolution in the mouse

    PubMed Central

    Kim, Yongsoo; Venkataraju, Kannan Umadevi; Pradhan, Kith; Mende, Carolin; Taranda, Julian; Turaga, Srinivas C.; Arganda-Carreras, Ignacio; Ng, Lydia; Hawrylycz, Michael J.; Rockland, Kathleen; Seung, H. Sebastian; Osten, Pavel

    2014-01-01

    Understanding how brain activation mediates behaviors is a central goal of systems neuroscience. Here we apply an automated method for mapping brain activation in the mouse in order to probe how sex-specific social behaviors are represented in the male brain. Our method uses the immediate early gene c-fos, a marker of neuronal activation, visualized by serial two-photon tomography: the c-fos-GFP-positive neurons are computationally detected, their distribution is registered to a reference brain and a brain atlas, and their numbers are analyzed by statistical tests. Our results reveal distinct and shared female and male interaction-evoked patterns of male brain activation representing sex discrimination and social recognition. We also identify brain regions whose degree of activity correlates to specific features of social behaviors and estimate the total numbers and the densities of activated neurons per brain areas. Our study opens the door to automated screening of behavior-evoked brain activation in the mouse. PMID:25558063

  17. Discovery of novel, high potent, ABC type PTP1B inhibitors with TCPTP selectivity and cellular activity.

    PubMed

    Liu, Peihong; Du, Yongli; Song, Lianhua; Shen, Jingkang; Li, Qunyi

    2016-08-01

    Protein tyrosine phosphatase 1B (PTP1B) as a key negative regulator of both insulin and leptin receptor pathways has been an attractive therapeutic target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. With the goal of enhancing potency and selectivity of the PTP1B inhibitors, a series of methyl salicylate derivatives as ABC type PTP1B inhibitors (P1-P7) were discovered. More importantly, compound P6 exhibited high potent inhibitory activity (IC50 = 50 nM) for PTP1B with 15-fold selectivity over T-cell PTPase (TCPTP). Further studies on cellular activities revealed that compound P6 could enhance insulin-mediated insulin receptor β (IRβ) phosphorylation and insulin-stimulated glucose uptake. PMID:27123900

  18. Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells

    PubMed Central

    Wintermeyer, P.; Gehring, S.; Eken, A.; Wands, J. R.

    2014-01-01

    SUMMARY Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)-based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H-2d) by hydrodynamic injection of a plasmid pUMVC3-hFLex expressing the secreted portion of the human Fms-like tyrosine kinase receptor-3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4+ cells and cytotoxic T-lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8+ generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co-stimulating molecules. Viral antigen-specific TH1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5-specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection. PMID:20002303

  19. Different levels of food restriction have opposite effects on adipocyte cellularity and lipoprotein-lipase activity in obese rats.

    PubMed

    Lemonnier, D; de Gasquet, P; Mackay, S; Planche, E; Alexiu, A; Rosselin, G; Loiseau, A

    1989-01-01

    The effects of several levels of chronic energy restriction on epididymal and perirenal adipose tissue cellularity and lipoprotein lipase activity, serum glucose and insulin and hepatic enzyme activities were studied in lean Fa/- and genetically obese fafa rats. The restricted rats were compared to rats fed ad libitum 24/24h or 8/24h. Restricting time of feeding was associated with increases in fat cell number in the lean, increases in perirenal adipose tissue fat cell size and serum insulin in the obese and increases in lipoprotein lipase activity in both phenotypes. Mild food restriction (-25%) had similar effects in the obese: perirenal adipose tissue fat cell size and serum insulin levels were even higher but fat cell hyperplasia was reduced. Restriction by 50% normalized lipoprotein lipase activity and markedly reduced fat cell size in the lean; in the obese, lipoprotein lipase activity and insulin levels were similar to or lower than those of the corresponding ad libitum 24/24h group but fat cell hypertrophy was not particularly affected. Restriction by 75% in the obese prevented adipocyte hyperplasia. Furthermore, lipoprotein lipase activity in adipose tissue was normalized, serum insulin and lipids being within normal limits. However, these animals had large adipocytes and were still fat.

  20. Complete solids retention activated sludge process.

    PubMed

    Amanatidou, E; Samiotis, G; Trikoilidou, E; Pekridis, G; Tsikritzis, L

    2016-01-01

    In a slaughterhouse's full-scale extended aeration activated sludge wastewater treatment plant (WWTP), operating under complete solids retention time, the evolution of mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS) concentration, food to micro-organisms ratio (F/M) and substrate utilization rate (SUR) were studied for over a year. Biomass growth phases in correlation to sludge biological and morphological characteristics were studied. Three distinguished growth phases were observed during the 425 days of monitoring. The imposed operational conditions led the process to extended biomass starvation conditions, minimum F/M, minimum SUR and predator species growth. MLSS and MLVSS reached a stabilization phase (plateau phase) where almost zero sludge accumulation was observed. The concept of degradation of the considered non-biodegradable particulate compounds in influent and in biomass (cell debris) was also studied. Comparison of evolution of observed sludge yields (Yobs) in the WWTP with Yobs predictions by activated sludge models verified the degradation concept for the considered non-biodegradable compounds. Control of the sedimentation process was achieved, by predicting the solids loading rate critical point using state point analysis and stirred/unstirred settling velocity tests and by applying a high return activated sludge rate. The nitrogen gas related sedimentation problems were taken into consideration.

  1. Active PZT fibers: a commercial production process

    NASA Astrophysics Data System (ADS)

    Strock, Harold B.; Pascucci, Marina R.; Parish, Mark V.; Bent, Aaron A.; Shrout, Thomas R.

    1999-07-01

    Lead Zirconate Titanate (PZT) active fibers, from 80 to 250 micrometers in diameter, are produced for the AFOSR/DARPA funded Active Fiber Composites Consortium (AFCC) Program and commercial customers. CeraNova has developed a proprietary ceramics-based technology to produce PZT mono-filaments of the required purity, composition, straightness, and piezoelectric properties for use in active fiber composite structures. CeraNova's process begins with the extrusion of continuous lengths of mono-filament precursor fiber from a plasticized mix of PZT-5A powder. The care that must be taken to avoid mix contamination is described using illustrations form problems experiences with extruder wear and metallic contamination. Corrective actions are described and example microstructures are shown. The consequences of inadequate lead control are also shown. Sintered mono- filament mechanical strength and piezoelectric properties data approach bulk values but the validity of such a benchmark is questioned based on variable correlation with composite performance measures. Comb-like ceramic preform structures are shown that are being developed to minimize process and handling costs while maintaining the required mono-filament straightness necessary for composite fabrication. Lastly, actuation performance data are presented for composite structures fabricated and tested by Continuum Control Corporation. Free strain actuation in excess of 2000 microstrain are observed.

  2. Brain activation during facial emotion processing.

    PubMed

    Gur, Ruben C; Schroeder, Lee; Turner, Travis; McGrath, Claire; Chan, Robin M; Turetsky, Bruce I; Alsop, David; Maldjian, Joseph; Gur, Raquel E

    2002-07-01

    Functional neuroimaging studies have helped identify neural systems involved in cognitive processing and more recently have indicated limbic activation to emotional stimuli. Some functional magnetic resonance imaging (fMRI) studies have reported increased amygdala response during exposure to emotional stimuli while others have not shown such activation. The present study was designed to test the hypothesis that activation of the amygdala is related to the relevance of the emotional valence of stimuli. Healthy young participants (7 men, 7 women) were studied in a high-field (4 tesla) scanner using blood oxygenation-level dependent (BOLD) signal changes in a blocked "box car" design. They viewed facial displays of happiness, sadness, anger, fear, and disgust as well as neutral faces obtained from professional actors and actresses of diverse ethnicity and age. Their task alternated between emotion discrimination (indicating whether the emotion was positive or negative) and age discrimination (indicating whether the poser was older or younger than 30). Blocks contained the same proportion of emotional and neutral faces. Limbic response was greater during the emotion than during the age discrimination conditions. The response was most pronounced in the amygdala, but was also present in the hippocampus and circumscribed voxels in other limbic regions. These results support the central role of the amygdala in emotion processing, and indicate its sensitivity to the task relevance of the emotional display.

  3. Cellular automata-based forecasting of the impact of accidental fire and toxic dispersion in process industries.

    PubMed

    Sarkar, Chinmoy; Abbasi, S A

    2006-09-01

    The strategies to prevent accidents from occurring in a process industry, or to minimize the harm if an accident does take place, always revolve around forecasting the likely accidents and their impacts. Based on the likely frequency and severity of the accidents, resources are committed towards preventing the accidents. Nearly all techniques of ranking hazardous units, be it the hazard and operability studies, fault tree analysis, hazard indice, etc.--qualitative as well as quantitative--depend essentially on the assessment of the likely frequency and the likely harm accidents in different units may cause. This fact makes it exceedingly important that the forecasting the accidents and their likely impact is done as accurately as possible. In the present study we introduce a new approach to accident forecasting based on the discrete modeling paradigm of cellular automata. In this treatment an accident is modeled as a self-evolving phenomena, the impact of which is strongly influenced by the size, nature, and position of the environmental components which lie in the vicinity of the accident site. The outward propagation of the mass, energy and momentum from the accident epicenter is modeled as a fast diffusion process occurring in discrete space-time coordinates. The quantum of energy and material that would flow into each discrete space element (cell) due to the accidental release is evaluated and the degree of vulnerability posed to the receptors if present in the cell is measured at the end of each time element. This approach is able to effectively take into account the modifications in the flux of energy and material which occur as a result of the heterogeneous environment prevailing between the accident epicenter and the receptor. Consequently, more realistic accident scenarios are generated than possible with the prevailing techniques. The efficacy of the approach has been illustrated with case studies.

  4. Cellular Distribution and Functions of Wild-Type and Constitutively Activated Dictyostelium PakBV⃞

    PubMed Central

    de la Roche, Marc; Mahasneh, Amjad; Lee, Sheu-Fen; Rivero, Francisco; Côté, Graham P.

    2005-01-01

    Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis. PMID:15509655

  5. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity

    PubMed Central

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-01-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe2+ chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods. PMID:26451354

  6. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    PubMed

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods. PMID:26451354

  7. P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

    PubMed

    Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

    2010-03-01

    We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. PMID:20004641

  8. A Cellular Resolution Map of Barrel Cortex Activity during Tactile Behavior.

    PubMed

    Peron, Simon P; Freeman, Jeremy; Iyer, Vijay; Guo, Caiying; Svoboda, Karel

    2015-05-01

    Comprehensive measurement of neural activity remains challenging due to the large numbers of neurons in each brain area. We used volumetric two-photon imaging in mice expressing GCaMP6s and nuclear red fluorescent proteins to sample activity in 75% of superficial barrel cortex neurons across the relevant cortical columns, approximately 12,000 neurons per animal, during performance of a single whisker object localization task. Task-related activity peaked during object palpation. An encoding model related activity to behavioral variables. In the column corresponding to the spared whisker, 300 layer (L) 2/3 pyramidal neurons (17%) each encoded touch and whisker movements. Touch representation declined by half in surrounding columns; whisker movement representation was unchanged. Following the emergence of stereotyped task-related movement, sensory representations showed no measurable plasticity. Touch direction was topographically organized, with distinct organization for passive and active touch. Our work reveals sparse and spatially intermingled representations of multiple tactile features. VIDEO ABSTRACT. PMID:25913859

  9. Changes in tRNA methyltransferase activity and cellular S-adenosylmethionine content following methionine deprivation.

