Sample records for cellular mrna degradation

  1. Hfq affects mRNA levels independently of degradation

    Directory of Open Access Journals (Sweden)

    Hajnsdorf Eliane


    Full Text Available Abstract Background The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. Results The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain. Conclusions The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript

  2. m6A RNA Modification Determines Cell Fate by Regulating mRNA Degradation. (United States)

    Guo, Minjun; Liu, Xinhui; Zheng, Xiaotong; Huang, Yinghui; Chen, Xuechai


    Emerging evidence suggests that epitranscriptional modifications influence multiple cellular processes. N6-methyladenosine (m6A), as the most abundant reversible methylation of mRNA, has also been reported to play critical roles in modulating embryonic stem cell differentiation and somatic cell reprogramming by regulating gene expression. This review examined the characteristics of m6A, including the distribution profile and currently discovered "writer," "eraser," and "reader" proteins. Moreover, the hypothesis is proposed that m6A could influence cell fate determination, and the underlying mechanisms are due to the related mRNA degradation, causing weakening of previous cell characteristics and eventually leading them to develop into the reverse direction (pluripotency or differentiation state). Accordingly, m6A modifications presented its potential role in cell fate determination, which provides new insights into understanding the mechanisms of various diseases.

  3. Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli. (United States)

    Chen, Huiyi; Shiroguchi, Katsuyuki; Ge, Hao; Xie, Xiaoliang Sunney


    An essential part of gene expression is the coordination of RNA synthesis and degradation, which occurs in the same cellular compartment in bacteria. Here, we report a genome-wide RNA degradation study in Escherichia coli using RNA-seq, and present evidence that the stereotypical exponential RNA decay curve obtained using initiation inhibitor, rifampicin, consists of two phases: residual RNA synthesis, a delay in the interruption of steady state that is dependent on distance relative to the mRNA's 5' end, and the exponential decay. This gives a more accurate RNA lifetime and RNA polymerase elongation rate simultaneously genome-wide. Transcripts typically have a single RNA decay constant along all positions, which is distinct between different operons, indicating that RNA stability is unlikely determined by local sequences. These measurements allowed us to establish a model for RNA processing involving co-transcriptional degradation, providing quantitative description of the macromolecular coordination in gene expression in bacteria on a system-wide level. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  4. Cellular volume is a global controller of mRNA abundance (United States)

    Padovan-Merhar, Olivia; Raj, Arjun


    Many researchers have observed large variability in the numbers of RNA and protein molecules from cell to cell, a phenomenon thought to result from random bursts of transcription. These findings hold even for genes involved in core cellular processes, raising questions as to how cells can function in the presence of such molecular noise. However, biochemical processes typically depend on concentrations of cellular constituents rather than absolute numbers, so we use RNA fluorescence in situ hybridization to measure mRNA counts and cellular volume in single cells. We find that while both mRNA numbers and volume vary widely between cells, mRNA density does not. Thus, for many genes, mRNA abundance is precisely controlled to match the volume of the cell, as though the genes know how big the cell is. We measure transcription on a global and single-gene scale, and find that transcriptional activity scales with volume, suggesting that density is regulated at a transcriptional level. We present a mathematical model explaining which transcriptional bursting parameters account for the presence or lack of density conservation. Our findings suggest that global properties of RNA dynamics require a reassessment of our understanding of cellular heterogeneity and stochastic gene expression.

  5. Simplified stochastic models with time delay for studying the degradation process of mRNA molecules. (United States)

    Tian, Tianhai


    Message RNA (mRNA) is the template for protein synthesis. It carries information from DNA in the nucleus to the ribosome sites of protein synthesis in the cell. The turnover process of mRNA is a chemical event with multiple small step reactions and the degradation of mRNA molecules is an important step in gene expression. A number of mathematical models have been proposed to study the dynamics of mRNA turnover, ranging from a one-step first order reaction model to the linear multi-component models. Although the linear multi-component models provide detailed dynamics of mRNA degradation, the simple first-order reaction model has been widely used in mathematical modelling of genetic regulatory networks. To illustrate the difference between these models, we first considered a stochastic model based on the multi-component model. Then a simpler stochastic model was proposed to approximate the linear multi-component model. We also discussed the delayed one-step reaction models with different types of time delay, including the constant delay, exponentially distributed delay and Erlang distributed delay. The comparison study suggested that the one-step reaction models failed to realise the dynamics of mRNA turnover accurately. Therefore, more sophisticated one-step reaction models are needed to study the dynamics of mRNA degradation.

  6. The role of IRE1alpha in the degradation of insulin mRNA in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Kathryn L Lipson

    Full Text Available BACKGROUND: The endoplasmic reticulum (ER is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR. IRE1alpha is a central component of the UPR. In pancreatic beta-cells, IRE1alpha also functions in the regulation of insulin biosynthesis. PRINCIPAL FINDINGS: Here we report that hyperactivation of IRE1alpha caused by chronic high glucose treatment or IRE1alpha overexpression leads to insulin mRNA degradation in pancreatic beta-cells. Inhibition of IRE1alpha signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1alpha retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates. CONCLUSIONS/SIGNIFICANCE: These results reveal a role of IRE1alpha in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of beta-cell homeostasis and may explain why the beta-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of "neo-autoantigens."

  7. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells. (United States)

    Nakagawa, K; Lokugamage, K G; Makino, S


    Coronaviruses have large positive-strand RNA genomes that are 5' capped and 3' polyadenylated. The 5'-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15-16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3'-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5' leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. © 2016 Elsevier Inc. All rights reserved.

  8. Interference of a speB 5' untranslated region partial deletion with mRNA degradation in Streptococcus pyogenes. (United States)

    Chen, Z; Mashburn-Warren, L; Merritt, J; Federle, M J; Kreth, J


    The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

    Directory of Open Access Journals (Sweden)

    Makio Saeki

    Full Text Available Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

  10. Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation.


    Hallahan, D E; Spriggs, D R; Beckett, M A; Kufe, D W; Weichselbaum, R. R.


    We report that tumor necrosis factor alpha (TNF-alpha) mRNA is increased after treatment with x-rays in certain human sarcoma cells. An increase in TNF-alpha mRNA is accompanied by the increased production of TNF-alpha protein. TNF-alpha enhances radiation lethality in both TNF-alpha-producing and -nonproducing tumor cells. These data suggest that, in addition to the direct cytotoxic effects of x-rays, production of TNF-alpha may add to radiation lethality through autocrine and paracrine mech...

  11. Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts. (United States)

    Golan-Lavi, Roni; Giacomelli, Chiara; Fuks, Garold; Zeisel, Amit; Sonntag, Johanna; Sinha, Sanchari; Köstler, Wolfgang; Wiemann, Stefan; Korf, Ulrike; Yarden, Yosef; Domany, Eytan


    Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts

    Directory of Open Access Journals (Sweden)

    Roni Golan-Lavi


    Full Text Available Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF. Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles.

  13. Interactions between mRNA export commitment, 3'-end quality control, and nuclear degradation

    DEFF Research Database (Denmark)

    Libri, Domenico; Dower, Ken; Boulay, Jocelyne


    Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional el...

  14. Smart Magnetic Nanosensors Synthesized through Layer-by-Layer Deposition of Molecular Beacons for Noninvasive and Longitudinal Monitoring of Cellular mRNA. (United States)

    Wang, Min; Hou, Xiaochun; Wiraja, Christian; Sun, Libo; Xu, Zhichuan J; Xu, Chenjie


    Noninvasive and longitudinal monitoring of gene expression in living cells is essential for understanding and monitoring cellular activities. Herein, a smart magnetic nanosensor is constructed for the real-time, noninvasive, and longitudinal monitoring of cellular mRNA expression through the layer-by-layer deposition of molecular beacons (MBs) and polyethylenimine on the iron oxide nanoparticles. The loading of MBs, responsible for the signal intensity and the tracking time, was easily tuned with the number of layers incorporated. The idea was first demonstrated with the magnetic nanosensors for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, which was efficiently internalized into the cells under the influence of magnetic field. This nanosensor allowed the continuous monitoring of the cellular GAPDH mRNA expression for 1 month. Then this platform was further utilized to incorporate two kinds of MBs for alkaline phosphatase (ALP) and GAPDH mRNAs, respectively. The multifunctional nanosensors permitted the simultaneous monitoring of the reference gene (GAPDH mRNA) and the early osteogenic differentiation marker (ALP mRNA) expression. When the fluorescence signal ratio between ALP mRNA MBs and GAPDH mRNA MBs was taken, the dynamic osteogenic differentiation process of MSCs was accurately monitored.

  15. Non-degradable contrast agent with selective phagocytosis for cellular and hepatic magnetic resonance imaging

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fei-Yan [Nanchang University, College of Chemistry (China); Gu, Zhe-Jia [Nanchang University, Institute for Advanced Study (China); Zhao, Dawen [UT Southwestern Medical Center, Department of Radiology (United States); Tang, Qun, E-mail: [Nanchang University, Institute for Advanced Study (China)


    Degradation is the long-existing toxic issue of metal-containing inorganic medicine. In this paper, we fully investigated the degradation of dextran-coated KMnF{sub 3} nanocube in the in vitro and in vivo surroundings. Different from the general decomposing and ion releasing events, this special agent is resistant to acidic environment, as well as ion exchange. Non-degradability was proved by simulated and real cellular experiments. Moreover, it can be engulfed in the macrophage cells and kept stable in the lysosome. Due to its stability and highly selective phagocytosis, implanted liver cancer can be clearly visualized after administration.

  16. Non-degradable contrast agent with selective phagocytosis for cellular and hepatic magnetic resonance imaging (United States)

    Chen, Fei-Yan; Gu, Zhe-Jia; Zhao, Dawen; Tang, Qun


    Degradation is the long-existing toxic issue of metal-containing inorganic medicine. In this paper, we fully investigated the degradation of dextran-coated KMnF3 nanocube in the in vitro and in vivo surroundings. Different from the general decomposing and ion releasing events, this special agent is resistant to acidic environment, as well as ion exchange. Non-degradability was proved by simulated and real cellular experiments. Moreover, it can be engulfed in the macrophage cells and kept stable in the lysosome. Due to its stability and highly selective phagocytosis, implanted liver cancer can be clearly visualized after administration.

  17. Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria (United States)

    Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel


    Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

  18. PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader (United States)

    Jarrige, Anne-Charlotte; Mathy, Nathalie; Portier, Claude


    Polynucleotide phosphorylase synthesis is autocontrolled at a post-transcriptional level in an RNase III-dependent mechanism. RNase III cleaves a long stem–loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase. The staggered cleavage by RNase III removes the upper part of the stem–loop structure, creating a duplex with a short 3′ extension. Mutations or high temperatures, which destabilize the cleaved stem–loop, decrease expression of pnp, while mutations that stabilize the stem increase expression. We propose that the dangling 3′ end of the duplex created by RNase III constitutes a target for polynucleotide phosphorylase, which binds to and degrades the upstream half of this duplex, hence inducing pnp mRNA instability. Consistent with this interpretation, a pnp mRNA starting at the downstream RNase III processing site exhibits a very low level of expression, regardless of the presence of polynucleotide phosphorylase. Moreover, using an in vitro synthesized pnp leader transcript, it is shown that polynucleotide phosphorylase is able to digest the duplex formed after RNase III cleavage. PMID:11726520

  19. PNPase autocontrols its expression by degrading a double-stranded structure in the pnp mRNA leader. (United States)

    Jarrige, A C; Mathy, N; Portier, C


    Polynucleotide phosphorylase synthesis is autocontrolled at a post-transcriptional level in an RNase III-dependent mechanism. RNase III cleaves a long stem-loop in the pnp leader, which triggers pnp mRNA instability, resulting in a decrease in the synthesis of polynucleotide phosphorylase. The staggered cleavage by RNase III removes the upper part of the stem-loop structure, creating a duplex with a short 3' extension. Mutations or high temperatures, which destabilize the cleaved stem-loop, decrease expression of pnp, while mutations that stabilize the stem increase expression. We propose that the dangling 3' end of the duplex created by RNase III constitutes a target for polynucleotide phosphorylase, which binds to and degrades the upstream half of this duplex, hence inducing pnp mRNA instability. Consistent with this interpretation, a pnp mRNA starting at the downstream RNase III processing site exhibits a very low level of expression, regardless of the presence of polynucleotide phosphorylase. Moreover, using an in vitro synthesized pnp leader transcript, it is shown that polynucleotide phosphorylase is able to digest the duplex formed after RNase III cleavage.