    PubMed

    Tisdale, M J

    1980-09-19

    Although homocysteine was unable to support growth of Walker carcinoma in media lacking methionine it did enable some proliferation of TLX5 lymphoma. In both cell lines there was an increase in growth rate in the presence of homocysteine at limiting methionine concentrations. The proliferation rate of Walker carcinoma was proportional to the methionine concentraion of the medium down to 0.5 microgram/ml, whereas growth of TLX5 lymphoma was only slightly reduced at such methionine concentrations. The difference in proliferative ability between the two cell lines was reflected in the level of S-adenosyl-L-methionine under conditions of methionine deprivation. In both cases transferance to a media in which methionine was growth limiting caused a rapid increase in the activity of tRNA methyltransferases to levels six to seven-fold greater than the control. The initial increase in methylase activity was not prevented by cycloheximide, although after 4 h there was a progressive decrease in activity which approached control values within 24 h. The increase in tRNA methyltransferase activity on removal of the normal level of methionine in the medium was also seen with human embryonic fibroblasts, which are able to proliferate normally in methionine-deficient, homocysteine-supplemented media. These results suggest that methyltransferase activity may be regulated in part by the S-adenosyl-methionine content of the cell.

  10. Synthesis, cellular uptake and structure-activity relationships for potent cytotoxic trichloridoiridium(III) polypyridyl complexes.

    PubMed

    Scharwitz, Michael A; Ott, Ingo; Gust, Ronald; Kromm, Anna; Sheldrick, William S

    2008-08-01

    The complexes fac-[IrCl(3)(DMSO)(pp)] 1a-5a may be prepared by stepwise reaction of IrCl(3) x 3H(2)O with the appropriate polypyridyl ligand (pp=bpy, phen, dpq, dppz, dppn) and DMSO in CH(3)OH solution in the dark. The fac isomers of 1a-5a are stable in light-protected CD(2)Cl(2) solution but, with the exception of 5a, isomerize rapidly to a mixture of the fac and mer isomers in the presence of light. In contrast, solutions of the fac isomers in the polar solvents D(2)O and CD(3)OD are stable under such conditions. The isomer mer-[IrCl(3)(DMSO-kappa S)(phen)] 2b was, however, isolated by slow evaporation of an H(2)O/CH(3)OH solution of 2a and characterized by X-ray structural analysis. UV/Vis and CD studies of the interaction of 1a-5a with calf thymus DNA are in accordance with an effective absence of intercalation. (1)H NMR studies indicate that the complexes react slowly with compounds containing soft S donor atoms (e. g. N-acetylmethionine) but do not react with the guanine base of 5'-GMP(2-). The complexes 2a-5a are potent in vitro cytotoxic agents toward the human cell lines MCF-7 and HT-29 and their IC(50) values are dependent on the size of the polypyridyl ligand in the order phen, dpq>dppz>dppn. For instance IC(50) values of 5.5 (0.9), 0.8 (0.3) and 0.21 (0.11)microM were established for 3a-5a against MCF-7 cells and 6.1 (0.7), 1.5 (0.2) and 1.3 (0.4)microM against HT-29 cells. These values correlate with the cellular uptake efficiency which, on exposure to 10 microM solutions, reaches its highest levels (19.3(0.8) and 37.4(8.9) ng Ir/mg protein for MCF-7 and HT-29, respectively) for the dppn compound 5a. PMID:18472166

  11. Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate

    PubMed Central

    Leight, Jennifer L.; Alge, Daniel L.; Maier, Andrew J.; Anseth, Kristi S.

    2014-01-01

    Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications. PMID:23830581

  12. Identification and quantification of phytochemical composition and anti-inflammatory, cellular antioxidant, and radical scavenging activities of 12 Plantago species.

    PubMed

    Zhou, Qin; Lu, Weiying; Niu, Yuge; Liu, Jie; Zhang, Xiaowei; Gao, Boyan; Akoh, Casimir C; Shi, Haiming; Yu, Liangli Lucy

    2013-07-10

    Twenty-eight seed samples of 12 Plantago species were investigated for their chemical compositions and anti-inflammatory, cellular antioxidant, and radical scavenging properties. A new UPLC-UV procedure was developed and applied to quantify acteoside and geniposidic acid, the characteristic constituents of the genus Plantago. The amounts of acteoside and geniposidic acid ranged from 0.07 to 15.96 mg/g and from 0.05 to 10.04 mg/g in the tested samples, respectively. Furthermore, 26 compounds were tentatively identified by UPLC/Q-TOF-MS analysis. The Plantago samples significantly differed in their phytochemical compositions. The extracts of Plantago seeds also showed inhibitory effects on LPS-induced IL-1β, IL-6, and COX-2 mRNA expression in RAW 264.7 mouse macrophage cells. Additionally, significant variations were observed among different samples on cellular antioxidant activities in HepG2 cells, as well as DPPH and hydroxyl radical scavenging capacities. The results from this study may be used to promote the use of the genus Plantago in improving human health. PMID:23767948

  13. Pilocarpine modulates the cellular electrical properties of mammalian hearts by activating a cardiac M3 receptor and a K+ current

    PubMed Central

    Wang, Huizhen; Shi, Hong; Lu, Yanjie; Yang, Baofeng; Wang, Zhiguo

    1999-01-01

    Pilocarpine, a muscarinic acetylcholine receptor (mAChR) agonist, is widely used for treatment of xerostomia and glaucoma. It can also cause many other cellular responses by activating different subtypes of mAChRs in different tissues. However, the potential role of pilocarpine in modulating cardiac function remained unstudied.We found that pilocarpine produced concentration-dependent (0.1–10 μM) decrease in sinus rhythm and action potential duration, and hyperpolarization of membrane potential in guinea-pig hearts. The effects were nearly completely reversed by 1 μM atropine or 2 nM 4DAMP methiodide (an M3-selective antagonist).Patch-clamp recordings in dispersed myocytes from guinea-pig and canine atria revealed that pilocarpine induces a novel K+ current with delayed rectifying properties. The current was suppressed by low concentrations of M3-selective antagonists 4DAMP methiodide (2–10 nM), 4DAMP mustard (4–20 nM, an ackylating agent) and p-F-HHSiD (20–200 nM). Antagonists towards other subtypes (M1, M2 or M4) all failed to alter the current.The affinity of pilocarpine (KD) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 2.2 μM (65% of the total binding) and that of 4DAMP methiodide was 2.8 nM (70% of total binding), consistent with the concentration of pilocarpine needed for the current induction and for the modulation of the cardiac electrical activity and the concentration of 4DAMP to block pilocarpine effects.Our data indicate, for the first time, that pilocarpine modulates the cellular electrical properties of the hearts, likely by activating a K+ current mediated by M3 receptors. PMID:10372814

  14. Functional relevance during lymphocyte migration and cellular localization of activated beta1 integrins.

    PubMed

    Gómez, M; Luque, A; del Pozo, M A; Hogg, N; Sánchez-Madrid, F; Cabañas, C

    1997-01-01

    The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the beta1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for beta1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common beta1 subunit of VLA proteins. Engagement of beta1 integrins through this LIBS epitope inhibited T lymphoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized beta1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-beta1 activating antibodies.

  15. Functionally Charged Polystyrene Particles Activate Immortalized Mouse Microglia (BV2): Cellular and Genomic Response

    EPA Science Inventory

    The effect of particle surface charge on the biological activation of immortalized mouse microglia (BV2) was examined. Same size (~850-950 nm) spherical polystyrene microparticles (SPM) with net negative (carboxyl, COOH-) or positive (dimethyl amino, CH3)2

  16. Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer.

    PubMed

    Dereli-Korkut, Zeynep; Gandhok, Harmeet; Zeng, Ling Ge; Waqas, Sidra; Jiang, Xuejun; Wang, Sihong

    2013-05-01

    Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.

  17. Identification and characterization of thiosemicarbazones with antifungal and antitumor effects: cellular iron chelation mediating cytotoxic activity.

    PubMed

    Opletalová, Veronika; Kalinowski, Danuta S; Vejsová, Marcela; Kunes, Jirí; Pour, Milan; Jampílek, Josef; Buchta, Vladimír; Richardson, Des R

    2008-09-01

    Thiosemicarbazones derived from acetylpyrazines were prepared by condensing an acetylpyrazine or a ring-substituted acetylpyrazine with thiosemicarbazide. Using the same procedure, N, N-dimethylthiosemicarbazones were synthesized from acetylpyrazines and N, N-dimethylthiosemicarbazide. A total of 20 compounds (16 novel) were chemically characterized and then tested for antifungal effects on eight strains of fungi and also for antitumor activity against SK-N-MC neuroepithelioma cells. The most effective compound identified in terms of both antifungal and antitumor activity was N, N-dimethyl-2-(1-pyrazin-2-ylethylidene)hydrazinecarbothioamide (5a). The mechanism of action of this and its related thiosemicarbazones was due, at least in part, to its ability to act as a tridentate ligand that binds metal ions. This was deduced from preparation of the related thiosemicarbazones [acetophenone thiosemicarbazone (6) and acetophenone N, N-dimethylthiosemicarbazone (7)] that do not possess a coordinating ring-N, which plays a vital role in metal ion chelation. Furthermore, 5a and several other thiosemicarbazones that showed high antiproliferative activity were demonstrated to have marked iron (Fe) chelation efficacy. In fact, these agents were highly effective at mobilizing (59)Fe from prelabeled SK-N-MC cells and preventing (59)Fe uptake from the serum Fe transport protein, transferrin. In contrast, compounds 6 and 7 that do not possess a tridentate metal-binding site showed little activity. Further studies examining ascorbate oxidation demonstrated that the Fe complexes of the most effective compounds were redox-inactive. Thus, in contrast to other thiosemicarbazones with potent antiproliferative activity, Fe chelation and mobilization rather than free radical generation played a significant role in the cytotoxic effects of the current ligands. PMID:18698850

  18. Comparison of the free and bound phenolic profiles and cellular antioxidant activities of litchi pulp extracts from different solvents

    PubMed Central

    2014-01-01

    Background The phenolic contents and antioxidant activities of fruits could be underestimated if the bound phenolic compounds are not considered. In the present study, the extraction efficiencies of various solvents were investigated in terms of the total content of the free and bound phenolic compounds, as well as the phenolic profiles and antioxidant activities of the extracts. Methods Five different solvent mixtures were used to extract the free phenolic compounds from litchi pulp. Alkaline and acidic hydrolysis methods were compared for the hydrolysis of bound phenolic compounds from litchi pulp residue. The phenolic compositions of the free and bound fractions from the litchi pulp were identified using HPLC-DAD. The antioxidant activities of the litchi pulp extracts were determined by oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Results Of the solvents tested, aqueous acetone extracted the largest amount of total free phenolic compounds (210.7 mg GAE/100 g FW) from litchi pulp, followed sequentially by aqueous mixtures of methanol, ethanol and ethyl acetate, and water itself. The acid hydrolysis method released twice as many bound phenolic compounds as the alkaline hydrolysis method. Nine phenolic compounds were detected in the aqueous acetone extract. In contrast, not all of these compounds were found in the other four extracts. The classification and content of the bound phenolic compounds released by the acid hydrolysis method were higher than those achieved by the alkaline hydrolysis. The aqueous acetone extract showing the highest ORAC value (3406.9 μmol TE/100 g FW) for the free phenolic extracts. For the CAA method, however, the aqueous acetone and methanol extracts (56.7 and 55.1 μmol QE/100 g FW) showed the highest levels of activity of the five extracts tested. The ORAC and CAA values of the bound phenolic compounds obtained by acid hydrolysis were 2.6- and 1.9-fold higher than those obtained using the

  19. Solid-Phase Synthesis, Characterization, and Cellular Activities of Collagen-Model Nanodiamond-Peptide Conjugates

    PubMed Central

    Knapinska, Anna M.; Tokmina-Roszyk, Dorota; Amar, Sabrina; Tokmina-Roszyk, Michal; Mochalin, Vadym N.; Gogotsi, Yury; Cosme, Patrick; Terentis, Andrew C.; Fields, Gregg B.