  20. Cellular Anti-Melanogenic Effects of a Euryale ferox Seed Extract Ethyl Acetate Fraction via the Lysosomal Degradation Machinery

    Directory of Open Access Journals (Sweden)

    Seung-Hwa Baek


    Full Text Available The aim of this study was to investigate the effect of ethyl acetate fraction of Euryale ferox seed extracts (Efse-EA on melanogenesis in immortalized mouse melanocyte cell line, melan-a. Efse-EA showed strong dose-dependent mushroom tyrosinase inhibitory activity. Treatment of melan-a cells with 30 μg/mL Efse-EA produced strong inhibition of cellular tyrosinase and melanin synthesis. Efse-EA significantly reduced the levels of melanogenesis-related proteins, such as tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor. Because Efse-EA treatment reduced tyrosinase protein levels without changing its mRNA expression, we investigated whether this decrease was related to proteasomal or lysosomal degradation of tyrosinase. We found that chloroquine, a lysosomal proteolysis inhibitor, almost completely abolished both the down-regulation of tyrosinase and the inhibition of melanin synthesis induced by Efse-EA. These results suggested that Efse-EA may contribute to the inhibition of melanogenesis by altering lysosomal degradation of tyrosinase, and that this extract may provide a new cosmetic skin-whitening agent.

  1. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    Directory of Open Access Journals (Sweden)

    Beatriz Alvarez-Castelao


    Full Text Available Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.

  2. Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue (United States)

    Yoshiba, Nagako; Yoshiba, Kunihiko; Ohkura, Naoto; Takei, Erika; Edanami, Naoki; Oda, Youhei; Hosoya, Akihiro; Nakamura, Hiroaki; Okiji, Takashi


    Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing. PMID:25805839

  3. Transcriptome kinetics is governed by a genome-wide coupling of mRNA production and degradation: a role for RNA Pol II.

    Directory of Open Access Journals (Sweden)

    Ophir Shalem


    Full Text Available Transcriptome dynamics is governed by two opposing processes, mRNA production and degradation. Recent studies found that changes in these processes are frequently coordinated and that the relationship between them shapes transcriptome kinetics. Specifically, when transcription changes are counter-acted with changes in mRNA stability, transient fast-relaxing transcriptome kinetics is observed. A possible molecular mechanism underlying such coordinated regulation might lay in two RNA polymerase (Pol II subunits, Rpb4 and Rpb7, which are recruited to mRNAs during transcription and later affect their degradation in the cytoplasm. Here we used a yeast strain carrying a mutant Pol II which poorly recruits these subunits. We show that this mutant strain is impaired in its ability to modulate mRNA stability in response to stress. The normal negative coordinated regulation is lost in the mutant, resulting in abnormal transcriptome profiles both with respect to magnitude and kinetics of responses. These results reveal an important role for Pol II, in regulation of both mRNA synthesis and degradation, and also in coordinating between them. We propose a simple model for production-degradation coupling that accounts for our observations. The model shows how a simple manipulation of the rates of co-transcriptional mRNA imprinting by Pol II may govern genome-wide transcriptome kinetics in response to environmental changes.

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

    Directory of Open Access Journals (Sweden)

    Enrico Giampieri

    Full Text Available 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.

  5. Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles. (United States)

    Sulheim, Einar; Baghirov, Habib; von Haartman, Eva; Bøe, Andreas; Åslund, Andreas K O; Mørch, Yrr; Davies, Catharina de Lange


    Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood-brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines. Fluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells. In the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible

  6. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, M; Kveiborg, Marie; Kassem, M


    -transformed human lung fibroblast cell line MRC5V2 have 20 to 40% higher levels of CBFA1 mRNA. Similar levels of CBFA1 mRNA are detectable in normal human skin fibroblasts, and these cells also exhibit an age-related decline to the same extent. In addition, the expression of topoisomerase I is reduced by 40......% in senescent osteoblasts, and the mRNA levels are significantly higher (40-70%) in transformed osteoblasts and fibroblasts. These changes in gene expression may be among the causes of impaired osteoblast functions, resulting in reduced bone formation during aging....

  7. Deep sequencing reveals direct targets of gammaherpesvirus-induced mRNA decay and suggests that multiple mechanisms govern cellular transcript escape.

    Directory of Open Access Journals (Sweden)

    Karen Clyde

    Full Text Available One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV, Epstein-Barr virus (EBV and murine herpesvirus 68 (MHV68, is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5 have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq. Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection.

  8. Deep sequencing reveals direct targets of gammaherpesvirus-induced mRNA decay and suggests that multiple mechanisms govern cellular transcript escape. (United States)

    Clyde, Karen; Glaunsinger, Britt A


    One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection.

  9. Conventional and unconventional mechanisms for capping viral mRNA. (United States)

    Decroly, Etienne; Ferron, François; Lescar, Julien; Canard, Bruno


    In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5' ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5'-caps and escape innate immunity.

  10. Susceptibility loci of CNOT6 in the general mRNA degradation pathway and lung cancer risk-A re-analysis of eight GWASs. (United States)

    Zhou, Fei; Wang, Yanru; Liu, Hongliang; Ready, Neal; Han, Younghun; Hung, Rayjean J; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S; Caporaso, Neil; Landi, Maria Teresa; Brüske, Irene; Risch, Angela; Ye, Yuanqing; Wu, Xifeng; Christiani, David C; Goodman, Gary; Chen, Chu; Amos, Christopher I; Wei, Qingyi


    mRNA degradation is an important regulatory step for controlling gene expression and cell functions. Genetic abnormalities involved in mRNA degradation genes were found to be associated with cancer risks. Therefore, we systematically investigated the roles of genetic variants in the general mRNA degradation pathway in lung cancer risk. Meta-analyses were conducted using summary data from six lung cancer genome-wide association studies (GWASs) from the Transdisciplinary Research in Cancer of the Lung and additional two GWASs from Harvard University and deCODE in the International Lung Cancer Consortium. Expression quantitative trait loci analysis (eQTL) was used for in silico functional validation of the identified significant susceptibility loci. This pathway-based analysis included 6816 single nucleotide polymorphisms (SNP) in 68 genes in 14 463 lung cancer cases and 44 188 controls. In the single-locus analysis, we found that 20 SNPs were associated with lung cancer risk with a false discovery rate threshold of lung cancer risk (odds ratio = 1.11, 95% confidence interval = 1.04-1.18) in the eight GWASs. In the eQTL analysis, we found that levels of CNOT6 mRNA expression were significantly correlated with the rs2453176 T allele, which provided additional biological basis for the observed positive association. The CNOT6 rs2453176 SNP may be a new functional susceptible locus for lung cancer risk. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  11. Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3′ Uridylated Intermediates Degraded by DIS3L2

    Directory of Open Access Journals (Sweden)

    Marshall P. Thomas


    Full Text Available Apoptosis is a tightly coordinated cell death program that damages mitochondria, DNA, proteins, and membrane lipids. Little is known about the fate of RNA as cells die. Here, we show that mRNAs, but not noncoding RNAs, are rapidly and globally degraded during apoptosis. mRNA decay is triggered early in apoptosis, preceding membrane lipid scrambling, genomic DNA fragmentation, and apoptotic changes to translation initiation factors. mRNA decay depends on mitochondrial outer membrane permeabilization and is amplified by caspase activation. 3′ truncated mRNA decay intermediates with nontemplated uridylate-rich tails are generated during apoptosis. These tails are added by the terminal uridylyl transferases (TUTases ZCCHC6 and ZCCHC11, and the uridylated transcript intermediates are degraded by the 3′ to 5′ exonuclease DIS3L2. Knockdown of DIS3L2 or the TUTases inhibits apoptotic mRNA decay, translation arrest, and cell death, whereas DIS3L2 overexpression enhances cell death. Our results suggest that global mRNA decay is an overlooked hallmark of apoptosis.

  12. Intragraft interleukin 2 mRNA expression during acute cellular rejection and left ventricular total wall thickness after heart transplantation

    NARCIS (Netherlands)

    de Groot-Kruseman, H A; Baan, C C; Hagman, E M; Mol, W M; Niesters, H G; Maat, A P; Zondervan, P E; Weimar, W; Balk, A H

    OBJECTIVE: To assess whether diastolic graft function is influenced by intragraft interleukin 2 (IL-2) messenger RNA (mRNA) expression in rejecting cardiac allografts. DESIGN: 16 recipients of cardiac allografts were monitored during the first three months after transplantation. The presence of IL-2

  13. Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study (United States)

    Wu, Yan-Yun; Cao, Huan-Huan; Kang, Ning; Gong, Ping; Ou, Guo-Min


    Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mm×1 mm×1 mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526±0.441) was lower than that in the healthy group (3.253±0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965±0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene. PMID:24008269

  14. mRNA expression levels in failing human hearts predict cellular electrophysiological remodeling: a population-based simulation study.

    Directory of Open Access Journals (Sweden)

    John Walmsley

    Full Text Available Differences in mRNA expression levels have been observed in failing versus non-failing human hearts for several membrane channel proteins and accessory subunits. These differences may play a causal role in electrophysiological changes observed in human heart failure and atrial fibrillation, such as action potential (AP prolongation, increased AP triangulation, decreased intracellular calcium transient (CaT magnitude and decreased CaT triangulation. Our goal is to investigate whether the information contained in mRNA measurements can be used to predict cardiac electrophysiological remodeling in heart failure using computational modeling. Using mRNA data recently obtained from failing and non-failing human hearts, we construct failing and non-failing cell populations incorporating natural variability and up/down regulation of channel conductivities. Six biomarkers are calculated for each cell in each population, at cycle lengths between 1500 ms and 300 ms. Regression analysis is performed to determine which ion channels drive biomarker variability in failing versus non-failing cardiomyocytes. Our models suggest that reported mRNA expression changes are consistent with AP prolongation, increased AP triangulation, increased CaT duration, decreased CaT triangulation and amplitude, and increased delay between AP and CaT upstrokes in the failing population. Regression analysis reveals that changes in AP biomarkers are driven primarily by reduction in I[Formula: see text], and changes in CaT biomarkers are driven predominantly by reduction in I(Kr and SERCA. In particular, the role of I(CaL is pacing rate dependent. Additionally, alternans developed at fast pacing rates for both failing and non-failing cardiomyocytes, but the underlying mechanisms are different in control and heart failure.

  15. PKCε promotes HuD-mediated neprilysin mRNA stability and enhances neprilysin-induced Aβ degradation in brain neurons.

    Directory of Open Access Journals (Sweden)

    Chol Seung Lim

    Full Text Available Amyloid-beta (Aβ peptide accumulation in the brain is a pathological hallmark of all forms of Alzheimer's disease. An imbalance between Aβ production and clearance from the brain may contribute to accumulation of neurotoxic Aβ and subsequent synaptic loss, which is the strongest correlate of the extent of memory loss in AD. The activity of neprilysin (NEP, a potent Aβ-degrading enzyme, is decreased in the AD brain. Expression of HuD, an mRNA-binding protein important for synaptogenesis and neuronal plasticity, is also decreased in the AD brain. HuD is regulated by protein kinase Cε (PKCε, and we previously demonstrated that PKCε activation decreases Aβ levels. We hypothesized that PKCε acts through HuD to stabilize NEP mRNA, modulate its localization, and support NEP activity. Conversely, loss of PKCε-activated HuD in AD leads to decreased NEP activity and accumulation of Aβ. Here we show that HuD is associated with NEP mRNA in cultures of human SK-N-SH cells. Treatment with bryostatin, a PKCε-selective activator, enhanced NEP association with HuD and increased NEP mRNA stability. Activation of PKCε also increased NEP protein levels, increased NEP phosphorylation, and induced cell surface expression. In addition, specific PKCε activation directly stimulated NEP activity, leading to degradation of a monomeric form of Aβ peptide and decreased Aβ neuronal toxicity, as measured by cell viability. Bryostatin treatment also rescued Aβ-mediated inhibition of HuD-NEP mRNA binding, NEP protein expression, and NEP cell membrane translocation. These results suggest that PKCε activation reduces Aβ by up-regulating, via the mRNA-binding protein HuD, Aβ-degrading enzymes such as NEP. Thus, PKCε activation may have therapeutic efficacy for AD by reducing neurotoxic Aβ accumulation as well as having direct anti-apoptotic and synaptogenic effects.