    2015-01-01

    Nanodiamonds (NDs) have received considerable attention as potential drug delivery vehicles. NDs are small (~5 nm diameter), can be surface modified in a controllable fashion with a variety of functional groups, and have little observed toxicity in vitro and in vivo. However, most biomedical applications of NDs utilize surface adsorption of biomolecules, as opposed to covalent attachment. Covalent modification provides reliable and reproducible ND–biomolecule ratios, and alleviates concerns over biomolecule desorption prior to delivery. The present study has outlined methods for the efficient solid-phase conjugation of ND to peptides and characterization of ND–peptide conjugates. Utilizing collagen-derived peptides, the ND was found to support or even enhance the cell adhesion and viability activities of the conjugated sequence. Thus, NDs can be incorporated into peptides and proteins in a selective manner, where the presence of the ND could potentially enhance the in vivo activities of the biomolecule it is attached to. PMID:25753561

  20. Interferon-γ regulates cellular metabolism and mRNA translation to potentiate macrophage activation.

    PubMed

    Su, Xiaodi; Yu, Yingpu; Zhong, Yi; Giannopoulou, Eugenia G; Hu, Xiaoyu; Liu, Hui; Cross, Justin R; Rätsch, Gunnar; Rice, Charles M; Ivashkiv, Lionel B

    2015-08-01

    Interferon-γ (IFN-γ) primes macrophages for enhanced microbial killing and inflammatory activation by Toll-like receptors (TLRs), but little is known about the regulation of cell metabolism or mRNA translation during this priming. We found that IFN-γ regulated the metabolism and mRNA translation of human macrophages by targeting the kinases mTORC1 and MNK, both of which converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-γ was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages showed that IFN-γ selectively modulated the macrophage translatome to promote inflammation, further reprogram metabolic pathways and modulate protein synthesis. These results show that IFN-γ-mediated metabolic reprogramming and translational regulation are key components of classical inflammatory macrophage activation.

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

    PubMed

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

    2016-09-01

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

  2. Transcriptional activation and repression by cellular DNA-binding protein C/EBP.

    PubMed Central

    Pei, D Q; Shih, C H

    1990-01-01

    A putative transcription factor, C/EBP, isolated from rat liver nuclei, has been shown to bind to at least two different sequence motifs: the CCAAT promoter domain and a core sequence [GTGG(T/A)(T/A)(T/A)G] common to many viral enhancers, including simian virus 40 and human hepatitis B virus. It has been proposed that C/EBP might function as a positive transcription factor by facilitating the communication between promoter and enhancer elements through its dual binding activities to DNA. Surprisingly, results from three different approaches suggest that C/EBP functions as a transcriptional repressor to hepatitis B virus and simian virus 40. Further investigation indicated that C/EBP can function as both a transcriptional activator and a repressor, depending on the reporter gene system. Images PMID:2157040

  3. The activation sequence of cellular protein handling systems after proteasomal inhibition in dopaminergic cells

    PubMed Central

    Xiong, Rui; Siegel, David; Ross, David

    2013-01-01

    Dysfunction of protein handling has been implicated in many neurodegenerative diseases and inhibition of the ubiquitin-proteasome system (UPS) has been linked to the formation of protein aggregates and proteinopathies in such diseases. While proteasomal inhibition could trigger an array of downstream protein handling changes including up-regulation of heat shock proteins (HSPs), induction of molecular chaperones, activation of the ER stress/unfolded protein response (UPR), autophagy and aggresome formation, little is known of the relationship of proteasomal inhibition to the sequence of activation of these diverse protein handling systems. In this study we utilized the reversible proteasome inhibitor MG132 and examined the activity of several major protein handling systems in the immortalized dopaminergic neuronal N27 cell line. In the early phase (up to 6 hours after proteasomal inhibition), MG132 induced time-dependent proteasomal inhibition which resulted in stimulation of the UPR, increased autophagic flux and stimulated heat shock protein response as determined by increased levels of phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), C/EBP homologous protein (CHOP)/GADD153, turnover of autophagy related microtubule-associated protein 1 light chain 3 (LC3) and increased levels of Hsp70 respectively. After prolonged proteasomal inhibition induced by MG132, we observed the formation of vimentin-caged aggresome-like inclusion bodies. A recovery study after MG132-induced proteasomal inhibition indicated that the autophagy-lysosomal pathway participated in the clearance of aggresomes. Our data characterizes the relationship between proteasome inhibition and activation of other protein handling systems. These data also indicated that the induction of alternate protein handling systems and their temporal relationships may be important factors that determine the extent of accumulation of misfolded proteins in cells as a result of

  4. Tissue plasminogen activator modulates the cellular and behavioral response to cocaine

    PubMed Central

    Maiya, Rajani; Zhou, Yan; Norris, Erin H.; Kreek, Mary Jeanne; Strickland, Sidney

    2009-01-01

    Cocaine exposure induces long-lasting molecular and structural adaptations in the brain. In this study, we show that tissue plasminogen activator (tPA), an extracellular protease involved in neuronal plasticity, modulates the biochemical and behavioral response to cocaine. When injected in the acute binge paradigm, cocaine enhanced tPA activity in the amygdala, which required activation of corticotropin-releasing factor type-1 (CRF-R1) receptors. Compared with WT mice, tPA−/− mice injected with cocaine displayed attenuated phosphorylation of ERK, cAMP response element binding protein (CREB), and dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) and blunted induction of immediate early genes (IEGs) c-Fos, Egr-1, and Homer 1a in the amygdala and the nucleus accumbens (NAc). tPA−/− mice also displayed significantly higher basal preprodynorphin (ppDyn) mRNA levels in the NAc in comparison to WT mice, and cocaine decreased ppDyn mRNA levels in tPA−/− mice only. Cocaine-induced locomotor sensitization and conditioned place preference (CPP) were attenuated in tPA−/− mice. Cocaine exposure also had an anxiolytic effect in tPA−/− but not WT mice. These results identify tPA as an important and novel component of the signaling pathway that modulates cocaine-induced changes in neuroadaptation and behavior. PMID:19181855

  5. Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors

    PubMed Central

    Meier-Stiegen, Franziska; Schwanbeck, Ralf; Bernoth, Kristina; Martini, Simone; Hieronymus, Thomas; Ruau, David; Zenke, Martin; Just, Ursula

    2010-01-01

    Background Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. Methodology/Principal Findings To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. Conclusion/Significance We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context. PMID:20628604

  6. Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells.

    PubMed

    Rhein, V; Baysang, G; Rao, S; Meier, F; Bonert, A; Müller-Spahn, F; Eckert, A

    2009-09-01

    Evidence suggests that amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Here we investigated the specific effects of Abeta on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Abeta protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

  7. 15 CFR 400.31 - Manufacturing and processing activity; criteria.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 15 Commerce and Foreign Trade 2 2012-01-01 2012-01-01 false Manufacturing and processing activity... ZONES BOARD Manufacturing and Processing Activity-Reviews § 400.31 Manufacturing and processing activity....” When evaluating zone and subzone manufacturing and processing activity, either as proposed in...

  8. 15 CFR 400.31 - Manufacturing and processing activity; criteria.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 2 2011-01-01 2011-01-01 false Manufacturing and processing activity... ZONES BOARD Manufacturing and Processing Activity-Reviews § 400.31 Manufacturing and processing activity....” When evaluating zone and subzone manufacturing and processing activity, either as proposed in...

  9. 15 CFR 400.31 - Manufacturing and processing activity; criteria.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Manufacturing and processing activity... ZONES BOARD Manufacturing and Processing Activity-Reviews § 400.31 Manufacturing and processing activity....” When evaluating zone and subzone manufacturing and processing activity, either as proposed in...

  10. Surface and semantic processing of cellular transport representations by high school students with low and high prior knowledge

    NASA Astrophysics Data System (ADS)

    Cook, Michelle Patrick

    The purpose of this study was to examine the influence of prior knowledge of cell transport processes on how students viewed and interpreted visual representations related to that topic. The participants were high school students (n=65) enrolled in Advanced Placement biology. Prior knowledge was assessed using a modified version of the Diffusion and Osmosis Diagnostic Test (Odom & Barrow, 1995). Eye movements were measured to reveal how students distribute their visual attention as they perceive and interpret graphics; in addition, interviews and questionnaires were employed to provide more interpretive data sources. The first manuscript of the study investigates the relationship between prior knowledge and students' ability to perceive salient features and interpret graphic representations of cellular transport. The results from eye tracking data, interviews, and questionnaire responses were triangulated and revealed differences in how high and low prior knowledge students attended to and interpreted various features of the graphic representations. Without adequate domain knowledge, low prior knowledge students focused on surface features of the graphics to build an understanding of the concepts represented. High prior knowledge students, with more abundant and better organized domain knowledge, were more likely to attend to thematically relevant content in the graphics and construct deeper understandings. The second manuscript of the study examines the influence of prior knowledge on how students transitioned among the macroscopic and molecular representations of selected graphics. Eye tracking and sequential analysis results indicated that high prior knowledge students transitioned more frequently between the molecular representations, where as low prior knowledge students transitioned more frequently between the macroscopic representations. In addition, low prior knowledge students transitioned more frequently between macroscopic and molecular representations

  11. Hijacking cellular garbage cans.

    PubMed

    Welsch, Sonja; Locker, Jacomine Krijnse

    2010-06-25

    Viruses are perfect opportunists that have evolved to modify numerous cellular processes in order to complete their replication cycle in the host cell. An article by Reggiori and coworkers in this issue of Cell Host & Microbe reveals how coronaviruses can divert a cellular quality control pathway that normally functions in degradation of mis-folded proteins to replicate the viral genome. PMID:20542246

  12. Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud.

    PubMed

    Risjani, Yenny; Yunianta; Couteau, Jerome; Minier, Christophe

    2014-05-01

    Since May 29, 2006, a mud volcano in the Brantas Delta of the Sidoarjo district has emitted mud that has inundated nearby villages. Pollution in this area has been implicated in detrimental effects on fish health. In fishes, leukocyte and phagocytic cells play a vital role in body defenses. We report for the first time the effect of "LUSI" volcano mud on the immune systems of fish in the Brantas Delta. The aim of this study was to find biomarkers to allow the evaluation of the effects of volcanic mud and anthropogenic pollution on fish health in the Brantas Delta. The study took places at the Brantas Delta, which was polluted by volcano mud, and at reference sites in Karangkates and Pasuruan. Leukocyte numbers were determined using a Neubauer hemocytometer and a light microscope. Differential leukocyte counts were determined using blood smears stained with May Grunwald-Giemsa, providing neutrophil, lymphocyte and monocyte counts. Macrophages were taken from fish kidney, and their phagocytic activity was measured. In vitro analyses revealed that leukocyte and differential leukocyte counts (DLC) were higher in Channa striata and Chanos chanos caught from the polluted area. Macrophage numbers were higher in Oreochromis mossambicus than in the other species, indicating that this species is more sensitive to pollution. In areas close to volcanic mud eruption, all specimens had lower phagocytic activity. Our results show that immune cells were changed and phagocytic activity was reduced in the polluted area indicating cytotoxicity and alteration of the innate immune system in fishes exposed to LUSI volcano mud and anthropogenic pollution.