  16. Proteasomal degradation resolves competition between cell polarization and cellular wound healing. (United States)

    Kono, Keiko; Saeki, Yasushi; Yoshida, Satoshi; Tanaka, Keiji; Pellman, David


    Cellular wound healing, enabling the repair of membrane damage, is ubiquitous in eukaryotes. One aspect of the wound healing response is the redirection of a polarized cytoskeleton and the secretory machinery to the damage site. Although there has been recent progress in identifying conserved proteins involved in wound healing, the mechanisms linking these components into a coherent response are not defined. Using laser damage in budding yeast, we demonstrate that local cell wall/membrane damage triggers the dispersal of proteins from the site of polarized growth, enabling their accumulation at the wound. We define a protein-kinase-C-dependent mechanism that mediates the destruction of the formin Bni1 and the exocyst component Sec3. This degradation is essential to prevent competition between the site of polarized growth and the wound. Mechanisms to overcome competition from a pre-existing polarized cytoskeleton may be a general feature of effective wound healing in polarized cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription. (United States)

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song; Lai, Michael M C


    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. 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 reported that RRP1B is

  18. MicroRNA hsa-miR-370-3p suppresses the expression and induction of CYP2D6 by facilitating mRNA degradation (United States)

    Zeng, Linjuan; Chen, Yinting; Wang, Yong; Yu, Li-Rong; Knox, Bridgett; Chen, Jiwei; Shi, Tieliu; Chen, Si; Ren, Zhen; Guo, Lei; Wu, Yuanfeng; Liu, David; Huang, Kaihong; Tong, Weida; Yu, Dianke; Ning, Baitang


    Cytochrome P450 2D6 (CYP2D6) participates in the metabolism of approximately 20–25% of prescribed drugs. Genetic polymorphisms influence the expression and/or activity of CYP2D6, and inter-individual differences in drug activation and elimination caused by CYP2D6 genetic variants were reported. However, little is known about the potential modulation of CYP2D6 expression by microRNAs (miRNAs). In the current study, by using in silico prediction of the stabilities of miRNA/mRNA complexes, we screened 38 miRNA candidates that may interact with the transcript of CYP2D6. An inverse correlation between the expression of miRNA hsa-miR-370-3p and the expression of CYP2D6 was observed in human liver tissue samples. Electrophoretic mobility shift assays confirmed that hsa-miR-370-3p was able to directly bind to its cognate target within the coding region of the CYP2D6 transcript. The transfection of hsa-miR-370-3p mimics into the HepG2CYP2D6 cell line, a genetically modified cell line that overexpresses exogenous CYP2D6, was able to suppress the expression of CYP2D6 significantly at both mRNA and protein levels. The transfection of hsa-miR-370-3p mimics was also able to inhibit endogenous mRNA expression and/or protein production of CYP2D6 in HepaRG cells. Furthermore, in HepaRG, HepG2, and Huh7 cells, dexamethasone-induced expression of CYP2D6 was inhibited by hsa-miR-370-3p mimics. To investigate whether the miRNA mediated suppression is caused by inhibiting protein translation or promoting mRNA degradation, an actinomycin D assay was used to measure the stability of CYP2D6 transcripts. The results indicated that hsa-miR-370-3p mimics facilitated significantly the degradation of CYP2D6 mRNA. In addition, proteomics analyses of proteins isolated from the miRNA/mRNA/protein complex suggested that a group of multifunctional proteins facilitated the interaction between hsa-miR-370-3p and CYP2D6, thereby promoting mRNA degradation. PMID:28552654

  19. Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris. (United States)

    Shen, Yu-Feng; Liu, Lei; Gong, Yu-Xin; Zhu, Bin; Liu, Guang-Lu; Wang, Gao-Xue


    This study investigated the effects of trifloxystrobin that one strobilurin used widely in the world as an effective fungicidal agent to control Asian soybean rust on aquatic unicellular algae Chlorella vulgaris. We determined the potential toxic effect of trifloxystrobin on C. vulgaris, and found median inhibition concentration (IC(50)) value 255.58 (95% confidence interval, 207.81-330.29)μgL(-1). In addition, the algal cells were obviously depressed or shrunk at different concentrations by electron microscopy. In the study, a real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL, and one energy gene, ATPs. The results showed that trifloxystrobin reduced the transcript abundances of the three genes and enhanced expression of ATPs after 48 and 96 h. The lowest abundances of psaB, psbC and rbcL transcripts in response to trifloxystrobin exposure were 58%, 79% and 60% of those of the control, respectively. For the potential toxic influences, trifloxystrobin could decrease the soluble protein and total antioxidant contents (T-AOC), and increase superoxide dismutase (SOD) and peroxidase (POD) activity with a gradual concentration-response relationship. Overall, the present study demonstrated that trifloxystrobin could affect the activities of antioxidant enzymes, disrupts photosynthesis in C. vulgaris, and damage cellular structure. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Tumor-Associated Macrophages Derived from Circulating Inflammatory Monocytes Degrade Collagen through Cellular Uptake

    DEFF Research Database (Denmark)

    Madsen, Daniel Hargbøl; Jürgensen, Henrik Jessen; Siersbæk, Majken Storm


    -ablation studies reveal that collagen-degrading TAMs originate from circulating CCR2+ monocytes. This study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation. Madsen et...... al. identify a population of tumor-associated macrophages with a distinct matrix catabolic signature as key effectors of collagen turnover during invasive tumor growth. These matrix-degrading macrophages are largely derived from CCR2+ monocytes reprogrammed by the tumor microenvironment and degrade...... tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all display vigorous endocytic collagen degradation. The cells engaged in this process are identified as tumor...

  1. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

    DEFF Research Database (Denmark)

    Hansen, M.C.; Nielsen, A.K.; Molin, Søren


    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When result...

  2. Monitoring alkane degradation by single BioBrick integration to an optimal cellular framework. (United States)

    Santala, Suvi; Karp, Matti; Santala, Ville


    Synthetic biology enables rewiring and reconstruction of desirable biochemical routes using well-characterized BioBricks. One goal is to optimize these biological systems in terms of robustness, functionality, and simplicity. Thus, in addition to optimizing the molecular level of the metabolic network, choosing an optimal "chassis" can have a great significance in the constructed system. As an example, this study presents a simplified system for monitoring and studying long-chain n-alkane degradation in Acinetobacter baylyi ADP1 online, provided by a single BioBrick insertion, bacterial luciferase luxAB. The system exploits the natural alkane degradation machinery of ADP1 and a sensitive response of bacterial luciferase to a specific intermediate, providing important aspects to natural alkane degradation kinetics. The study suggests the monitoring system to be applicable in the field of environmental biotechnology and emphasizes the utility of ADP1 as a host in both model systems and applications.

  3. Concordant association of insulin degrading enzyme gene (IDE variants with IDE mRNA, Abeta, and Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Minerva M Carrasquillo


    Full Text Available The insulin-degrading enzyme gene (IDE is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD.We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5x10(-8, fold-increase = 2.12,; the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003. Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2-C (p = 0.006 and HepG2 (p = 0.02 cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879, we identified a proxy for rs6583817 that associated significantly with decreased plasma Abeta40 levels (ss = -0.124, p = 0.011 and total measured plasma Abeta levels (b = -0.130, p = 0.009. Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03, and the eleven IDE haplotypes (global p = 0.02 also showed significant association.Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Abeta40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect.

  4. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1* (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard


    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  5. The melanoma-associated transmembrane glycoprotein Gpnmb controls trafficking of cellular debris for degradation and is essential for tissue repair (United States)

    Li, Bing; Castano, Ana P.; Hudson, Thomas E.; Nowlin, Brian T.; Lin, Shuei-Liong; Bonventre, Joseph V.; Swanson, Kenneth D.; Duffield, Jeremy S.


    Kidney damage due to injury rarely resolves completely, and there are currently no therapies capable of promoting repair. In addition to understanding mechanisms by which tissues are damaged, illuminating mechanisms of repair and regeneration is also of great importance. Here we show that the melanoma-associated, transmembrane glycoprotein, Gpnmb, is up-regulated 15-fold following ischemic damage in kidney tissue and by more than 10-fold in macrophages and 3-fold in surviving epithelial cells. Gpnmb-expressing macrophages and epithelial cells were found to contain apoptotic bodies at 3 times the rate of nonexpressing cells. Either mutation of Gpnmb or ablation of inflammatory macrophages prevents normal repair of the kidney. Significantly, the kidneys from postischemic Gpnmb mutant mice exhibited a 5-fold increase in apoptotic cellular debris compared to wild-type mice. These mice also experienced an 85% increase in mortality following bilateral ischemic kidney. Finally, we demonstrate that Gpnmb is a phagocytic protein that is necessary for recruitment of the autophagy protein LC3 to the phagosome where these proteins are colocalized and for lysosomal fusion with the phagosome and hence bulk degradation of their content. Therefore, Gpnmb is a novel prorepair gene that is necessary for crosstalk between the macroautophagic degradation pathway and phagocytosis.—Li, B., Castano, A. P., Hudson, T. E., Nowlin, B. T., Lin, S.-L., Bonventre, J. V., Swanson, K. D., Duffield, J. S. The melanoma-associated transmembrane glycoprotein Gpnmb controls trafficking of cellular debris for degradation and is essential for tissue repair. PMID:20709912

  6. Cellular retinoic-acid-binding-protein and retinol-binding-protein mRNA expression in the cells of the rat seminiferous tubules and their regulation by retinoids. (United States)

    Faraonio, R; Galdieri, M; Colantuoni, V


    The levels of the mRNA corresponding to the intracellular binding proteins for retinoic acid and retinol (CRABP1 and CRBP1, respectively) were studied in primary cultures of somatic and germ cells of the rat seminiferous tubules. We show that the CRABP1 mRNA is expressed in Sertoli and germ cells and a single molecular species of mRNA is detected. CRBP1 mRNA is detected in Sertoli and peritubular cells. The regulation of the expression of both genes by retinoids was studied in Sertoli cells. CRABP1 mRNA levels are not affected by either retinoic acid or retinol, whereas both compounds positively regulate CRBP1 mRNA synthesis in a dose-dependent manner. A fivefold increase in CRBP1 mRNA levels was observed 32-48 h after addition of either agent. These results demonstrate that in Sertoli cells the expression of CRABP1 is not affected by retinoids, similar to the situation observed in vivo and in other in-vitro cultures. CRBP1-gene expression is, instead, induced and the variations in CRBP1-mRNA levels may regulate the intracellular concentrations of retinoids, as a response to changes in the vitamin-A nutritional status.

  7. Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution. (United States)

    Eibinger, Manuel; Zahel, Thomas; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd


    Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave

  8. Cellular uptake and degradation behaviour of biodegradable poly(ethylene glycol-graft-methyl methacrylate) nanoparticles crosslinked with dimethacryloyl hydroxylamine. (United States)

    Scheler, Stefan; Kitzan, Martina; Fahr, Alfred


    Crosslinked polymers with hydrolytically cleavable linkages are highly interesting materials for the design of biodegradable drug carriers. The aim of this study was to investigate if nanoparticles made of such polymers have the potential to be used also for intracellular drug delivery. PEGylated nanoparticles were prepared by copolymerization of methacrylic acid esters and N,O-dimethacryloylhydroxylamine (DMHA). The particles were stable at pH 5.0. At pH 7.4 and 9.0 the degradation covered a time span of about 14 days, following first-order kinetics with higher crosslinked particles degrading slower. Cellular particle uptake and cytotoxicity were tested with L929 mouse fibroblasts. The particle uptake rate was found to correlate linearly with the surface charge and to increase as the zeta potential becomes less negative. Coating of the particle surface with polysorbate 80 drops the internalization rate close to zero and the charge dependence disappears. This indicates the existence of a second effect apart from surface charge. A similar pattern of correlation with zeta potential and coating was also found for the degree of membrane damage while there was no effect of polysorbate on the cell metabolism which increased as the negative charge decreased. It is discussed whether exocytotic processes may explain this behaviour. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Sterilization of collagen scaffolds designed for peripheral nerve regeneration: Effect on microstructure, degradation and cellular colonization. (United States)

    Monaco, Graziana; Cholas, Rahmatullah; Salvatore, Luca; Madaghiele, Marta; Sannino, Alessandro


    In this study we investigated the impact of three different sterilization methods, dry heat (DHS), ethylene oxide (EtO) and electron beam radiation (β), on the properties of cylindrical collagen scaffolds with longitudinally oriented pore channels, specifically designed for peripheral nerve regeneration. Scanning electron microscopy, mechanical testing, quantification of primary amines, differential scanning calorimetry and enzymatic degradation were performed to analyze possible structural and chemical changes induced by the sterilization. Moreover, in vitro proliferation and infiltration of the rat Schwann cell line RSC96 within the scaffolds was evaluated, up to 10days of culture. No major differences in morphology and compressive stiffness were observed among scaffolds sterilized by the different methods, as all samples showed approximately the same structure and stiffness as the unsterilized control. Proliferation, infiltration, distribution and morphology of RSC96 cells within the scaffolds were also comparable throughout the duration of the cell culture study, regardless of the sterilization treatment. However, we found a slight increase of chemical crosslinking upon sterilization (EtOsterilized scaffolds. The results demonstrated that β irradiation impaired the scaffold properties to a greater extent, whereas EtO exposure appeared as the most suitable method for the sterilization of the proposed scaffolds. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. BTG/Tob family members Tob1 and Tob2 inhibit proliferation of mouse embryonic stem cells via Id3 mRNA degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yuanfan; Wang, Chenchen [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Peking University Stem Cell Research Center, China National Center for International Research, Peking University Health Science Center, Beijing 100191 (China); SARI Center for Stem Cell and Nanomedicine, Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Shanghai 200120 (China); Wu, Jenny [SARI Center for Stem Cell and Nanomedicine, Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Shanghai 200120 (China); Li, Lingsong, E-mail: [Department of Cell Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Peking University Stem Cell Research Center, China National Center for International Research, Peking University Health Science Center, Beijing 100191 (China); SARI Center for Stem Cell and Nanomedicine, Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Shanghai 200120 (China)


    The mammalian BTG/Tob family is a group of proteins with anti-proliferative ability, and there are six members including BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2. Among them, Tob subfamily members, specifically Tob1/Tob and Tob2, have the most extensive C-terminal regions. As previously reported, overexpression of BTG/Tob proteins is associated with the inhibition of G1 to S-phase cell cycle progression and decreased cell proliferation in a variety of cell types. Tob subfamily proteins have similar anti-proliferative effects on cell cycle progression in cultured tumor cells. An important unresolved question is whether or not they have function in rapidly proliferating cells, such as embryonic stem cells (ESCs). Tob1 and Tob2 were expressed ubiquitously in mouse ESCs (mESCs), suggesting a possible role in early embryonic development and mESCs. To address the above question and explore the possible functions of the Tob subfamily in ESCs, we established ESCs from different genotypic knockout inner cell mass (ICM). We found that Tob1{sup −/−}, Tob2{sup −/−}, and Tob1/2 double knockout (DKO, Tob1{sup −/−} & Tob2{sup −/−}) ESCs grew faster than wild type (WT) ESCs without losing pluripotency, and we provide a possible mechanistic explanation for these observations: Tob1 and Tob2 inhibit the cell cycle via degradation of Id3 mRNA, which is a set of directly targeted genes of BMP4 signaling in mESCs that play critical roles in the maintenance of ESC properties. Together, our data suggest that BTG/Tob family protein Tob1 and Tob2 regulation cell proliferation does not compromise the basic properties of mESCs. - Highlights: • We established mouse Tob1/2 double knockout embryonic stem cells. • Tob1 and Tob2 inhibit the proliferation of ESCs without effect on pluripotency. • Tob1 and Tob2 involved in the degradation of Id3 in mESCs.

  11. Dexamethasone alters the hepatic inflammatory cellular profile without changes in matrix degradation during liver repair following biliary decompression. (United States)

    Muratore, Christopher S; Harty, Mark W; Papa, Elaine F; Tracy, Thomas F


    , whereas MMP9, 13, and 14 were unchanged compared with sham controls. Despite substantial cellular and molecular changes during repair, collagen resorption was the same in both groups Dexamethasone has clear effects on both the hepatic macrophage populations and infiltrating neutrophils following biliary decompression. Altered MMP and TIMP gene expression might suggest that steroids have the potential to modify matrix metabolism during repair. Nevertheless, successful resorption of collagen fibrosis proceeded presumably through other MMP activating mechanisms. We conclude that steroids do not impede the rapid intrinsic repair mechanisms of matrix degradation required for successful repair.

  12. Changes in Cellular mRNA Stability, Splicing, and Polyadenylation through HuR Protein Sequestration by a Cytoplasmic RNA Virus

    Directory of Open Access Journals (Sweden)

    Michael D. Barnhart


    Full Text Available The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3′ UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3′ UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

  13. A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types. (United States)

    Gagnon, David; Archambault, Jacques


    A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

  14. Developing the conceptual instructional design with inquiry-based instruction model of secondary students at the 10th grade level on digestion system and cellular degradation issue (United States)

    Rotjanakunnatam, Boonthida; Chayaburakul, Kanokporn


    The aims of this research study was to develop the conceptual instructional design with the Inquiry-Based Instruction Model (IBIM) of secondary students at the 10th grade level on Digestion System and Cellular Degradation issue using both oxygen and oxygen-degrading cellular nutrients were designed instructional model with a sample size of 45 secondary students at the 10th Grade level. Data were collected by asking students to do a questionnaire pre and post learning processes. The questionnaire consists of two main parts that composed of students' perception questionnaire and the questionnaire that asked the question answer concept for the selected questionnaire. The 10-item Conceptual Thinking Test (CTT) was assessed students' conceptual thinking evaluation that it was covered in two main concepts, namely; Oxygen degradation nutrients and degradation nutrients without oxygen. The data by classifying students' answers into 5 groups and measuring them in frequency and a percentage of students' performances of their learning pre and post activities with the Inquiry-Based Instruction Model were analyzed as a tutorial. The results of this research found that: After the learning activities with the IBIM, most students developed concepts of both oxygen and oxygen-degrading cellular nutrients in the correct, complete and correct concept, and there are a number of students who have conceptual ideas in the wrong concept, and no concept was clearly reduced. However, the results are still found that; some students have some misconceptions, such as; the concept of direction of electron motion and formation of the ATP of bioactivities of life. This cause may come from the nature of the content, the complexity, the continuity, the movement, and the time constraints only in the classroom. Based on this research, it is suggested that some students may take some time, and the limited time in the classroom to their learning activity with content creation content binding and

  15. Recognition of nonsense mRNA: towards a unified model. (United States)

    Mühlemann, Oliver


    Among the different cellular surveillance mechanisms that ensure accurate gene expression, nonsense-mediated mRNA decay rapidly degrades mRNAs harbouring PTCs (premature translation-termination codons) and thereby prevents the accumulation of potentially deleterious proteins with C-terminal truncations. In the present article, I review recent data from yeast, fluitflies, nematode worms and human cells and endeavour to merge these results into a unified model for recognition of nonsense mRNA. According to this model, the distinction between translation termination at PTCs and at 'normal' termination codons relies on the physical distance between the terminating ribosome and PABP [poly(A)-binding protein]. Correct translation termination is promoted by a PABP-mediated signal to the terminating ribosome, whereas the absence of this signal leads to the assembly of an mRNA decay-promoting protein complex including the conserved NMD factors UPF (up-frameshift) 1-3.

  16. Engineering hyaluronic acid hydrogel degradation to control cellular interactions and adult stem cell fate in 3D (United States)

    Khetan, Sudhir

    The design and implementation of extracellular matrix (ECM)-mimetic hydrogels for tissue engineering (TE) applications requires an intensive understanding of cell-material interactions, including matrix remodeling and stem cell differentiation. However, the influence of microenvironmental cues, e.g., matrix biodegradability, on cell behavior in vitro has not been well studied in the case of direct cell encapsulation within 3-dimensional (3D) hydrogels. To address these issues, a facile sequential crosslinking technique was developed that provides spatial and temporal control of 3D hydrogel degradability to investigate the importance of material design on cell behavior. Specifically, hydrogels were synthesized from hyaluronic acid (HA) macromers in a sequential process: (1) a primary Michael-type addition crosslinking using cell adhesive and matrix metalloprotease (MMP)-degradable oligopeptides to consume a portion of total reactive groups and resulting in "-UV" hydrogels permissive to cell-mediated degradation, followed by (2) a secondary, light initiated free-radical crosslinking to consume remaining reactive groups and "switch" the network to a non-degradable structure ("+UV") via the addition of non-degradable kinetic chains. Using this approach, we demonstrated control of encapsulated hMSC spreading by varying the crosslink type (i.e., the relative hydrogel biodegradability), including with spatial control. Upon incubation with bipotential soluble differentiation factors, these same degradation-mediated spreading cues resulted in an hMSC differentiation fate switch within -UV versus +UV environments. Follow-up studies demonstrated that degradation-mediated traction generation, rather than matrix mechanics or cell morphology, is the critical biophysical signal determining hMSC fate. Sequentially crosslinked HA hydrogels were also studied for the capacity to support remodeling by in vivo and ex vivo tissues, including with spatial control, toward tissue

  17. S-Adenosylmethionine Synthesis Is Regulated by Selective N6-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1

    Directory of Open Access Journals (Sweden)

    Hiroki Shima


    Full Text Available S-adenosylmethionine (SAM is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N6-methyladenosine (m6A in the 3′ UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3′ UTR. Knockdown of METTL16 and the m6A reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3′ UTR revealed that these m6As were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

  18. Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Damgaard, Christian Kroun; Tomecki, Rafal


    Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes...... another RNase II/R domain protein-hDIS3L2. Here, we show that hDIS3L2 is an exosome-independent cytoplasmic mRNA 3'-5' exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA-dependent manner and can, like hXRN1, be found on polysomes....... The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P-bodies) upon hDIS3L2 depletion, which also increases half-lives of investigated mRNAs. Consistently, RNA sequencing (RNA-seq) analyses demonstrate that depletion of hDIS3L2, like...

  19. Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA. (United States)

    Saha, Chaitrali; Hegde, Pushpa; Friboulet, Alain; Bayry, Jagadeesh; Kaveri, Srinivas V


    Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

  20. Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

    Directory of Open Access Journals (Sweden)

    Chaitrali Saha

    Full Text Available Extensive use of Viscum album (VA preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2, one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

  1. Multifunctional nanoparticles, nanocages and degradable polymers as a potential novel generation of non-invasive molecular and cellular imaging systems. (United States)

    Masotti, Andrea


    In recent years, polymeric scaffolds have been used in several biomedical applications for delivery of drugs or other biologically relevant molecules. Polymeric nanostructures display different (and in some cases more powerful) properties respect to bulk materials. This, lead academic researchers and industry to cooperate in developing pioneering nanostructured materials for industrial and biomedical applications. Moreover, the preparation and use of systems with multiple (multifunctional) properties (i.e., bioconjugation with superparamagnetic, fluorescent or targeting molecules) is positioned to become a viable and innovative tool for application in several clinical fields. Other nanostructured systems like nanocages and degradable nanoparticles, are emerging as potential innovative systems that could be exploited as multifunctional delivery vectors. This brief critical review is aimed at collecting and discussing some recent patents dealing with the preparation and use of multifunctional nanoparticles, nanocages and degradable nanoparticles in biomedicine and non-invasive bioimaging applications. Perspectives for a potential use of these multifunctional nanosystems in pediatries have been also discussed.

  2. Cellular inhibitor of apoptosis 1 (cIAP-1) degradation by caspase 8 during TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. (United States)

    Guicciardi, Maria Eugenia; Mott, Justin L; Bronk, Steven F; Kurita, Satoshi; Fingas, Christian D; Gores, Gregory J


    TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies. Copyright © 2010 Elsevier Inc. All rights reserved.

  3. Characterization of the microchemical structure of seed endosperm within a cellular dimension among six barley varieties with distinct degradation kinetics, using ultraspatially resolved synchrotron-based infrared microspectroscopy. (United States)

    Liu, Na; Yu, Peiqiang


    Barley varieties have similar chemical composition but exhibit different rumen degradation kinetics and nutrient availability. These biological differences may be related to molecular, structural, and chemical makeup among the seed endosperm tissue. No detailed study was carried out. The objectives of this study were: (1) to use a molecular spectroscopy technique, synchrotron-based Fourier transform infrared microspectroscopy (SFTIRM), to determine the microchemical-structural features in seed endosperm tissue of six developed barley varieties; (2) to study the relationship among molecular-structural characteristics, degradation kinetics, and nutrient availability in six genotypes of barley. The results showed that inherent microchemical-structural differences in the endosperm among the six barley varieties were detected by the synchrotron-based analytical technique, SFTIRM, with the univariate molecular spectral analysis. The SFTIRM spectral profiles differed (P barley samples in terms of the peak ratio and peak area and height intensities of amides I (ca. 1650 cm(-1)) and II (ca. 1550 cm(-1)), cellulosic compounds (ca. 1240 cm(-1)), CHO component peaks (the first peak at the region ca. 1184-1132 cm(-1), the second peak at ca. 1132-1066 cm(-1), and the third peak at ca. 1066-950 cm(-1)). With the SFTIRM technique, the structural characteristics of the cereal seeds were illuminated among different cultivars at an ultraspatial resolution. The structural differences of barley seeds may be one reason for the various digestive behaviors and nutritive values in ruminants. The results show weak correlations between the functional groups' spectral data (peak area, height intensities, and ratios) and rumen biodegradation kinetics (rate and extent of nutrient degradation). Weak correlations may indicate that limited variations of these six barley varieties might not be sufficient to interpret the relationship between spectroscopic information and the nutrient value of

  4. Arsenic Induces Polyadenylation of Canonical Histone mRNA by Down-regulating Stem-Loop-binding Protein Gene Expression* (United States)

    Brocato, Jason; Fang, Lei; Chervona, Yana; Chen, Danqi; Kiok, Kathrin; Sun, Hong; Tseng, Hsiang-Chi; Xu, Dazhong; Shamy, Magdy; Jin, Chunyuan; Costa, Max


    The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3′-end. Instead, the histone mRNAs display a stem-loop structure at their 3′-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis. PMID:25266719

  5. Interferon gamma, interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats: dynamics of cellular mRNA expression in the central nervous system and lymphoid cells

    DEFF Research Database (Denmark)

    Issazadeh-Navikas, Shohreh; Mustafa, M; Ljungdahl, A


    The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon gamma (IFN-gamma), interleukin 4 (IL-4) and transforming growth factor beta (TGF...