  13. Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud.

    PubMed

    Risjani, Yenny; Yunianta; Couteau, Jerome; Minier, Christophe

    2014-05-01

    Since May 29, 2006, a mud volcano in the Brantas Delta of the Sidoarjo district has emitted mud that has inundated nearby villages. Pollution in this area has been implicated in detrimental effects on fish health. In fishes, leukocyte and phagocytic cells play a vital role in body defenses. We report for the first time the effect of "LUSI" volcano mud on the immune systems of fish in the Brantas Delta. The aim of this study was to find biomarkers to allow the evaluation of the effects of volcanic mud and anthropogenic pollution on fish health in the Brantas Delta. The study took places at the Brantas Delta, which was polluted by volcano mud, and at reference sites in Karangkates and Pasuruan. Leukocyte numbers were determined using a Neubauer hemocytometer and a light microscope. Differential leukocyte counts were determined using blood smears stained with May Grunwald-Giemsa, providing neutrophil, lymphocyte and monocyte counts. Macrophages were taken from fish kidney, and their phagocytic activity was measured. In vitro analyses revealed that leukocyte and differential leukocyte counts (DLC) were higher in Channa striata and Chanos chanos caught from the polluted area. Macrophage numbers were higher in Oreochromis mossambicus than in the other species, indicating that this species is more sensitive to pollution. In areas close to volcanic mud eruption, all specimens had lower phagocytic activity. Our results show that immune cells were changed and phagocytic activity was reduced in the polluted area indicating cytotoxicity and alteration of the innate immune system in fishes exposed to LUSI volcano mud and anthropogenic pollution. PMID:24631200

  14. Plumbagin-silver nanoparticle formulations enhance the cellular uptake of plumbagin and its antiproliferative activities.

    PubMed

    Appadurai, Prakash; Rathinasamy, Krishnan

    2015-10-01

    Colloidal silver nanoparticles (AgNPs) have attracted much attention in recent years as diagnostics and new drug delivery system in cancer medicine. To study the effects of plumbagin (PLB), a relatively non-toxic napthaquinone isolated from the roots of Plumbago indica in human cervical cancer cell line and developed a formulation to enhance its cytotoxic activities. Silver nanoparticles were synthesised by chemical reduction method and complexed with PLB. Both the AgNPs and the complex PLB-AgNPs were characterised by dynamic light scattering, high-resolution scanning electron microscopy and transmission electron microscopy. The amount of PLB and PLB-AgNPs internalised was determined by ultra-violet-visible spectrophotometer. Cell inhibition was determined by sulphorhodamine B assay. Mitotic index was determined by Wright-Giemsa staining. Apoptosis induction was assessed by western blot using cleaved poly adenosine diphosphate-ribose polymerase antibody. The scanning electron microscope analysis indicated an average particle size of 32±8 nm in diameter. Enhanced internalisation of PLB into the HeLa cells was observed in PLB-AgNPs. PLB inhibited proliferation of cells with IC50 value of about 18±0.6 µM and blocked the cells at mitosis in a concentration-dependent manner. PLB also inhibited the post-drug exposure clonogenic survival of cells and induced apoptosis. The antiproliferative, antimitotic and apoptotic activities were also found to be increased when cells were treated with PLB-AgNPs. The authors results support the idea that AgNP could be a promising and effective drug delivery system for enhanced activity of PLB in cancer treatment. PMID:26435279

  15. PREFACE: Selected papers from the Fourth Annual q-bio Conference on Cellular Information Processing Selected papers from the Fourth Annual q-bio Conference on Cellular Information Processing

    NASA Astrophysics Data System (ADS)

    Nemenman, Ilya; Faeder, James R.; Hlavacek, William S.; Jiang, Yi; Wall, Michael E.; Zilman, Anton

    2011-10-01

    Summary This special issue consists of 11 original papers that elaborate on work presented at the Fourth Annual q-bio Conference on Cellular Information Processing, which was held on the campus of St John's College in Santa Fe, New Mexico, USA, 11-14 August 2010. Now in its fourth year, the q-bio conference has changed considerably over time. It is now well established and a major event in systems biology. The 2010 conference saw attendees from all continents (except Antarctica!) sharing novel results and participating in lively discussions at both the oral and poster sessions. The conference was oversubscribed and grew to 27 contributed talks, 16 poster spotlights and 137 contributed posters. We deliberately decreased the number of invited speakers to 21 to leave more space for contributed presentations, and the attendee feedback confirmed that the choice was a success. Although the q-bio conference has grown and matured, it has remained true to the original goal of being an intimate and dynamic event that brings together modeling, theory and quantitative experimentation for the study of cell regulation and information processing. Funded in part by a grant from NIGMS and by DOE funds through the Los Alamos National Laboratory Directed Research and Development program, the conference has continued to exhibit youth and vigor by attracting (and partially supporting) over 100 undergraduate, graduate and postdoctoral researchers. The associated q-bio summer school, which precedes the conference each year, further emphasizes the development of junior scientists and makes q-bio a singular event in its impact on the future of quantitative biology. In addition to an increased international presence, the conference has notably diversified its demographic representation within the USA, including increased participation from the southeastern corner of the country. One big change in the conference this year is our new publication partner, Physical Biology. Although we are very

  16. Proteasome-Mediated Processing of Def1, a Critical Step in the Cellular Response to Transcription Stress

    PubMed Central

    Wilson, Marcus D.; Harreman, Michelle; Taschner, Michael; Reid, James; Walker, Jane; Erdjument-Bromage, Hediye; Tempst, Paul; Svejstrup, Jesper Q.

    2013-01-01

    Summary DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function. PMID:23993092

  17. GST activity and membrane lipid saturation prevents mesotrione-induced cellular damage in Pantoea ananatis.

    PubMed

    Prione, Lilian P; Olchanheski, Luiz R; Tullio, Leandro D; Santo, Bruno C E; Reche, Péricles M; Martins, Paula F; Carvalho, Giselle; Demiate, Ivo M; Pileggi, Sônia A V; Dourado, Manuella N; Prestes, Rosilene A; Sadowsky, Michael J; Azevedo, Ricardo A; Pileggi, Marcos

    2016-12-01

    Callisto(®), containing the active ingredient mesotrione (2-[4-methylsulfonyl-2-nitrobenzoyl]1,3-cyclohenanedione), is a selective herbicide that controls weeds in corn crops and is a potential environmental contaminant. The objective of this work was to evaluate enzymatic and structural changes in Pantoea ananatis, a strain isolated from water, in response to exposure to this herbicide. Despite degradation of mesotrione, probably due a glutathione-S-transferase (GST) pathway in Pantoea ananatis, this herbicide induced oxidative stress by increasing hydrogen peroxide production. Thiol fragments, eventually produced after mesotrione degradation, could be involved in increased GST activity. Nevertheless, there was no peroxidation damage related to this production, as malondialdehyde (MDA) synthesis, which is due to lipid peroxidation, was highest in the controls, followed by the mesotrione- and Callisto(®)-treated cultures at log growth phase. Therefore, P. ananatis can tolerate and grow in the presence of the herbicide, probably due an efficient control of oxidative stress by a polymorphic catalase system. MDA rates depend on lipid saturation due to a pattern change to a higher level of saturation. These changes are likely related to the formation of GST-mesotrione conjugates and mesotrione degradation-specific metabolites and to the presence of cytotoxic adjuvants. These features may shift lipid membrane saturation, possibly providing a protective effect to bacteria through an increase in membrane impermeability. This response system in P. ananatis provides a novel model for bacterial herbicide tolerance and adaptation in the environment. PMID:27620734

  18. PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

    PubMed Central

    Kim, Yoosik; Lee, Jung Hyun; Park, Jong-Eun; Cho, Jun; Yi, Hyerim; Kim, V. Narry

    2014-01-01

    dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as CYCLINS A and B and POLO-LIKE KINASE 1 and phosphorylation of HISTONE H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells. PMID:24939934

  19. GST activity and membrane lipid saturation prevents mesotrione-induced cellular damage in Pantoea ananatis.

    PubMed

    Prione, Lilian P; Olchanheski, Luiz R; Tullio, Leandro D; Santo, Bruno C E; Reche, Péricles M; Martins, Paula F; Carvalho, Giselle; Demiate, Ivo M; Pileggi, Sônia A V; Dourado, Manuella N; Prestes, Rosilene A; Sadowsky, Michael J; Azevedo, Ricardo A; Pileggi, Marcos

    2016-12-01

    Callisto(®), containing the active ingredient mesotrione (2-[4-methylsulfonyl-2-nitrobenzoyl]1,3-cyclohenanedione), is a selective herbicide that controls weeds in corn crops and is a potential environmental contaminant. The objective of this work was to evaluate enzymatic and structural changes in Pantoea ananatis, a strain isolated from water, in response to exposure to this herbicide. Despite degradation of mesotrione, probably due a glutathione-S-transferase (GST) pathway in Pantoea ananatis, this herbicide induced oxidative stress by increasing hydrogen peroxide production. Thiol fragments, eventually produced after mesotrione degradation, could be involved in increased GST activity. Nevertheless, there was no peroxidation damage related to this production, as malondialdehyde (MDA) synthesis, which is due to lipid peroxidation, was highest in the controls, followed by the mesotrione- and Callisto(®)-treated cultures at log growth phase. Therefore, P. ananatis can tolerate and grow in the presence of the herbicide, probably due an efficient control of oxidative stress by a polymorphic catalase system. MDA rates depend on lipid saturation due to a pattern change to a higher level of saturation. These changes are likely related to the formation of GST-mesotrione conjugates and mesotrione degradation-specific metabolites and to the presence of cytotoxic adjuvants. These features may shift lipid membrane saturation, possibly providing a protective effect to bacteria through an increase in membrane impermeability. This response system in P. ananatis provides a novel model for bacterial herbicide tolerance and adaptation in the environment.

  20. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    SciTech Connect

    Boel, Gaby-Fleur . E-mail: boel@mail.dife.de; Jurrmann, Nadine; Brigelius-Flohe, Regina

    2005-06-24

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4{sup IRAK-1-K239S}). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4{sup IRAK-1-K239S} cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.

  1. PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator.

    PubMed

    Kim, Yoosik; Lee, Jung Hyun; Park, Jong-Eun; Cho, Jun; Yi, Hyerim; Kim, V Narry

    2014-06-15

    dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as cyclins A and B and Polo-like kinase 1 and phosphorylation of histone H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.