  6. An RNA element in human interleukin 6 confers escape from degradation by the gammaherpesvirus SOX protein. (United States)

    Hutin, Stephanie; Lee, Yeon; Glaunsinger, Britt A


    Several viruses express factors to silence host gene expression via widespread mRNA degradation. This phenotype is the result of the coordinated activity of the viral endonuclease SOX and the cellular RNA degradation enzyme Xrn1 during lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection. While most cellular transcripts are highly downregulated, a subset of host mRNA escapes turnover via unknown mechanisms. One of the most prominent escapees is the interleukin 6 (IL-6) mRNA, which accumulates robustly during KSHV lytic infection and is not subjected to SOX-induced degradation. Here we reveal that the IL-6 mRNA contains a dominant, cis-acting ∼100-nucleotide element within its 3' untranslated region (UTR) that renders it directly refractory to cleavage by SOX. This element specifically interacts with a cellular protein complex both in SOX-transfected cells and in KSHV-infected B cells. Using a directed RNA pulldown approach, we identified two components of this complex to be the AU-rich element (ARE) binding proteins AUF1 and HuR. Depletion of these proteins significantly reduced the protective capacity of the IL-6 RNA element in SOX-expressing cells. These findings suggest that SOX activity may be directly counteracted by select RNA regulatory complexes and reveal a novel mechanism contributing to the robust expression of IL-6 during KSHV replication.

  7. The number of regulatory T cells in transbronchial lung allograft biopsies is related to FoxP3 mRNA levels in bronchoalveolar lavage fluid and to the degree of acute cellular rejection

    DEFF Research Database (Denmark)

    Krustrup, Dorrit; Madsen, Caroline B; Iversen, Martin


    the number of lymphocytes expressing FoxP3 in transbronchial biopsies from lung allografts with the FoxP3 expression in bronchoalveolar lavage fluid (BALF). In addition, we aimed to correlate the number of FoxP3+ cells in transbronchial biopsies with the degree of acute cellular rejection in lung allografts....

  8. Functional and cellular adaptations of rodent skeletal muscle to weightlessness (United States)

    Caiozzo, Vincent J.; Haddad, Fadia; Baker, Michael J.; Baldwin, Kenneth M.


    This paper describes the affects of microgravity upon three key cellular levels (functional, protein, and mRNA) that are linked to one another. It is clear that at each of these levels, microgravity produces rapid and substantial alterations. One of the key challenges facing the life science community is the development of effective countermeasures that prevent the loss of muscle function as described in this paper. The development of optimal countermeasures, however, awaits a clearer understanding of events occurring at the levels of transcription, translation, and degradation.

  9. Depletion of cellular iron by curcumin leads to alteration in histone acetylation and degradation of Sml1p in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Gajendra Kumar Azad

    Full Text Available Curcumin, a naturally occurring polyphenolic compound, is known to possess diverse pharmacological properties. There is a scarcity of literature documenting the exact mechanism by which curcumin modulates its biological effects. In the present study, we have used yeast as a model organism to dissect the mechanism underlying the action of curcumin. We found that the yeast mutants of histone proteins and chromatin modifying enzymes were sensitive to curcumin and further supplementation of iron resulted in reversal of the changes induced by curcumin. Additionally, treatment of curcumin caused the iron starvation induced expression of FET3, FRE1 genes. We also demonstrated that curcumin induces degradation of Sml1p, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. The degradation of Sml1p was mediated through proteasome and vacuole dependent protein degradation pathways. Furthermore, curcumin exerts biological effect by altering global proteome profile without affecting chromatin architecture. These findings suggest that the medicinal properties of curcumin are largely contributed by its cumulative effect of iron starvation and epigenetic modifications.

  10. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  11. Hijacking cellular garbage cans. (United States)

    Welsch, Sonja; Locker, Jacomine Krijnse


    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. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  12. Self-amplifying mRNA vaccines. (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J


    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Flavopiridol induces cellular FLICE-inhibitory protein degradation by the proteasome and promotes TRAIL-induced early signaling and apoptosis in breast tumor cells. (United States)

    Palacios, Carmen; Yerbes, Rosario; López-Rivas, Abelardo


    The cyclin-dependent kinase inhibitor flavopiridol is undergoing clinical trials as an antitumor drug. We show here that pretreatment of different human breast cancer cell lines with flavopiridol facilitates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In breast tumor cells, apoptosis induction by TRAIL is blocked at the level of apical caspase-8 activation. Flavopiridol treatment enhances TRAIL-induced formation of death-inducing signaling complex and early processing of procaspase-8. Subsequently, a TRAIL-induced, mitochondria-operated pathway of apoptosis is activated in cells treated with flavopiridol. Down-regulation of cellular FLICE-inhibitory proteins (c-FLIP; c-FLIP(L) and c-FLIP(S)) is observed on flavopiridol treatment. c-FLIP loss and apoptosis sensitization by flavopiridol are both prevented in cells treated with an inhibitor of the ubiquitin-proteasome system. Furthermore, targeting c-FLIP directly with small interfering RNA oligonucleotides also sensitizes various human breast tumor cell lines to TRAIL-induced apoptosis. Our results indicate that flavopiridol sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway, and this combination treatment could be regarded as a potential therapeutic tool against breast tumors.

  14. Cellular automata

    CERN Document Server

    Codd, E F


    Cellular Automata presents the fundamental principles of homogeneous cellular systems. This book discusses the possibility of biochemical computers with self-reproducing capability.Organized into eight chapters, this book begins with an overview of some theorems dealing with conditions under which universal computation and construction can be exhibited in cellular spaces. This text then presents a design for a machine embedded in a cellular space or a machine that can compute all computable functions and construct a replica of itself in any accessible and sufficiently large region of t

  15. Conceptual modeling in systems biology fosters empirical findings: the mRNA lifecycle.

    Directory of Open Access Journals (Sweden)

    Dov Dori

    Full Text Available One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM, a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency.

  16. Applying the breaks on gene expression - mRNA deadenylation by Pop2p

    DEFF Research Database (Denmark)

    Andersen, Kasper Røjkjær; Jonstrup, Anette Thyssen; Van, Lan Bich

    When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems to be the ......When driving a car, control of the brakes is just as important as control of the accelerator pedal. Likewise, in gene expression, regulation of mRNA degradation is as important as regulation of its synthesis (Mühlemann, 2005). The rate-determining step of mRNA decay in eukaryotes seems...

  17. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. (United States)

    Kedersha, N; Anderson, P


    Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2alpha, a key regulatory step in translational initiation. The activation of one or more of the eIF2alpha kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNA(Met), the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNA(Met) allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2alpha kinase system, mRNA stability and cellular chaperone levels.

  18. Loss of the scavenger mRNA decapping enzyme DCPS causes syndromic intellectual disability with neuromuscular defects

    NARCIS (Netherlands)

    Ng, Calista K. L.; Shboul, Mohammad; Taverniti, Valerio; Bonnard, Carine; Lee, Hane; Eskin, Ascia; Nelson, Stanley F.; Al-Raqad, Mohammed; Altawalbeh, Samah; Séraphin, Bertrand; Reversade, Bruno


    mRNA decay is an essential and active process that allows cells to continuously adapt gene expression to internal and environmental cues. There are two mRNA degradation pathways: 3' to 5' and 5' to 3'. The DCPS protein is the scavenger mRNA decapping enzyme which functions in the last step of the 3'

  19. Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)* (United States)

    Lee, Sunhee; Challa-Malladi, Madhavi; Bratton, Shawn B.; Wright, Casey W.


    Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal. PMID:25246529

  20. Nuclear factor-κB-inducing kinase (NIK) contains an amino-terminal inhibitor of apoptosis (IAP)-binding motif (IBM) that potentiates NIK degradation by cellular IAP1 (c-IAP1). (United States)

    Lee, Sunhee; Challa-Malladi, Madhavi; Bratton, Shawn B; Wright, Casey W


    Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation. (United States)

    Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A


    Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus.IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

  2. Protein degradation systems in platelets. (United States)

    Kraemer, B F; Weyrich, A S; Lindemann, S


    Protein synthesis and degradation are essential processes that allow cells to survive and adapt to their surrounding milieu. In nucleated cells, the degradation and/or cleavage of proteins is required to eliminate aberrant proteins. Cells also degrade proteins as a mechanism for cell signalling and complex cellular functions. Although the last decade has convincingly shown that platelets synthesise proteins, the roles of protein degradation in these anucleate cytoplasts are less clear. Here we review what is known about protein degradation in platelets placing particular emphasis on the proteasome and the cysteine protease calpain.

  3. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao


    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  4. Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy. (United States)

    Nashimoto, Yuji; Takahashi, Yasufumi; Zhou, Yuanshu; Ito, Hidenori; Ida, Hiroki; Ino, Kosuke; Matsue, Tomokazu; Shiku, Hitoshi


    Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

  5. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    Directory of Open Access Journals (Sweden)

    Amandine Bonnet


    Full Text Available Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs. mRNPs are then exported through nuclear pore complexes (NPCs, which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed.

  6. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

    Directory of Open Access Journals (Sweden)

    Marta Maria Gaglia


    Full Text Available Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV. Previous studies indicated that cleavage of messenger RNAs (mRNA by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

  7. Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX (United States)

    Gaglia, Marta Maria; Rycroft, Chris H.; Glaunsinger, Britt A.


    Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. PMID:26646420

  8. Regulation by small RNAs via coupled degradation: mean-field and variational approaches

    CERN Document Server

    Platini, Thierry; Kulkarni, Rahul V


    Regulatory genes called small RNAs (sRNAs) are known to play critical roles in cellular responses to changing environments. For several sRNAs, regulation is effected by coupled stoichiometric degradation with messenger RNAs (mRNAs). The nonlinearity inherent in this regulatory scheme indicates that exact analytical solutions for the corresponding stochastic models are intractable. Here, we present a variational approach to analyze a well-studied stochastic model for regulation by sRNAs via coupled degradation. The proposed approach is efficient and provides accurate estimates of mean mRNA levels as well as higher order terms. Results from the variational ansatz are in excellent agreement with data from stochastic simulations for a wide range of parameters, including regions of parameter space where mean-field approaches break down. The proposed approach can be applied to quantitatively model stochastic gene expression in complex regulatory networks.

  9. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro


    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  10. Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues. (United States)

    Bahar Halpern, Keren; Itzkovitz, Shalev


    A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique based on single molecule Fluorescence in-situ Hybridization (smFISH) to measure transcription and degradation rates in intact mammalian tissues. The technique is based on dual-color libraries targeting the introns and exons of the genes of interest, enabling visualization and quantification of both nascent and mature mRNA. We present a software, TransQuant, that facilitates quantifying these rates from smFISH images. Our approach enables assessment of both transcription and degradation rates of any gene of interest while controlling for the inherent heterogeneity of intact tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Live Cell Imaging of Endogenous mRNA Using RNA-Based Fluorescence "Turn-On" Probe. (United States)

    Ong, Wei Qiang; Citron, Y Rose; Sekine, Sayaka; Huang, Bo


    Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence "turn-on" probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live E. coli imaging.

  12. Rae1/YacP, a new endoribonuclease involved in ribosome-dependent mRNA decay in Bacillus subtilis. (United States)

    Leroy, Magali; Piton, Jérémie; Gilet, Laetitia; Pellegrini, Olivier; Proux, Caroline; Coppée, Jean-Yves; Figaro, Sabine; Condon, Ciarán


    The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo. © 2017 The Authors.

  13. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Directory of Open Access Journals (Sweden)

    Beatriz M. A. Fontoura


    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  14. Functions of NQO1 in Cellular Protection and CoQ10 Metabolism and its Potential Role as a Redox Sensitive Molecular Switch

    Directory of Open Access Journals (Sweden)

    David Ross


    Full Text Available NQO1 is one of the two major quinone reductases in mammalian systems. It is highly inducible and plays multiple roles in cellular adaptation to stress. A prevalent polymorphic form of NQO1 results in an absence of NQO1 protein and activity so it is important to elucidate the specific cellular functions of NQO1. Established roles of NQO1 include its ability to prevent certain quinones from one electron redox cycling but its role in quinone detoxification is dependent on the redox stability of the hydroquinone generated by two-electron reduction. Other documented roles of NQO1 include its ability to function as a component of the plasma membrane redox system generating antioxidant forms of ubiquinone and vitamin E and at high levels, as a direct superoxide reductase. Emerging roles of NQO1 include its function as an efficient intracellular generator of NAD+ for enzymes including PARP and sirtuins which has gained particular attention with respect to metabolic syndrome. NQO1 interacts with a growing list of proteins, including intrinsically disordered proteins, protecting them from 20S proteasomal degradation. The interactions of NQO1 also extend to mRNA. Recent identification of NQO1 as a mRNA binding protein have been investigated in more detail using SERPIN1A1 (which encodes the serine protease inhibitor α-1-antitrypsin as a target mRNA and indicate a role of NQO1 in control of translation of α-1-antitrypsin, an important modulator of COPD and obesity related metabolic syndrome. NQO1 undergoes structural changes and alterations in its ability to bind other proteins as a result of the cellular reduced/oxidized pyridine nucleotide ratio. This suggests NQO1 may act as a cellular redox switch potentially altering its interactions with other proteins and mRNA as a result of the prevailing redox environment.

  15. In situ hybridization of cytokine mRNA using alkaline phosphatase-labelled oligodeoxynucleotide probes

    DEFF Research Database (Denmark)

    Clausen, Bettina Hjelm; Fenger, Christina; Finsen, B.


    In situ hybridization is a powerful tool for visualizing cellular gene expression in morphologically preserved brain tissue giving precise information on the regional expression of specific mRNA sequences in cells of diverse phenotype. Here, we describe a sensitive, simple, and robust method usin...... alkaline phosphatase (AP)-labelled oligodeoxynucleotide probes to detect cytokine mRNA in the acutely injured or inflamed mouse CNS....

  16. The BTG2 protein is a general activator of mRNA deadenylation. (United States)

    Mauxion, Fabienne; Faux, Céline; Séraphin, Bertrand


    BTG2 is a prototype member of the BTG/Tob family of antiproliferative proteins, originally identified as a primary response gene induced by growth factors and tumour promoters. Its expression has been linked to diverse cellular processes such as cell-cycle progression, differentiation or apoptosis. BTG2 has also been shown to interact with the Pop2/Caf1 deadenylase. Here, we demonstrate that BTG2 is a general activator of mRNA decay, thereby contributing to gene expression control. Detailed characterizations of BTG2 show that it enhances deadenylation of all transcripts tested. Our results demonstrate that Caf1 nuclease activity is required for efficient deadenylation in mammalian cells and that the deadenylase activities of both Caf1 and its Ccr4 partner are required for Btg2-induced poly(A) degradation. General activation of deadenylation may represent a new mode of global regulation of gene expression, which could be important to allow rapid resetting of protein production during development or after specific stresses. This may constitute a common function for BTG/Tob family members.

  17. Phenotype fingerprinting suggests the involvement of single-genotype consortia in degradation of aromatic compounds by Rhodopseudomonas palustris.

    Directory of Open Access Journals (Sweden)

    Tatiana V Karpinets

    Full Text Available Anaerobic degradation of complex organic compounds by microorganisms is crucial for development of innovative biotechnologies for bioethanol production and for efficient degradation of environmental pollutants. In natural environments, the degradation is usually accomplished by syntrophic consortia comprised of different bacterial species. This strategy allows consortium organisms to reduce efforts required for maintenance of the redox homeostasis at each syntrophic level. Cellular mechanisms that maintain the redox homeostasis during the degradation of aromatic compounds by one organism are not fully understood. Here we present a hypothesis that the metabolically versatile phototrophic bacterium Rhodopseudomonas palustris forms its own syntrophic consortia, when it grows anaerobically on p-coumarate or benzoate as a sole carbon source. We have revealed the consortia from large-scale measurements of mRNA and protein expressions under p-coumarate, benzoate and succinate degrading conditions using a novel computational approach referred as phenotype fingerprinting. In this approach, marker genes for known R. palustris phenotypes are employed to determine the relative expression levels of genes and proteins in aromatics versus non-aromatics degrading condition. Subpopulations of the consortia are inferred from the expression of phenotypes and known metabolic modes of the R. palustris growth. We find that p-coumarate degrading conditions may lead to at least three R. palustris subpopulations utilizing p-coumarate, benzoate, and CO2 and H2. Benzoate degrading conditions may also produce at least three subpopulations utilizing benzoate, CO2 and H2, and N2 and formate. Communication among syntrophs and inter-syntrophic dynamics in each consortium are indicated by up-regulation of transporters and genes involved in the curli formation and chemotaxis. The N2-fixing subpopulation in the benzoate degrading consortium has preferential activation of the

  18. Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells

    DEFF Research Database (Denmark)

    Petersen, C M; Christensen, E I; Andresen, B S


    Protein kinase C activating phorbol esters downregulated membrane CD4 by endocytosis in U-937 and human T-cells. Half-time for internalization (approximately 15 min at 50 ng/ml PMA) was determined by FACS. CD4-bound 125I-labeled anti-CD4 mAb was rapidly degraded in PMA-activated cells, whereas...... degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation...... in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal...

  19. Translation with frameshifting of ribosome along mRNA transcript

    CERN Document Server

    Li, Jingwei


    Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

  20. Translational repression of mRNA for eucaryotic elongation factors in Friend erythroleukemia cells. (United States)

    Slobin, L I; Jordan, P


    Poly(A)-containing mRNA was prepared from polyribosomes and postpolyribosomal messenger ribonucleoprotein particles (mRNP) from Friend erythroleukemic cells. Both mRNA types were translated in vitro and the 35S-labeled translation products examined by two-dimensional gel electrophoresis. Among the most abundant untranslated mRNA species was the mRNA coding for eucaryotic elongation factor Tu (eEF-Tu). In addition, the mRNA for eucaryotic elongation factor Ts was also present in Friend cells in untranslated form. Calculations based on translation assays indicate that eEF-Tu represents about 15% of the translation products of RNP mRNA and that approximately 40% of the eEF-Tu synthesized in vitro is encoded by translationally repressed mRNA. This repressed mRNA can be activated by addition of cycloheximide to cell cultures. At the level of 0.1 micrograms/ml, cycloheximide was found to inhibit cellular protein synthesis by about 50% while augmenting the relative rate of eEF-Tu synthesis 1.6-fold. This result suggested that eEF-Tu mRNA might initiate poorly. However, addition of supersaturating levels of mRNA to a reticulocyte lysate augmented eEF-Tu synthesis about twofold, while generally depressing the synthesis of other proteins by about 40%. Thus the storage of large amounts of eEF-Tu mRNA in vivo is unlikely to be due directly to the ineffectiveness of the mRNA in competing for the initiation machinery of the cell. The results presented in this report suggest that the supply of active eEF-Tu in erythroleukemic cells is controlled, at least in part, by a translational mechanism.

  1. Integration of miRNA and mRNA expression profiles reveals microRNA-regulated networks during muscle wasting in cardiac cachexia

    DEFF Research Database (Denmark)

    Moraes, Leonardo N; Fernandez, Geysson J; Vechetti-Júnior, Ivan J


    Cardiac cachexia (CC) is a common complication of heart failure (HF) associated with muscle wasting and poor patient prognosis. Although different mechanisms have been proposed to explain muscle wasting during CC, its pathogenesis is still not understood. Here, we described an integrative analysis...... between miRNA and mRNA expression profiles of muscle wasting during CC. Global gene expression profiling identified 1,281 genes and 19 miRNAs differentially expressed in muscle wasting during CC. Several of these deregulated genes are known or putative targets of the altered miRNAs, including miR-29a-3p......, miR-29b-3p, miR-210-5p, miR-214, and miR-489. Gene ontology analysis on integrative mRNA/miRNA expression profiling data revealed miRNA interactions affecting genes that regulate extra-cellular matrix (ECM) organization, proteasome protein degradation, citric acid cycle and respiratory electron...

  2. 3D-engineering of Cellularized Conduits for Peripheral Nerve Regeneration (United States)

    Hu, Yu; Wu, Yao; Gou, Zhiyuan; Tao, Jie; Zhang, Jiumeng; Liu, Qianqi; Kang, Tianyi; Jiang, Shu; Huang, Siqing; He, Jiankang; Chen, Shaochen; Du, Yanan; Gou, Maling


    Tissue engineered conduits have great promise for bridging peripheral nerve defects by providing physical guiding and biological cues. A flexible method for integrating support cells into a conduit with desired architectures is wanted. Here, a 3D-printing technology is adopted to prepare a bio-conduit with designer structures for peripheral nerve regeneration. This bio-conduit is consisted of a cryopolymerized gelatin methacryloyl (cryoGelMA) gel cellularized with adipose-derived stem cells (ASCs). By modeling using 3D-printed “lock and key” moulds, the cryoGelMA gel is structured into conduits with different geometries, such as the designed multichannel or bifurcating and the personalized structures. The cryoGelMA conduit is degradable and could be completely degraded in 2-4 months in vivo. The cryoGelMA scaffold supports the attachment, proliferation and survival of the seeded ASCs, and up-regulates the expression of their neurotrophic factors mRNA in vitro. After implanted in a rat model, the bio-conduit is capable of supporting the re-innervation across a 10 mm sciatic nerve gap, with results close to that of the autografts in terms of functional and histological assessments. The study describes an indirect 3D-printing technology for fabricating cellularized designer conduits for peripheral nerve regeneration, and could lead to the development of future nerve bio-conduits for clinical use.

  3. Poly(D,L-lactide-co-glycolide)/hydroxyapatite core-shell nanospheres. Part 4: a change of the surface properties during degradation process and the corresponding in vitro cellular response. (United States)

    Vukomanović, M; Sarčev, I; Petronijević, B; Skapin, S D; Ignjatović, N; Uskoković, D


    The surface properties of PLGA/HAp core-shell nanoparticles loaded with clindamycin obtained by an ultrasonic processing method and their changes under the simulated physiological conditions during the degradation process (when the morphology is changed starting from the nanospheres, over micrometer-sized plate-like films to a porous network) were investigated. The dynamic change of the surface properties of this material obtained in a water environment showed an increase of the surface area (up to 70 m(2)/g) and an improved wettability (estimated water contact angle was in the range between 40° and 60°) suggesting the possibility for its good interaction with cells. The in vitro tests are in a good correlation with this hypothesis, showing a high level of cytocompatibility of the material with the mouse L929 and human lung MRC-5 fibroblasts. The fibroblasts were able to achieve the contact with the material's surface and to attach onto it. The significance of HAp, as the bioceramic phase within the PLGA/HAp core-shell nanoparticles, may be brought into relationship with its role in improving the surface properties of PLGA/HAp obtained during the degradation process. These properties are closely related to the bioactivity and biocompatibility of this material, which are highly relevant for its biomedical application. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. The yeast POP2 gene encodes a nuclease involved in mRNA deadenylation. (United States)

    Daugeron, M C; Mauxion, F; Séraphin, B


    The major mRNA degradation pathway involves deadenylation of the target molecule followed by decapping and, finally, 5'-->3' exonuclease digestion of the mRNA body. While yeast factors involved in the decapping and exonuclease degradation steps have been identified, the nature of the factor(s) involved in the deadenylation step remained elusive. Database searches for yeast proteins related to the mammalian deadenylase PARN identified the Pop2 protein (Pop2p) as a potential deadenylase. While Pop2p was previously identified as a factor affecting transcription, we identified a non-canonical RNase D sequence signature in its sequence. Analysis of the fate of a reporter mRNA in a pop2 mutant demonstrates that Pop2p is required for efficient mRNA degradation in vivo. Characterisation of mRNA degradation intermediates accumulating in this mutant supports the involvement of Pop2p in mRNA deadenylation in vivo. Similar phenotypes are observed in yeast strains lacking the Ccr4 protein, which is known to be associated with Pop2p. A recombinant Pop2p fragment encompassing the putative catalytic domain degrades poly(A) in vitro demonstrating that Pop2p is a nuclease. We also demonstrate that poly(A) is a better competitor than poly(G) or poly(C) of the Pop2p nuclease activity. Altogether, our study indicates that Pop2p is a nuclease subunit of the yeast deadenylase and suggests that Pop2p homologues in other species may have similar functions.

  5. Antagonistic Effects of Cellular Poly(C) Binding Proteins on Vesicular Stomatitis Virus Gene Expression ▿ (United States)

    Dinh, Phat X.; Beura, Lalit K.; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K.