  2. Broadband activation by white-opsin lowers intensity threshold for cellular stimulation

    PubMed Central

    Batabyal, Subrata; Cervenka, Gregory; Birch, David; Kim, Young-tae; Mohanty, Samarendra

    2015-01-01

    Photoreceptors, which initiate the conversion of ambient light to action potentials via retinal circuitry, degenerate in retinal diseases such as retinitis pigmentosa and age related macular degeneration leading to loss of vision. Current prosthetic devices using arrays consisting of electrodes or LEDs (for optogenetic activation of conventional narrow-band opsins) have limited spatial resolution and can cause damage to retinal circuits by mechanical or photochemical (by absorption of intense narrow band light) means. Here, we describe a broad-band light activatable white-opsin for generating significant photocurrent at white light intensity levels close to ambient daylight conditions. White-opsin produced an order of magnitude higher photocurrent in response to white light as compared to narrow-band opsin channelrhodopsin-2, while maintaining the ms-channel kinetics. High fidelity of peak-photocurrent (both amplitude and latency) of white-opsin in response to repetitive white light stimulation of varying pulse width was observed. The significantly lower intensity stimulation required for activating white-opsin sensitized cells may facilitate ambient white light-based restoration of vision for patients with widespread photoreceptor degeneration. PMID:26658483

  3. Physics of active jamming during collective cellular motion in a monolayer.

    PubMed

    Garcia, Simon; Hannezo, Edouard; Elgeti, Jens; Joanny, Jean-François; Silberzan, Pascal; Gov, Nir S

    2015-12-15

    Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell-cell and cell-substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data.

  4. Physics of active jamming during collective cellular motion in a monolayer

    PubMed Central

    Garcia, Simon; Hannezo, Edouard; Elgeti, Jens; Joanny, Jean-François; Silberzan, Pascal; Gov, Nir S.

    2015-01-01

    Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell−cell and cell−substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data. PMID:26627719

  5. Physics of active jamming during collective cellular motion in a monolayer.

    PubMed

    Garcia, Simon; Hannezo, Edouard; Elgeti, Jens; Joanny, Jean-François; Silberzan, Pascal; Gov, Nir S

    2015-12-15

    Although collective cell motion plays an important role, for example during wound healing, embryogenesis, or cancer progression, the fundamental rules governing this motion are still not well understood, in particular at high cell density. We study here the motion of human bronchial epithelial cells within a monolayer, over long times. We observe that, as the monolayer ages, the cells slow down monotonously, while the velocity correlation length first increases as the cells slow down but eventually decreases at the slowest motions. By comparing experiments, analytic model, and detailed particle-based simulations, we shed light on this biological amorphous solidification process, demonstrating that the observed dynamics can be explained as a consequence of the combined maturation and strengthening of cell-cell and cell-substrate adhesions. Surprisingly, the increase of cell surface density due to proliferation is only secondary in this process. This analysis is confirmed with two other cell types. The very general relations between the mean cell velocity and velocity correlation lengths, which apply for aggregates of self-propelled particles, as well as motile cells, can possibly be used to discriminate between various parameter changes in vivo, from noninvasive microscopy data. PMID:26627719

  6. Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism.

    PubMed

    Lussey-Lepoutre, Charlotte; Hollinshead, Kate E R; Ludwig, Christian; Menara, Mélanie; Morin, Aurélie; Castro-Vega, Luis-Jaime; Parker, Seth J; Janin, Maxime; Martinelli, Cosimo; Ottolenghi, Chris; Metallo, Christian; Gimenez-Roqueplo, Anne-Paule; Favier, Judith; Tennant, Daniel A

    2015-11-02

    The tricarboxylic acid (TCA) cycle is a central metabolic pathway responsible for supplying reducing potential for oxidative phosphorylation and anabolic substrates for cell growth, repair and proliferation. As such it thought to be essential for cell proliferation and tissue homeostasis. However, since the initial report of an inactivating mutation in the TCA cycle enzyme complex, succinate dehydrogenase (SDH) in paraganglioma (PGL), it has become clear that some cells and tissues are not only able to survive with a truncated TCA cycle, but that they are also able of supporting proliferative phenotype observed in tumours. Here, we show that loss of SDH activity leads to changes in the metabolism of non-essential amino acids. In particular, we demonstrate that pyruvate carboxylase is essential to re-supply the depleted pool of aspartate in SDH-deficient cells. Our results demonstrate that the loss of SDH reduces the metabolic plasticity of cells, suggesting vulnerabilities that can be targeted therapeutically.

  7. Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

    PubMed Central

    Mani, Timmy; Liu, Degang; Zhou, Donghui; Li, Liwei; Knabe, William Eric; Wang, Fang; Oh, Kyungsoo; Meroueh, Samy O.

    2014-01-01

    The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here we evaluate the binding and biological activity of a new class of pyrrolidinone (3) and piperidinone (4) compounds, along with derivatives of previously-identified pyrazole (1) and propylamine (2) compounds. Competition assays revealed that the compounds displaced a fluorescently-labeled peptide (AE147-FAM) with inhibition constant Ki ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics simulations and free energy calculations suggested pyrazole-based 1a and piperidinone-based 4 adopt different binding modes, despite their similar two-dimensional structures. In cells, compounds 1b and 1f showed significant inhibition of breast MDA-MB-231 and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but 4b exhibited no cytotoxicity even at concentrations of 100 μM. 1f impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while 4b inhibited only invasion. 1f inhibited gelatinase (MMP-9) activity in a concentration-dependent manner, while 4b showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinone and piperidinone (3 and 4b) had no effect. Annexin V staining suggested that the effect of pyrazole-based 1f on proliferation was due to cell killing through an apoptotic mechanism. PMID:24115356

  8. Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates

    NASA Astrophysics Data System (ADS)

    Park, Soonhyang; Chibli, Hicham; Wong, Jody; Nadeau, Jay L.

    2011-05-01

    We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.

  9. Retrovolution: HIV-driven evolution of cellular genes and improvement of anticancer drug activation.

    PubMed

    Rossolillo, Paola; Winter, Flore; Simon-Loriere, Etienne; Gallois-Montbrun, Sarah; Negroni, Matteo

    2012-08-01

    In evolution strategies aimed at isolating molecules with new functions, screening for the desired phenotype is generally performed in vitro or in bacteria. When the final goal of the strategy is the modification of the human cell, the mutants selected with these preliminary screenings may fail to confer the desired phenotype, due to the complex networks that regulate gene expression in higher eukaryotes. We developed a system where, by mimicking successive infection cycles with HIV-1 derived vectors containing the gene target of the evolution in their genome, libraries of gene mutants are generated in the human cell, where they can be directly screened. As a proof of concept we created a library of mutants of the human deoxycytidine kinase (dCK) gene, involved in the activation of nucleoside analogues used in cancer treatment, with the aim of isolating a variant sensitizing cancer cells to the chemotherapy compound Gemcitabine, to be used in gene therapy for anti-cancer approaches or as a poorly immunogenic negative selection marker for cell transplantation approaches. We describe the isolation of a dCK mutant, G12, inducing a 300-fold sensitization to Gemcitabine in cells originally resistant to the prodrug (Messa 10K), an effect 60 times stronger than the one induced by the wt enzyme. The phenotype is observed in different tumour cell lines irrespective of the insertion site of the transgene and is due to a change in specificity of the mutated kinase in favour of the nucleoside analogue. The mutations characterizing G12 are distant from the active site of the enzyme and are unpredictable on a rational basis, fully validating the pragmatic approach followed. Besides the potential interest of the G12 dCK variant for therapeutic purposes, the methodology developed is of interest for a large panel of applications in biotechnology and basic research. PMID:22927829

  10. Recent progress with microtubule stabilizers: new compounds, binding modes and cellular activities

    PubMed Central

    Rohena, Cristina C.

    2014-01-01

    Nature has yielded numerous classes of chemically distinct microtubule stabilizers. Several of these, including paclitaxel (Taxol) and docetaxel (Taxotere), are important drugs used in the treatment of cancer. New microtubule stabilizers and novel formulations of these agents continue to provide advances in cancer therapy. In this review we cover recent progress from late 2008 to August 2013 in the chemistry and biology of these diverse microtubule stabilizers focusing on the wide range of organisms that produce these compounds, their mechanisms of inhibiting microtubule-dependent processes, mechanisms of drug resistance, and their interactions with tubulin including their distinct binding sites and modes. A new potential role for microtubule stabilizers in neurodegenerative diseases is reviewed. PMID:24481420

  11. Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides

    PubMed Central

    2004-01-01

    In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861–866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes α-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1–3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER

  12. Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils.

    PubMed

    Hulme-Moir, K Lisa; Clark, Phillip

    2011-07-15

    Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA. PMID:21596444

  13. Exogenous coenzyme Q10 modulates MMP-2 activity in MCF-7 cell line as a breast cancer cellular model

    PubMed Central

    2010-01-01

    Background/Aims Matrix Metalloproteinases 2 is a key molecule in cellular invasion and metastasis. Mitochondrial ROS has been established as a mediator of MMP activity. Coenzyme Q10 contributes to intracellular ROS regulation. Coenzyme Q10 beneficial effects on cancer are still in controversy but there are indications of Coenzyme Q10 complementing effect on tamoxifen receiving breast cancer patients. Methods In this study we aimed to investigate the correlation of the effects of co-incubation of coenzyme Q10 and N-acetyl-L-cysteine (NAC) on intracellular H2O2 content and Matrix Metalloproteinase 2 (MMP-2) activity in MCF-7 cell line. Results and Discussion Our experiment was designed to assess the effect in a time and dose related manner. Gelatin zymography and Flowcytometric measurement of H2O2 by 2'7',-dichlorofluorescin-diacetate probe were employed. The results showed that both coenzyme Q10 and N-acetyl-L-cysteine reduce MMP-2 activity along with the pro-oxidant capacity of the MCF-7 cell in a dose proportionate manner. Conclusions Collectively, the present study highlights the significance of Coenzyme Q10 effect on the cell invasion/metastasis effecter molecules. PMID:21118526

  14. Correlated activity supports efficient cortical processing

    PubMed Central

    Hung, Chou P.; Cui, Ding; Chen, Yueh-peng; Lin, Chia-pei; Levine, Matthew R.

    2015-01-01

    Visual recognition is a computational challenge that is thought to occur via efficient coding. An important concept is sparseness, a measure of coding efficiency. The prevailing view is that sparseness supports efficiency by minimizing redundancy and correlations in spiking populations. Yet, we recently reported that “choristers”, neurons that behave more similarly (have correlated stimulus preferences and spontaneous coincident spiking), carry more generalizable object information than uncorrelated neurons (“soloists”) in macaque inferior temporal (IT) cortex. The rarity of choristers (as low as 6% of IT neurons) indicates that they were likely missed in previous studies. Here, we report that correlation strength is distinct from sparseness (choristers are not simply broadly tuned neurons), that choristers are located in non-granular output layers, and that correlated activity predicts human visual search efficiency. These counterintuitive results suggest that a redundant correlational structure supports efficient processing and behavior. PMID:25610392

  15. Antisperm antibody-mediated alterations in the cellular activity of human trophoblast cells in culture.

    PubMed

    Sinha, D; Chattopadhyay, S

    1994-04-01

    Immune recognition of the fetus is well documented, yet the immunological basis of pregnancy loss awaits elucidation. Identification of trophoblast membrane epitopes as non-self either by preformed immunoglobulins or by circulating immunocompetent cells would lead to immunological rejection of the tissue. Such an event may occur in cases of cross-reacting antibodies developed as a consequence of exposure of sperm surface antigens. This hypothesis was tested by developing specific antibodies in rabbits against intact sperm surface antigens. These were subjected to different schedules of IgG purification and characterization. By means of nuclide precursor incorporation, the effect of antisperm antibody on DNA, RNA and protein synthesis of trophoblast cells in culture were studied. The results showed that the antibody inhibits incorporation into cells but after a delay of 72 hours some cells gradually recover. The interaction also led to a reduced rate of hCG production. Lysosomal enzyme activity was inhibited in the spent medium of antibody-treated cells but lysosome rich fractions showed no effect. This indicated that the major effect of the antibody was growth inhibitory rather than cytolytic. PMID:7520885

  16. Cellular membranes, the sites for the antifungal activity of the herbicide sethoxydim.

    PubMed

    Pakdaman, B S; Goltapeh, E Mohammadi; Sepehrifar, R; Pouriesa, M; Fard, M Rahimi; Moradi, F; Modarres, S A M

    2007-08-01

    The fungicidal effect of sethoxydim on the canola (Brassica napus var. Olifera) white stem rot pathogen (Sclerotinia sclerotiorum) encouraged us to conduct a series of studies on the mechanism of the antifungal activity of this herbicide commonly applied in Iranian fields under canola cultivation. Present preliminary studies on the changes in the level of Malondialdehyde (MDA) as the main product generated through peroxidation of polyunsaturated fatty acids indicated the disintegration of the fungal bilayer of plasma membrane as the result of the herbicidal treatment. Also, it was demonstrated that the amount of hydrogen peroxide in the treated samples was higher than the control samples with no herbicidal treatment. Therefore, our present results confirm the disintegration of the plasma membrane as one of the mechanism for the antifungal impact of sethoxydim. As with weed plants, the phytotoxic impact of this herbicide has been attributed to the inhibition of the first enzyme in the lipid biosynthesis pathway, acetyl-CoA carboxylase, therefore, it would be very interesting to study on this subject and the relations between the sensitivity of different fungi and their DNA and protein sequences of acetyl-CoA carboxylase. PMID:19070118

  17. Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation

    PubMed Central

    Ho, Nathan K.; Ossa, Juan C.; Silphaduang, Uma; Johnson, Roger; Johnson-Henry, Kathene C.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system. PMID:22526675

  18. Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

    PubMed

    Châtel, Amélie; Faucet-Marquis, Virginie; Gourlay-Francé, Catherine; Pfohl-Leszkowicz, Annie; Vincent-Hubert, Françoise

    2015-04-01

    The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution. PMID:24951272

  19. The putative tumor activator ARHGEF3 promotes nasopharyngeal carcinoma cell pathogenesis by inhibiting cellular apoptosis

    PubMed Central

    Guo, Lin; Lin, Huan-Xin; Chen, Jie-Wei; Liao, Yi-Ji; Kung, Hsiang-Fu; Zeng, Yi-Xin; Xie, Dan

    2016-01-01

    Nasopharyngeal carcinoma (NPC) is one of the most prevalent forms of highly invasive malignancy in Southern China and Southeast Asia. The pathogenesis of NPC is a multistep process driven by the acquisition of numerous genetic abnormalities. We investigated the potential oncogenic role of the Rho-guanine nucleotide exchange factor 3 gene, ARHGEF3, in NPC pathogenesis. Expression levels of ARHGEF3 were frequently up-regulated in NPC cell lines and tissues. In a large cohort of clinical NPC tissues high expression of ARHGEF3 was positively associated with an increased T status, distant metastasis, and a more advanced clinical stage (P < 0.05). Survival analysis revealed that ARHGEF3 expression was a significant and independent prognosis factor for NPC patients. In NPC cell lines, knockdown of ARHGEF3 was sufficient to inhibit cell growth, motility, and invasion in vitro, whereas ectopic overexpression of ARHGEF3 substantially enhanced NPC cells tumorigenesis and metastasis in vivo. Depletion of ARHGEF3 in NPC cells dramatically promoted caspase-3 induced apoptosis and an anti-apoptosis factor, BIRC8, was identified as a critical downstream target of the ARHGEF3. Our findings suggest that increased expression of ARHGEF3 plays a critical oncogenic role in NPC pathogenesis by preventing cell apoptosis through the up-regulation of BIRC8, and ARHGEF3 might be employed as a novel prognostic marker and effective therapeutic target for human NPC. PMID:27028992

  20. Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

    PubMed

    Châtel, Amélie; Faucet-Marquis, Virginie; Gourlay-Francé, Catherine; Pfohl-Leszkowicz, Annie; Vincent-Hubert, Françoise

    2015-04-01

    The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution.

  1. Cellular Trojan horse based polymer nanoreactors with light-sensitive activity.

    PubMed

    Baumann, Patric; Spulber, Mariana; Dinu, Ionel Adrian; Palivan, Cornelia G

    2014-08-01

    production, stability, and Trojan horse activity inside cells support our light-sensitive nanoreactors for medical applications which require ROS to be generated with precise time and space control.

  2. Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro

    PubMed Central

    Pogany, Judit

    2015-01-01

    complex, the viral RdRp becomes activated through an incompletely understood process that makes the RdRp capable of RNA synthesis. By using TBSV RdRp, we show that the co-opted cellular Hsp70 chaperone and neutral phospholipids facilitate RdRp activation in vitro. In contrast, phosphatidylglycerol (PG) has a dominant inhibitory effect on in vitro RdRp activation and RdRp-viral RNA interaction, suggesting that the membranous microdomain surrounding the RdRp greatly affects its ability for RNA synthesis. Thus, the activation of the viral RdRp likely depends on multiple host components in infected cells. PMID:25762742

  3. Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.

    PubMed

    Condori, Jose; Acosta, Walter; Ayala, Jorge; Katta, Varun; Flory, Ashley; Martin, Reid; Radin, Jonathan; Cramer, Carole L; Radin, David N

    2016-02-01

    cleaved and rapidly degraded. The activated β-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.

  4. Activation of nuclear factor-kappaB and not activator protein-1 in cellular response to nickel compounds.

    PubMed Central

    Huang, Yi; Davidson, Gerard; Li, Jingxia; Yan, Yan; Chen, Fei; Costa, Max; Chen, Lung Chi; Huang, Chuanshu

    2002-01-01

    The predominant exposure route for nickel compounds is by inhalation, and several studies have indicated the correlation between nickel exposure and respiratory cancers. The tumor-promoting effects of nickel compounds are thought to be associated with their transactivation of transcription factors. We have investigated the possible activation of activator protein-1 (AP-1) and nuclear factor KB (NF-kappaB) in mouse C141 epidermal cells and fibroblasts 3T3 and B82, and human bronchoepithelial BEAS-2B cells in response to nickel compound exposure. Our results show that NF-kappaB activity is induced by nickel exposure in 3T3 and BEAS-2B cells. Conversely, similar nickel treatment of these cells did not induce AP-1 activity, suggesting that nickel tumorigenesis occurs through NF-kappaB and not AP-1. We also investigated the role of NF-kappaB in the induction of Cap43 by nickel compounds using dominant negative mutant Ikappabeta kinase b-KM BEAS-2B transfectants. PMID:12426142

  5. Ethanol Extract of Ganoderma lucidum Augments Cellular Anti-oxidant Defense through Activation of Nrf2/HO-1

    PubMed Central

    Lee, Yoo-hwan; Kim, Jung-hee; Song, Choon-ho; Jang, Kyung-jeon; kim, Cheol-hong; Kang, Ji- Sook; Choi, Yung-hyun

    2016-01-01

    Objectives: The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, H2O2) and elucidated the underlying mechanisms in a C2C12 myoblast cell line. Methods: Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and H2O2 in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and H2O2-induced growth inhibition. Results: The results showed that EGL effectively inhibited H2O2-induced growth and the generation of ROS. EGL markedly suppressed H2O2-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 (p-γH2AX), a widely used marker of DNA damage, suggesting that EGL prevented H2O2-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against H2O2-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner. Conclusion: Collectively, these results demonstrate that EGL augments the

  6. Phyto-mediated nanostructured carriers based on dual vegetable actives involved in the prevention of cellular damage.

    PubMed

    Istrati, D; Lacatusu, I; Bordei, N; Badea, G; Oprea, O; Stefan, L M; Stan, R; Badea, N; Meghea, A

    2016-07-01

    The growing scientific interest in exploitation of vegetable bioactives has raised a number of questions regarding their imminent presence in pharmaceutical formulations. This study intends to demonstrate that a dual combination between vegetable oil (e.g. thistle oil, safflower oil, sea buckthorn oil) and a carrot extract represents an optimal approach to formulate safe carrier systems that manifest cell regeneration effect and promising antioxidant and anti-inflammatory activity. Inclusion of both natural actives into lipid carriers imparted a strong negative charge on the nanocarrier surface (up to -45mV) and displayed average sizes of 70nm to 140nm. The entrapment efficiency of carrot extract into nanostructured carriers ranged between 78.3 and 88.3%. The in vitro release study has demonstrated that the entrapment of the extract represents a viable way for an equilibrated release of carotenoids. Besides the excellent antioxidant properties (e.g. scavenging up to 98% of the free oxygen radicals), the results of cellular integrity (e.g. cell viability of 133%) recommend these nanocarriers based on dual carrot extract-bioactive oil as a promising trend for the treatment of certain disorders in which oxidative stress plays a prominent role. In addition, the lipid nanocarriers based on safflower oil and sea buckthorn oil demonstrated an anti-inflammatory effect on LPS induced THP-1 macrophages, by inhibiting the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α. PMID:27127051

  7. Phyto-mediated nanostructured carriers based on dual vegetable actives involved in the prevention of cellular damage.

    PubMed

    Istrati, D; Lacatusu, I; Bordei, N; Badea, G; Oprea, O; Stefan, L M; Stan, R; Badea, N; Meghea, A

    2016-07-01

    The growing scientific interest in exploitation of vegetable bioactives has raised a number of questions regarding their imminent presence in pharmaceutical formulations. This study intends to demonstrate that a dual combination between vegetable oil (e.g. thistle oil, safflower oil, sea buckthorn oil) and a carrot extract represents an optimal approach to formulate safe carrier systems that manifest cell regeneration effect and promising antioxidant and anti-inflammatory activity. Inclusion of both natural actives into lipid carriers imparted a strong negative charge on the nanocarrier surface (up to -45mV) and displayed average sizes of 70nm to 140nm. The entrapment efficiency of carrot extract into nanostructured carriers ranged between 78.3 and 88.3%. The in vitro release study has demonstrated that the entrapment of the extract represents a viable way for an equilibrated release of carotenoids. Besides the excellent antioxidant properties (e.g. scavenging up to 98% of the free oxygen radicals), the results of cellular integrity (e.g. cell viability of 133%) recommend these nanocarriers based on dual carrot extract-bioactive oil as a promising trend for the treatment of certain disorders in which oxidative stress plays a prominent role. In addition, the lipid nanocarriers based on safflower oil and sea buckthorn oil demonstrated an anti-inflammatory effect on LPS induced THP-1 macrophages, by inhibiting the secretion of two pro-inflammatory cytokines, IL-6 and TNF-α.

  8. Multi-scale continuum modeling of biological processes: from molecular electro-diffusion to sub-cellular signaling transduction

    NASA Astrophysics Data System (ADS)

    Cheng, Y.; Kekenes-Huskey, P.; Hake, J. E.; Holst, M. J.; McCammon, J. A.; Michailova, A. P.