    Immunoprecipitation and subsequent mass spectrometry analysis of the cellular proteins from cells expressing the vesicular stomatitis virus (VSV) P protein identified the poly(C) binding protein 2 (PCBP2) as one of the P protein-interacting proteins. To investigate the role of PCBP2 in the viral life cycle, we examined the effects of depletion or overexpression of this protein on VSV growth. Small interfering RNA-mediated silencing of PCBP2 promoted VSV replication. Conversely, overexpression of PCBP2 in transfected cells suppressed VSV growth. Further studies revealed that PCBP2 negatively regulates overall viral mRNA accumulation and subsequent genome replication. Coimmunoprecipitation and immunofluorescence microscopic studies showed that PCBP2 interacts and colocalizes with VSV P protein in virus-infected cells. The P-PCBP2 interaction did not result in reduced levels of protein complex formation with the viral N and L proteins, nor did it induce degradation of the P protein. In addition, PCBP1, another member of the poly(C) binding protein family with homology to PCBP2, was also found to interact with the P protein and inhibit the viral mRNA synthesis at the level of primary transcription without affecting secondary transcription or genome replication. The inhibitory effects of PCBP1 on VSV replication were less pronounced than those of PCBP2. Overall, the results presented here suggest that cellular PCBP2 and PCBP1 antagonize VSV growth by affecting viral gene expression and highlight the importance of these two cellular proteins in restricting virus infections. PMID:21752917

  6. Large-scale mRNA expression profiling in the common ice plant, Mesembryanthemum crystallinum, performing C3 photosynthesis and Crassulacean acid metabolism (CAM). (United States)

    Cushman, John C; Tillett, Richard L; Wood, Joshua A; Branco, Joshua M; Schlauch, Karen A


    The common ice plant (Mesembryanthemum crystallinum L.) has emerged as a useful model for molecular genetic studies of Crassulacean acid metabolism (CAM) because CAM can be induced in this species by water deficit or salinity stress. Non-redundant sequence information from expressed sequence tag data was used to fabricate a custom oligonucleotide microarray to compare large-scale mRNA expression patterns in M. crystallinum plants conducting C(3) photosynthesis versus CAM. Samples were collected every 4 h over a 24 h time period at the start of the subjective second day from plants grown under constant light and temperature conditions in order to capture variation in mRNA expression due to salinity stress and circadian clock control. Of 8455 genes, a total of 2343 genes (approximately 28%) showed a significant change as judged by analysis of variance (ANOVA) in steady-state mRNA abundance at one or more time points over the 24 h period. Of these, 858 (10%) and 599 (7%) exhibited a greater than two-fold ratio (TFR) increase or decrease in mRNA abundance, respectively. Functional categorization of these TFR genes revealed that many genes encoding products that function in CAM-related C(4) acid carboxylation/decarboxylation, glycolysis/gluconeogenesis, polysaccharide, polyol, and starch biosynthesis/degradation, protein degradation, transcriptional activation, signalling, stress response, and transport facilitation, and novel, unclassified proteins exhibited stress-induced increases in mRNA abundance. In contrast, salt stress resulted in a significant decrease in transcript abundance for genes encoding photosynthetic functions, protein synthesis, and cellular biogenesis functions. Many genes with CAM-related functions exhibited phase shifts in their putative circadian expression patterns following CAM induction. This report establishes an extensive catalogue of gene expression patterns for future investigations aimed at understanding the complex, transcriptional

  7. Kaposi's sarcoma-associated herpesvirus ORF57 protein binds and protects a nuclear noncoding RNA from cellular RNA decay pathways.

    Directory of Open Access Journals (Sweden)

    Brooke B Sahin


    Full Text Available The control of RNA stability is a key determinant in cellular gene expression. The stability of any transcript is modulated through the activity of cis- or trans-acting regulatory factors as well as cellular quality control systems that ensure the integrity of a transcript. As a result, invading viral pathogens must be able to subvert cellular RNA decay pathways capable of destroying viral transcripts. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV ORF57 protein binds to a unique KSHV polyadenylated nuclear RNA, called PAN RNA, and protects it from degradation by cellular factors. ORF57 increases PAN RNA levels and its effects are greatest on unstable alleles of PAN RNA. Kinetic analysis of transcription pulse assays shows that ORF57 protects PAN RNA from a rapid cellular RNA decay process, but ORF57 has little effect on transcription or PAN RNA localization based on chromatin immunoprecipitation and in situ hybridization experiments, respectively. Using a UV cross-linking technique, we further demonstrate that ORF57 binds PAN RNA directly in living cells and we show that binding correlates with function. In addition, we define an ORF57-responsive element (ORE that is necessary for ORF57 binding to PAN RNA and sufficient to confer ORF57-response to a heterologous intronless beta-globin mRNA, but not its spliced counterparts. We conclude that ORF57 binds to viral transcripts in the nucleus and protects them from a cellular RNA decay pathway. We propose that KSHV ORF57 protein functions to enhance the nuclear stability of intronless viral transcripts by protecting them from a cellular RNA quality control pathway.

  8. Emerging links between m6A and misregulated mRNA methylation in cancer. (United States)

    Jaffrey, Samie R; Kharas, Michael G


    N 6-methyladenosine (m6A) in mRNA has emerged as a crucial epitranscriptomic modification that controls cellular differentiation and pluripotency. Recent studies are pointing to a role for the RNA methylation program in cancer self-renewal and cell fate, making this a new and promising therapeutic avenue for investigation.

  9. Dynamics of Translation of Single mRNA Molecules in Vivo

    NARCIS (Netherlands)

    Yan, Xiaowei; Hoek, Tim A.; Vale, Ronald D.; Tanenbaum, Marvin E.


    Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of

  10. Deformation heterogeneity in cellular Al alloys

    Energy Technology Data Exchange (ETDEWEB)

    Bastawros, A.-F. [Iowa State Univ., Ames, IA (United States). Dept. of Aerospace Engineering and Mechanics; Evans, A.G. [Princeton Univ., NJ (United States). Materials Inst.


    Cell ellipticity and non-planar membranes are thought to be dominant degradation factors of cellular metals. The mechanisms operating at the cell/membrane level in both closed and open cell Al alloys have been established by following the evolution of deformation with cell-level resolution using digital image correlation analysis. (orig.)

  11. Polysome Fractionation to Analyze mRNA Distribution Profiles. (United States)

    Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam


    Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

  12. Toxin MqsR Cleaves Single-Stranded mRNA with Various 5 Ends (United States)


    purified by the mini quick spin column for RNA as per manufacturer’s protocol (Roche, Indianapolis, IN). The RNA concentration was determined from the CPM...were visu- alized with a Typhoon 9410 phosphorimager (GE Healthcare , Tyrone, PA). Results To explore how toxin MqsR degrades mRNA, we designed four in

  13. Matrin 3 binds and stabilizes mRNA.

    Directory of Open Access Journals (Sweden)

    Maayan Salton

    Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  14. Impact of folic acid supplementation on single- and double-stranded RNA degradation in human colostrum and mature milk. (United States)

    Kocic, Gordana; Bjelakovic, Ljiljana; Bjelakovic, Bojko; Jevtoci-Stoimenov, Tatjana; Sokolovic, Dusan; Cvetkovic, Tatjana; Kocic, Hristina; Stojanovic, Svetlana; Langerholc, Tomaz; Jonovic, Marina


    Sufficient intake of folic acid is necessary for normal embryogenesis, fetal, and neonatal development. Folic acid facilitates nucleic acid internalization, and protects cellular DNA from nuclease degradation. Human milk contains enzymes, antimicrobial proteins, and antibodies, along with macrophages, that protect against infections and allergies. However, little to no information is available on the effects of folic acid supplementation on degradation of nucleic acids in human milk. In the present study, we aimed to determine the RNase activity (free and inhibitor-bound) in colostrum and mature milk, following folic acid supplementation. The study design included a total of 59 women, 27 of whom received 400 μg of folic acid daily periconceptionally and after. Folic acid supplementation increased the free RNase and polyadenylase activity following lactation. However, the increased RNase activity was not due to de novo enzyme synthesis, as the inhibitor-bound (latent) RNase activity was significantly lower and disappeared after one month. Folic acid reduced RNase activity by using double-stranded RNA as substrate. Data suggests that folic acid supplementation may improve viral RNAs degradation and mRNA degradation, but not dsRNA degradation, preserving in this way the antiviral defense.

  15. A single molecule approach to mRNA transport by a class V myosin. (United States)

    Sladewski, Thomas E; Trybus, Kathleen M


    mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. We show that an in vitro single molecule approach, using purified recombinant full-length proteins and synthesized mRNA, provides insight into the mechanism by which localizing mRNAs are carried to their destination. A messenger ribonucleoprotein (mRNP) complex was reconstituted from a budding yeast class V myosin motor complex (Myo4p-She3p), an mRNA-binding adaptor protein (She2p), and a localizing mRNA (ASH1). The motion of the mRNP was tracked with high spatial (∼10 nm) and temporal (70 ms) resolution. Using this "bottom-up" methodology, we show that mRNA triggers the assembly of a high affinity double-headed motor-mRNA complex that moves continuously for long distances on actin filaments at physiologic ionic strength. Without mRNA, the myosin is monomeric and unable to move continuously on actin. This finding reveals an elegant strategy to ensure that only cargo-bound motors are activated for transport. Increasing the number of localization elements ("zip codes") in the mRNA enhanced both the frequency of motile events and their run length, features which likely enhance cellular localization. Future in vitro reconstitution of mRNPs with kinesin and dynein motors should similarly yield mechanistic insight into mRNA transport by microtubule-based motors.

  16. The Dynamics of mRNA Turnover Revealed by Single-Molecule Imaging in Single Cells. (United States)

    Horvathova, Ivana; Voigt, Franka; Kotrys, Anna V; Zhan, Yinxiu; Artus-Revel, Caroline G; Eglinger, Jan; Stadler, Michael B; Giorgetti, Luca; Chao, Jeffrey A


    RNA degradation plays a fundamental role in regulating gene expression. In order to characterize the spatiotemporal dynamics of RNA turnover in single cells, we developed a fluorescent biosensor based on dual-color, single-molecule RNA imaging that allows intact transcripts to be distinguished from stabilized degradation intermediates. Using this method, we measured mRNA decay in single cells and found that individual degradation events occur independently within the cytosol and are not enriched within processing bodies. We show that slicing of an mRNA targeted for endonucleolytic cleavage by the RNA-induced silencing complex can be observed in real time in living cells. This methodology provides a framework for investigating the entire life history of individual mRNAs from birth to death in single cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Some Issues in WSN, MANET and Cellular Security (United States)


    gracefully degrade/elax security services? - What metrics to use when degrading? " Whither RFID /Sensor hybrid? MANETs Oblivious Routing - Fixed node...private? Most routing protools can’t hack it.. 4 2 Cellular (Wireless Devices) * Secure Association/Pairing - Heterogeneous devices - No PKI, no

  18. Regulation of mRNA translation during mitosis. (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D


    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  19. GLUT3 protein and mRNA in autopsy muscle specimens (United States)

    Stuart, C. A.; Wen, G.; Jiang, J.


    GLUT3 is expressed in rat muscle, but this glucose transporter protein has not been identified previously in adult human skeletal muscle. We quantified the rapidity of disappearance of mRNA and protein from human skeletal muscle at room temperature and at 4 degrees C. Fifty percent of the immunologically detectable GLUT3 protein disappeared by 1 hour at 20 degrees C and by 2 hours at 4 degrees C. mRNA for GLUT3 was decreased 50% by 2.2 hours at 20 degrees C and by 24 hours at 4 degrees C. Half of the measurable mRNAs for GLUT4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), alpha-actin, and beta-myosin disappeared by 0.8 to 2.1 hours at 20 degrees C and by 5.0 to 16.6 hours at 4 degrees C. Previous conclusions that GLUT3 is not expressed in human muscle were likely drawn because of artifacts related to degradation of GLUT3 protein in the specimens prior to study. Because of the rapid degradation of protein and mRNA, autopsy specimens of muscle must be obtained within 6 hours of death, and even then, protein and mRNA data will likely dramatically underestimate their expression in fresh muscle. Some previously published conclusions and recommendations regarding autopsy specimens are not stringent enough to consistently yield useful protein and mRNA.

  20. Reduced stability of mRNA secondary structure near the translation-initiation site in dsDNA viruses


    Wilke Claus O; Zhou Tong


    Abstract Background Recent studies have demonstrated a selection pressure for reduced mRNA secondary-structure stability near the start codon of coding sequences. This selection pressure can be observed in bacteria, archaea, and eukaryotes, and is likely caused by the requirement of efficient translation initiation in cellular organism. Results Here, we surveyed the complete genomes of 650 dsDNA virus strains for signals of reduced stability of mRNA secondary structure near the start codon. O...

  1. Prostaglandins stimulate renin secretion and renin mRNA in mouse renal juxtaglomerular cells

    DEFF Research Database (Denmark)

    Jensen, B L; Schmid, C; Kurtz, A


    identified PGI2 and PGE2 as stimulators of renin secretion; the effects were dose and time dependent. PGE2 also increased renin mRNA accumulation time and dose dependent. PGE2 and PGI2 activated adenylate cyclase concentration dependent in granular cells. PGE2 stimulations of renin secretion and renin m......RNA were nonadditive to those of forskolin and were inhibited by endothelin. The findings are compatible with cellular actions through adenosine 3',5'-cyclic monophosphate (cAMP). On total RNA harvested from whole kidneys, from microdisected glomeruli with attached afferent arterioles and from mesangial...