    2012-01-01

    This paper presents a brief review of multi-scale modeling at the molecular to cellular scale, with new results for heart muscle cells. A finite element-based simulation package (SMOL) was used to investigate the signaling transduction at molecular and sub-cellular scales (http://mccammon.ucsd.edu/smol/, http://FETK.org) by numerical solution of the time-dependent Smoluchowski equations and a reaction-diffusion system. At the molecular scale, SMOL has yielded experimentally validated estimates of the diffusion-limited association rates for the binding of acetylcholine to mouse acetylcholinesterase using crystallographic structural data. The predicted rate constants exhibit increasingly delayed steady-state times, with increasing ionic strength, and demonstrate the role of an enzyme's electrostatic potential in influencing ligand binding. At the sub-cellular scale, an extension of SMOL solves a nonlinear, reaction-diffusion system describing Ca2+ ligand buffering and diffusion in experimentally derived rodent ventricular myocyte geometries. Results reveal the important role of mobile and stationary Ca2+ buffers, including Ca2+ indicator dye. We found that alterations in Ca2+-binding and dissociation rates of troponin C (TnC) and total TnC concentration modulate sub-cellular Ca2+ signals. The model predicts that reduced off-rate in the whole troponin complex (TnC, TnI, TnT) versus reconstructed thin filaments (Tn, Tm, actin) alters cytosolic Ca2+ dynamics under control conditions or in disease-linked TnC mutations. The ultimate goal of these studies is to develop scalable methods and theories for the integration of molecular-scale information into simulations of cellular-scale systems.

  9. The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking.

    PubMed Central

    Lusson, J; Benjannet, S; Hamelin, J; Savaria, D; Chrétien, M; Seidah, N G

    1997-01-01

    In order to define the functional importance of the conserved RRGDL motif in the P-domain of the mammalian proprotein convertases(PCs) we generated and cellularly expressed three mutant PC1 vaccinia-virus (VV) recombinants: ARGDL-PC1, RAGDL-PC1 and RRGEL-PC1. Functionally, these mutants caused a decreased level of processing of pro-opiomelanocortin (POMC) into beta-lipotropic pituitary hormone (beta-LPH), especially in the constitutively secreting BSC40 cells. Pulse-chase analyses demonstrated that, in part, this effect was due to both an increased degradation of the mutant PC1s within the endoplasmic reticulum and to a diminished level of zymogen processing in the same compartment. In addition, within cells containing secretory granules such as PC12 and GH4C1 cells, such mutations prevented the C-terminal auto-processing of PC1 into the fully mature 66 kDa form stored in the secretory granules of regulated cells. Since the 66 kDa PC1 is the most active form of the enzyme, it is proposed that the RRGDL sequence is critical for the generation of maximal intracellular PC1 activity. In regulated cells, co-expression of POMC with PC1 or its mutants together with the general PC inhibitor alpha1-antitrypsin Portland (alpha1-PDX), which acts primarily within the constitutive secretory pathway, demonstrated that the latter completely inhibited the formation of beta-LPH by PC1 mutants, whereas it only partially inhibited the ability of wild-type PC1 to process POMC. This suggests that RRGDL mutations prevent PC1 from entering secretory granules and hence the formation of the 66 kDa PC1, and result in the mis-sorting of PC1 mutants towards the constitutive secretory pathway. This conclusion was further supported by immunocytochemical data demonstrating that RRGDL mutants exhibit an intracellular localization pattern different from that of the granule-associated wild-type PC1,but similar to that of the Golgi-localized convertase PC5-B. PMID:9307023

  10. p-process nucleosynthesis: Activation experiments

    NASA Astrophysics Data System (ADS)

    Glorius, J.; Knörzer, M.; Müller, S.; Pietralla, N.; Sauerwein, A.; Sonnabend, K.; Wälzlein, C.; Wiescher, M.

    2011-04-01

    For the astrophysical p process a complex reaction network has to be solved. In the order of 10,000 theoretically predicted reaction rates are needed for simulations of this network. For reactions involving α particles or protons, the predictions in the framework of the Hauser-Feshbach (HF) model were found to deviate from experimental results partially by a factor of 5 or even more. To optimize the predictive power of the applied HF codes, the nuclear physics input has to be improved. For this purpose, the reactions 166ErTm(p,n) as well as the reaction 170Yb(γ,n) have been measured with the activation method at low energies. The data can provide a further test of HF predictions but can also be used to optimize input parameters of the afore mentioned codes. Preliminary results of the experiments are presented and compared to theoretical predictions using the standard settings of the HF codes NON-SMOKER and TALYS.

  11. Rapid TCR-mediated SHP-1 S591 phosphorylation regulates SHP-1 cellular localization and phosphatase activity

    PubMed Central

    Liu, Yin; Kruhlak, Michael J.; Hao, Jian-Jiang; Shaw, Stephen

    2007-01-01

    Since the tyrosine phosphatase SHP-1 plays a major role in regulating T-cell signaling, we investigated regulation thereof by Ser/Thr phosphorylation. We found that TCR stimulation induced fast (≤1min) and transient phosphorylation of SHP-1 S591 in both Jurkat and human peripheral blood T-cells (PBT). Phosphorylation of S591 in T-cells could be mediated artificially by a constitutive active PKC-theta construct, but the dose dependence of inhibition by PKC inhibitors indicated that PKCs were not the relevant basophilic kinase in the physiologic response. S591 phosphorylation inhibited phosphatase function since a S591D mutant had lower activity than the S591A mutant. Additional evidence that S591 phosphorylation alters SHP-1 function was provided by studies of Jurkat cells stably expressing SHP-1 wildtype or mutants. In those cells, S591D mutation reduced the capacity of transfected SHP-1 to inhibit TCR-induced phosphorylation of PLC-γ1. Interestingly, SHP-1 Y536 phosphorylation (previously shown to augment phosphatase activity) was also induced in PBT by TCR signal but at a much later time compared to S591 (~30 min). S591 phosphorylation also altered cellular distribution of SHP-1 because: 1) SHP-1 in lipid rafts and a sheared membrane fraction was hypo-phosphorylated; 2) In stably transfected Jurkat cell lines, S591D mutant protein had reduced presence in both lipid raft and the sheared membrane fraction; 3) S591 phosphorylation prevented nuclear localization of a C-terminal GFP tagged SHP-1 construct. Our studies also shed light on an additional mechanism regulating SHP-1 nuclear localization, namely conformational autoinhibition. These findings highlight elegant regulation of SHP-1 by sequential phosporylation of serine then tyrosine. PMID:17575265

  12. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    PubMed

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer. PMID:27260468

  13. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    PubMed

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer.

  14. Regulation of HTLV-1 tax stability, cellular trafficking and NF-κB activation by the ubiquitin-proteasome pathway.

    PubMed

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-10-23

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.

  15. Cellular and subcellular distributions of delta opioid receptor activation sites in the ventral oral pontine tegmentum of the cat.

    PubMed

    Alvira-Botero, Maria Ximena; Garzón, Miguel

    2006-12-01

    The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

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

    PubMed Central

    2010-01-01

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

  17. Cellular Functions of Tissue Transglutaminase

    PubMed Central

    Nurminskaya, Maria V.; Belkin, Alexey M.

    2013-01-01

    Transglutaminase 2 (TG2 or tissue transglutaminase) is a highly complex multifunctional protein that acts as transglutaminase, GTPase/ATPase, protein disulfide isomerase, and protein kinase. Moreover, TG2 has many well-documented nonenzymatic functions that are based on its noncovalent interactions with multiple cellular proteins. A vast array of biochemical activities of TG2 accounts for its involvement in a variety of cellular processes, including adhesion, migration, growth, survival, apoptosis, differentiation, and extracellular matrix organization. In turn, the impact of TG2 on these processes implicates this protein in various physiological responses and pathological states, contributing to wound healing, inflammation, autoimmunity, neurodegeneration, vascular remodeling, tumor growth and metastasis, and tissue fibrosis. TG2 is ubiquitously expressed and is particularly abundant in endothelial cells, fibroblasts, osteoblasts, monocytes/macrophages, and smooth muscle cells. The protein is localized in multiple cellular compartments, including the nucleus, cytosol, mitochondria, endolysosomes, plasma membrane, and cell surface and extracellular matrix, where Ca2+, nucleotides, nitric oxide, reactive oxygen species, membrane lipids, and distinct protein–protein interactions in the local microenvironment jointly regulate its activities. In this review, we discuss the complex biochemical activities and molecular interactions of TG2 in the context of diverse subcellular compartments and evaluate its wide ranging and cell type-specific biological functions and their regulation. PMID:22364871

  18. Multiphoton microscopy can visualize zonal damage and decreased cellular metabolic activity in hepatic ischemia-reperfusion injury in rats

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Liu, Xin; Burczynski, Frank J.; Fletcher, Linda M.; Gobe, Glenda C.; Roberts, Michael S.

    2011-11-01

    Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery. In orthotopic transplantation, the donor liver is exposed to periods of ischemia and when oxygenated blood is reintroduced to the liver, oxidative stress may develop and lead to graft failure. The aim of this project was to investigate whether noninvasive multiphoton and fluorescence lifetime imaging microscopy, without external markers, were useful in detecting early liver damage caused by I/R injury. Localized hepatic ischemia was induced in rats for 1 h followed by 4 h reperfusion. Multiphoton and fluorescence lifetime imaging microscopy was conducted prior to ischemia and up to 4 h of reperfusion and compared to morphological and biochemical assessment of liver damage. Liver function was significantly impaired at 2 and 4 h of reperfusion. Multiphoton microscopy detected liver damage at 1 h of reperfusion, manifested by vacuolated cells and heterogeneous spread of damage over the liver. The damage was mainly localized in the midzonal region of the liver acinus. In addition, fluorescence lifetime imaging showed a decrease in cellular metabolic activity. Multiphoton and fluorescence lifetime imaging microscopy detected evidence of early I/R injury both structurally and functionally. This provides a simple noninvasive technique useful for following progressive liver injury without external markers.

  19. Structure of modified ε-polylysine micelles and their application in improving cellular antioxidant activity of curcuminoids

    PubMed Central

    Yu, Hailong; Li, Ji; Shi, Ke; Huang, Qingrong

    2011-01-01

    The micelle structure of octenyl succinic anhydride modified ε-polylysine (M-EPL), an anti-microbial surfactant prepared from natural peptide ε-polylysine in aqueous solution has been studied using synchrotron small-angle X-ray scattering (SAXS). Our results revealed that M-EPLs formed spherical micelles with individual size of 24–26 Å in aqueous solution which could further aggregate to form a larger dimension with averaged radius of 268–308 Å. Furthermore, M-EPL micelle was able to encapsulate curcuminoids, a group of poorly-soluble bioactive compounds from turmeric with poor oral bioavailability, and improve their water solubility. Three loading methods, including solvent evaporation, dialysis, and high-speed homogenization were compared. The results indicated that the dialysis method generated the highest loading capacity and curcuminoids water solubility. The micelle encapsulation was confirmed as there were no free curcuminoid crystals detected in the differential scanning calorimetry analysis. It was also demonstrated that M-EPL encapsulation stabilized curcuminoids against hydrolysis at pH 7.4 and the encapsulated curcuminoids showed elevated cellular antioxidant activity compared with free curcuminoids. This work suggested that M-EPL could be used as new biopolymer micelles for delivering poorly soluble drugs/phytochemicals and improving their bioactivities. PMID:21894323

  20. The influence of temperature on the electrical resistivity of the cellular polypropylene and the effect of activation energy.

    PubMed

    Vila, Floran; Dhima, Pranvera; Mandija, Florian

    2013-01-01

    In this paper, we determine the surface and volume electrical resistivity of the 50 μm thick cellular polypropylen (VHD50), for the temperature range 393-453 K. For this we use a contemporary methodology, which consist of a voltage measurement across the sample, with a known current flowing through it. This methodology includes a three-electrode system, which allows us to estimate the resistivity of the samples, based on their corresponding total resistances. The electric fields applied for a time interval of 1 min are of the order of 200 kVm (-1). The order of magnitude of surface and volume electrical resistivity is 10(13) Ω and 10(11) Ωm, respectively. For both types of the resistivity, the temperature dependence is an increasing or decreasing exponential function, depending on the type of the activation energy, (its average value for the temperature range mentioned above is 41,20 kJmol (-1)), totally confirmed by the corresponding theoretical interpretation, conditioned by the ionic conduction. The methodology and equipment used, as well as the satisfying accordance with the results, found out directly or indirectly with the consulted literature, confirm the high accuracy of experimental measurements. PMID:25674393

  1. Structure of modified [epsilon]-polylysine micelles and their application in improving cellular antioxidant activity of curcuminoids

    SciTech Connect

    Yu, Hailong; Li, Ji; Shi, Ke; Huang, Qingrong

    2015-10-15

    The micelle structure of octenyl succinic anhydride modified {var_epsilon}-polylysine (M-EPL), an anti-microbial surfactant prepared from natural peptide {var_epsilon}-polylysine in aqueous solution has been studied using synchrotron small-angle X-ray scattering (SAXS). Our results revealed that M-EPLs formed spherical micelles with individual size of 24-26 {angstrom} in aqueous solution which could further aggregate to form a larger dimension with averaged radius of 268-308 {angstrom}. Furthermore, M-EPL micelle was able to encapsulate curcuminoids, a group of poorly-soluble bioactive compounds from turmeric with poor oral bioavailability, and improve their water solubility. Three loading methods, including solvent evaporation, dialysis, and high-speed homogenization were compared. The results indicated that the dialysis method generated the highest loading capacity and curcuminoids water solubility. The micelle encapsulation was confirmed as there were no free curcuminoid crystals detected in the differential scanning calorimetry analysis. It was also demonstrated that M-EPL encapsulation stabilized curcuminoids against hydrolysis at pH 7.4 and the encapsulated curcuminoids showed elevated cellular antioxidant activity compared with free curcuminoids. This work suggested that M-EPL could be used as new biopolymer micelles for delivering poorly soluble drugs/phytochemicals and improving their bioactivities.

  2. Effects of Activating Mutations on EGFR Cellular Protein Turnover and Amino Acid Recycling Determined Using SILAC Mass Spectrometry

    PubMed Central

    Greig, Michael J.; Niessen, Sherry; Weinrich, Scott L.; Feng, Jun Li; Shi, Manli; Johnson, Ted O.

    2015-01-01

    Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates. PMID:26689952

  3. In Rasmussen Encephalitis, Hemichannels Associated with Microglial Activation are linked to Cortical Pyramidal Neuron Coupling: A Possible Mechanism for Cellular Hyperexcitability

    PubMed Central

    Cepeda, Carlos; Chang, Julia W.; Owens, Geoffrey C.; Huynh, My N.; Chen, Jane Y.; Tran, Conny; Vinters, Harry V.; Levine, Michael S.; Mathern, Gary W.

    2014-01-01

    Aims Rasmussen encephalitis (RE) is a rare but devastating condition, mainly in children, characterized by sustained brain inflammation, atrophy of one cerebral hemisphere, epilepsy and progressive cognitive deterioration. The etiology of RE-induced seizures associated with the inflammatory process remains unknown. Methods Cortical tissue samples from children undergoing surgical resections for the treatment of RE (n=16) and non-RE (n=12) were compared using electrophysiological, morphological, and immunohistochemical techniques to examine neuronal properties and the relationship with microglial activation using the specific microglia/macrophage calcium-binding protein, IBA1 in conjunction with connexins and pannexin expression. Results Compared with non-RE cases, pyramidal neurons from RE cases displayed increased cell capacitance and reduced input resistance. However, neuronal somatic areas were not increased in size. Instead, intracellular injection of biocytin led to increased dye-coupling between neurons from RE cases. By Western blot, expression of IBA1 and pannexin was increased while connexin 32 was decreased in RE cases compared with non-RE cases. IBA1 immunostaining overlapped with pannexin and connexin 36 in RE cases. Conclusions In RE, these results support the notion that a possible mechanism for cellular hyperexcitability may be related to increased intercellular coupling from pannexin linked to increased microglial activation. Such findings suggest that a possible anti-seizure treatment for RE may involve the use of gap junction blockers. PMID:25438677

  4. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

    PubMed

    Yu, Ker; Toral-Barza, Lourdes; Shi, Celine; Zhang, Wei-Guo; Lucas, Judy; Shor, Boris; Kim, Jamie; Verheijen, Jeroen; Curran, Kevin; Malwitz, David J; Cole, Derek C; Ellingboe, John; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Gibbons, James J; Abraham, Robert T; Nowak, Pawel; Zask, Arie

    2009-08-01

    The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

  5. Perfluorinated chemicals: Differential toxicity, inhibition of aromatase activity and alteration of cellular lipids in human placental cells

    SciTech Connect

    Gorrochategui, Eva; Pérez-Albaladejo, Elisabet; Casas, Josefina; Lacorte, Sílvia; Porte, Cinta

    2014-06-01

    The cytotoxicity of eight perfluorinated chemicals (PFCs), namely, perfluorobutanoic acid (PFBA), perfluorohexanoic acid (PFHxA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorododecanoic acid (PFDoA), perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHxS) and perfluorooctanesulfonate (PFOS) was assessed in the human placental choriocarcinoma cell line JEG-3. Only the long chain PFCs – PFOS, PFDoA, PFNA, PFOA – showed significant cytotoxicity in JEG-3 cells with EC50 values in the range of 107 to 647 μM. The observed cytotoxicity was to some extent related to a higher uptake of the longer chain PFCs by cells (PFDoA > PFOS ≫ PFNA > PFOA > PFHxA). Moreover, this work evidences a high potential of PFOS, PFOA and PFBS to act as aromatase inhibitors in placental cells with IC50s in the range of 57–80 μM, the inhibitory effect of PFBS being particularly important despite the rather low uptake of the compound by cells. Finally, exposure of JEG-3 cells to a mixture of the eight PFCs (0.6 μM each) led to a relative increase (up to 3.4-fold) of several lipid classes, including phosphatidylcholines (PCs), plasmalogen PC and lyso plasmalogen PC, which suggests an interference of PFCs with membrane lipids. Overall, this work highlights the ability of the PFC mixture to alter cellular lipid pattern at concentrations well below those that generate toxicity, and the potential of the short chain PFBS, often considered a safe substitute of PFOS, to significantly inhibit aromatase activity in placental cells. - Highlights: • Eight perfluorinated chemicals of different chain lengths have been selected. • Long chain ones – PFOS, PFDoA, PFNA, PFOA – were cytotoxic in placenta cells. • The uptake of long chain perfluorinated chemicals by cells was comparatively higher. • PFOS, PFOA and the short chain PFBS significantly inhibited aromatase activity. • A mixture of perfluorinated chemicals significantly altered placenta cell

  6. Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4.

    PubMed

    Chen, Qiongzhen; Tu, Hui; Huang, Fei; Wang, Yicheng; Dong, Weiliang; Wang, Wenhui; Li, Zhoukun; Wang, Fei; Cui, Zhongli

    2016-06-01

    LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para-nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL-ΔpnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL-ΔpnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, γ-aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL-ΔpnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources. PMID:27190157

  7. The NS1 Protein of Influenza A Virus Interacts with Cellular Processing Bodies and Stress Granules through RNA-Associated Protein 55 (RAP55) during Virus Infection

    PubMed Central

    Mok, Bobo Wing-Yee; Song, Wenjun; Wang, Pui; Tai, Hung; Chen, Yixin; Zheng, Min; Wen, Xi; Lau, Siu-Ying; Wu, Wai Lan; Matsumoto, Ken

    2012-01-01

    The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism. PMID:22973032

  8. Carbon dioxide enrichment alleviates heat stress by improving cellular redox homeostasis through an ABA-independent process in tomato plants.

    PubMed

    Li, X; Ahammed, G J; Zhang, Y Q; Zhang, G Q; Sun, Z H; Zhou, J; Zhou, Y H; Xia, X J; Yu, J Q; Shi, K

    2015-01-01

    Plant responses to elevated CO₂ and high temperature are critically regulated through a complex network of phytohormones and redox homeostasis. However, the involvement of abscisic acid (ABA) in plant adaptation to heat stress under elevated CO₂ conditions has not been thoroughly studied. This study investigated the interactive effects of elevated CO₂ (800 μmol·mol(-1) ) and heat stress (42 °C for 24 h) on the endogenous level of ABA and the cellular redox state of two genotypes of tomato with different ABA biosynthesis capacities. Heat stress significantly decreased maximum photochemical efficiency of PSII (Fv/Fm) and leaf water potential, but also increased levels of malondialdehyde (MDA) and electrolyte leakage (EL) in both genotypes. Heat-induced damage was more severe in the ABA-deficient mutant notabilis (not) than in its parental cultivar Ailsa Craig (Ailsa), suggesting that a certain level of endogenous ABA is required to minimise the heat-induced oxidative damage to the photosynthetic apparatus. Irrespective of genotype, the enrichment of CO₂ remarkably stimulated Fv/Fm, MDA and EL in heat-stressed plants towards enhanced tolerance. In addition, elevated CO₂ significantly strengthened the antioxidant capacity of heat-stressed tomato seedlings towards a reduced cellular redox state for a prolonged period, thereby mitigating oxidative stress. However, elevated CO₂ and heat stress did not alter the endogenous level of ABA or the expression of its biosynthetic gene NCED2 in either genotype, indicating that ABA is not involved in elevated CO₂ -induced heat stress alleviation. The results of this study suggest that elevated CO₂ alleviated heat stress through efficient regulation of the cellular redox poise in an ABA-independent manner in tomato plants.

  9. Asymmetrical hemisphere activation enhances global-local processing.

    PubMed

    Gable, Philip A; Poole, Bryan D; Cook, Mary S

    2013-12-01

    Decades of research focusing on the neurophysiological underpinnings related to global-local processing of hierarchical stimuli have associated global processing with the right hemisphere and local processing with the left hemisphere. The current experiment sought to expand this research by testing the causal contributions of hemisphere activation to global-local processing. To manipulate hemisphere activation, participants engaged in contralateral hand contractions. Then, EEG activity and attentional scope were measured. Right-hand contractions caused greater relative left-cortical activity than left-hand contractions. Participants were more narrowly focused after left-hemisphere activation than after right-hemisphere activation. Moreover, N1 amplitudes to local targets in the left hemisphere were larger after left-hemisphere activation than after right-hemisphere activation. Consistent with past research investigating hemispheric asymmetry and attentional scope, the current results suggest that manipulating left (right) hemisphere activity enhanced local (global) attentional processing.

  10. Transcriptional activation of the herpes simplex virus type 1 UL38 promoter conferred by the cis-acting downstream activation sequence is mediated by a cellular transcription factor.

    PubMed Central

    Guzowski, J F; Singh, J; Wagner, E K

    1994-01-01

    The herpes simplex virus (HSV) type 1 strict late (gamma) UL38 promoter contains three cis-acting transcriptional elements: a TATA box, a specific initiator element, and the downstream activation sequence (DAS). DAS is located between positions +20 and +33 within the 5' untranslated leader region and strongly influences transcript levels during productive infection. In this communication, we further characterize DAS and investigate its mechanism of action. DAS function has a strict spacing requirement, and DAS contains an essential 6-bp core element. A similarly positioned element from the gamma gC gene (UL44) has partial DAS function within the UL38 promoter context, and the promoter controlling expression of the gamma US11 transcript contains an identically located element with functional and sequence similarity to UL38 DAS. These data suggest that downstream elements are a common feature o