  2. Molecular approaches for forensic cell type identification: On mRNA, miRNA, DNA methylation and microbial markers. (United States)

    Sijen, Titia


    Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development. (United States)

    Kim, Kee K; Adelstein, Robert S; Kawamoto, Sachiyo


    Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin-proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo. Published by Elsevier Inc.

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

    Directory of Open Access Journals (Sweden)

    Alexei A. Bogdanov, Jr.


    Full Text Available 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 GFPnegative cells (2.5-3.0% had seven times higher NIRF intensity than the majority of GFP-positive or GFPnegative 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.

  5. The functional half-life of an mRNA depends on the ribosome spacing in an early coding region

    DEFF Research Database (Denmark)

    Pedersen, Margit; Nissen, Søren; Mitarai, Namiko


    Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5'-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described....... Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual...... codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts...

  6. Wireless Cellular Mobile Communications


    V. Zalud


    In this article is briefly reviewed the history of wireless cellular mobile communications, examined the progress in current second generation (2G) cellular standards and discussed their migration to the third generation (3G). The European 2G cellular standard GSM and its evolution phases GPRS and EDGE are described somewhat in detail. The third generation standard UMTS taking up on GSM/GPRS core network and equipped with a new advanced access network on the basis of code division multiple ac...

  7. Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Fiona Chalmers


    Full Text Available Background: The mammalian endoplasmic reticulum (ER continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR.  This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis.  The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α, double-stranded RNA-activated protein kinase-like ER kinase (PERK or activating transcription factor 6 (ATF6.  Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity.  The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1, as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR.  Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.

  8. The Human T-Lymphotropic Virus Type 1 Tax Protein Inhibits Nonsense-Mediated mRNA Decay by Interacting with INT6/EIF3E and UPF1 (United States)

    Mocquet, Vincent; Neusiedler, Julia; Rende, Francesca; Cluet, David; Robin, Jean-Philippe; Terme, Jean-Michel; Duc Dodon, Madeleine; Wittmann, Jürgen; Morris, Christelle; Le Hir, Hervé; Ciminale, Vincenzo


    In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. PMID:22553336

  9. Nano-flares: Probes for Transfection and mRNA Detection in Living Cells (United States)

    Seferos, Dwight S.; Giljohann, David A.; Hill, Haley D.; Prigodich, Andrew E.


    We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular “nano-flares” for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells. PMID:18034495

  10. Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

    DEFF Research Database (Denmark)

    Weischenfeldt, Joachim Lütken; Waage, Johannes Eichler; Tian, Geng


    a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes......ABSTRACT: BACKGROUND: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation...... of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. RESULTS: We developed...

  11. Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF. (United States)

    Sanford, Jeremy R; Coutinho, Pedro; Hackett, Jamie A; Wang, Xin; Ranahan, William; Caceres, Javier F


    The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.

  12. Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells. (United States)

    Rao, T R; Slobin, L I


    When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.

  13. Validation of two reference genes for mRNA level studies of murine disease models in neurobiology

    DEFF Research Database (Denmark)

    Meldgaard, Michael; Fenger, Christina; Lambertsen, Kate Lykke


    Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantificatio...

  14. Heterogeneous cellular networks

    CERN Document Server

    Hu, Rose Qingyang


    A timely publication providing coverage of radio resource management, mobility management and standardization in heterogeneous cellular networks The topic of heterogeneous cellular networks has gained momentum in industry and the research community, attracting the attention of standardization bodies such as 3GPP LTE and IEEE 802.16j, whose objectives are looking into increasing the capacity and coverage of the cellular networks. This book focuses on recent progresses,  covering the related topics including scenarios of heterogeneous network deployment, interference management i

  15. Nominal Cellular Automata

    Directory of Open Access Journals (Sweden)

    Tommaso Bolognesi


    Full Text Available The emerging field of Nominal Computation Theory is concerned with the theory of Nominal Sets and its applications to Computer Science. We investigate here the impact of nominal sets on the definition of Cellular Automata and on their computational capabilities, with a special focus on the emergent behavioural properties of this new model and their significance in the context of computation-oriented interpretations of physical phenomena. A preliminary investigation of the relations between Nominal Cellular Automata and Wolfram's Elementary Cellular Automata is also carried out.

  16. Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Koller, K.J.; Wolff, R.S.; Warden, M.K.; Zoeller, R.T.


    Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone, replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a /sup 35/S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatments with exogenous L-triiodothyronine (T/sub 3/) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T/sub 3/ replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-..beta.. mRNA levels were elevated after thyroidectomy and reduced by T/sub 3/ treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.

  17. Reversible methylation of m6Am in the 5′ cap controls mRNA stability (United States)

    Mauer, Jan; Luo, Xiaobing; Blanjoie, Alexandre; Jiao, Xinfu; Grozhik, Anya V.; Patil, Deepak P.; Linder, Bastian; Pickering, Brian F.; Vasseur, Jean-Jacques; Chen, Qiuying; Gross, Steven S.; Elemento, Olivier; Debart, Françoise; Kiledjian, Megerditch; Jaffrey, Samie R.


    Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. PMID:28002401

  18. The C. elegans mRNA decapping enzyme shapes morphology of cilia. (United States)

    Adachi, Takeshi; Nagahama, Keigo; Izumi, Susumu


    Cilia and flagella are evolutionarily conserved organelles that protrude from cell surfaces. Most cilia and flagella are single rod-shaped but some cilia show a variety of shapes. For example, human airway epithelial cells are multiciliated, flagella of crayfish spermatozoon are star-like shaped, and fruit fly spermatozoon extends long flagella. In Caenorhabditis elegans, cilia display morphological diversity of shapes (single, dual rod-type and wing-like and highly-branched shapes). Here we show that DCAP-1 and DCAP-2, which are the homologues of mammalian DCP1 and DCP2 mRNA decapping enzymes, respectively, are involved in formation of dual rod-type and wing-like shaped cilia in C. elegans. mRNA decapping enzyme catalyzes hydrolysis of 5' cap structure of mRNA, which leads to degradation of mRNA. Rescue experiments showed that DCAP-2 acts not in glial cells surrounding cilia but in neurons. This is the first evidence to demonstrate that mRNA decapping is involved in ciliary shape formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Kinetics of lipid-nanoparticle-mediated intracellular mRNA delivery and function (United States)

    Zhdanov, Vladimir P.


    mRNA delivery into cells forms the basis for one of the new and promising ways to treat various diseases. Among suitable carriers, lipid nanoparticles (LNPs) with a size of about 100 nm are now often employed. Despite high current interest in this area, the understanding of the basic details of LNP-mediated mRNA delivery and function is limited. To clarify the kinetics of mRNA release from LNPs, the author uses three generic models implying (i) exponential, (ii) diffusion-controlled, and (iii) detachment-controlled kinetic regimes, respectively. Despite the distinct differences in these kinetics, the associated transient kinetics of mRNA translation to the corresponding protein and its degradation are shown to be not too sensitive to the details of the mRNA delivery by LNPs (or other nanocarriers). In addition, the author illustrates how this protein may temporarily influence the expression of one gene or a few equivalent genes. The analysis includes positive or negative regulation of the gene transcription via the attachment of the protein without or with positive or negative feedback in the gene expression. Stable, bistable, and oscillatory schemes have been scrutinized in this context.

  20. Down regulation of a matrix degrading cysteine protease cathepsin L, by acetaldehyde: role of C/EBPα.

    Directory of Open Access Journals (Sweden)

    Riyaz A Mir

    Full Text Available BACKGROUND: The imbalance between extra cellular matrix (ECM synthesis and degradation is critical aspect of various hepatic pathologies including alcohol induced liver fibrosis. This study was carried out to investigate the effect of acetaldehyde on expression of an extra cellular matrix degrading protease cathepsin L (CTSL in HepG2 cells. METHODOLOGY AND RESULTS: We measured the enzymatic activity, protein and, mRNA levels of CTSL in acetaldehyde treated and untreated cells. The binding of CAAT enhancer binding protein α (C/EBP α to CTSL promoter and its key role in the transcription from this promoter and conferring responsiveness to acetaldehyde was established by site directed mutagenesis, electrophoretic mobility shift assay (EMSA, chromatin immunoprecipitation (ChIP assays and siRNA technology. Acetaldehyde treatment significantly decreased CTSL activity and protein levels in HepG2 cells. A similar decrease in the mRNA levels and promoter activity was also observed. This decrease by acetaldehyde was attributed to the fall in the liver enriched transcription factor C/EBP α levels and it's binding to the CTSL promoter. Mutagenesis of C/EBP α binding motifs revealed the key role of this factor in CTSL transcription as well as conferring responsiveness to acetaldehyde. The siRNA mediated silencing of the C/EBP α expression mimicked the effect of acetaldehyde on CTSL levels and its promoter activity. It also abolished the responsiveness of this promoter to acetaldehyde. CONCLUSION: Acetaldehyde down regulates the C/EBP α mediated CTSL expression in hepatic cell lines. The decreased expression of CTSL may at least in part contribute to ECM deposition in liver which is a hallmark of alcoholic liver fibrosis.

  1. Relative degradation of nuclear and mitochondrial DNA: an experimental approach. (United States)

    Foran, David R


    Single copy nuclear loci often cannot be amplified from degraded remains, necessitating the analysis of mitochondrial DNA (mtDNA). The success in analyzing mtDNA is generally thought to result from its higher copy number in the cell; however, other factors, such as cellular location or molecular features, may be equally or more important in the superior preservation of mtDNA. To explore and compare mtDNA and nuclear DNA degradation, mouse tissues (muscle, liver, and brain) were allowed to degrade at different temperatures, and the relative degradation of a mitochondrial gene, a single copy nuclear gene, and a multi-copy nuclear gene was assayed using real-time polymerase chain reaction. The tissues were also homogenized, allowing the three loci to degrade in the same cellular environment. Gene copy number and cellular location both influence DNA recovery. In some instances, multi-copy loci could be recovered when the single copy locus could not; however, the pattern of relative DNA degradation changed between whole and homogenized tissues. The overall results indicate that DNA degradation is influenced by multiple factors-including cellular location, chromatin structure, and transcriptional activity-factors that could be used to exploit loci for more robust forensic analysis from degraded biological material.

  2. Cellular magnesium homeostasis. (United States)

    Romani, Andrea M P


    Magnesium, the second most abundant cellular cation after potassium, is essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways, as attested by more than 1000 entries in the literature. Despite significant recent progress, however, our understanding of how cells regulate Mg(2+) homeostasis and transport still remains incomplete. For example, the occurrence of major fluxes of Mg(2+) in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli has been extensively documented. Yet, the mechanisms ultimately responsible for magnesium extrusion across the cell membrane have not been cloned. Even less is known about the regulation in cellular organelles. The present review is aimed at providing the reader with a comprehensive and up-to-date understanding of the mechanisms enacted by eukaryotic cells to regulate cellular Mg(2+) homeostasis and how these mechanisms are altered under specific pathological conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Modeling cellular systems

    CERN Document Server

    Matthäus, Franziska; Pahle, Jürgen


    This contributed volume comprises research articles and reviews on topics connected to the mathematical modeling of cellular systems. These contributions cover signaling pathways, stochastic effects, cell motility and mechanics, pattern formation processes, as well as multi-scale approaches. All authors attended the workshop on "Modeling Cellular Systems" which took place in Heidelberg in October 2014. The target audience primarily comprises researchers and experts in the field, but the book may also be beneficial for graduate students.

  4. Aberrant herpesvirus-induced polyadenylation correlates with cellular messenger RNA destruction.

    Directory of Open Access Journals (Sweden)

    Yeon J Lee


    Full Text Available Regulation of messenger RNA (mRNA stability plays critical roles in controlling gene expression, ensuring transcript fidelity, and allowing cells to respond to environmental cues. Unregulated enhancement of mRNA turnover could therefore dampen cellular responses to such signals. Indeed, several herpesviruses instigate widespread destruction of cellular mRNAs to block host gene expression and evade immune detection. Kaposi's sarcoma-associated herpesvirus (KSHV promotes this phenotype via the activity of its viral SOX protein, although the mechanism of SOX-induced mRNA turnover has remained unknown, given its apparent lack of intrinsic ribonuclease activity. Here, we report that KSHV SOX stimulates cellular transcriptome turnover via a unique mechanism involving aberrant polyadenylation. Transcripts in SOX-expressing cells exhibit extended poly(A polymerase II-generated poly(A tails and polyadenylation-linked mRNA turnover. SOX-induced polyadenylation changes correlate with its RNA turnover function, and inhibition of poly(A tail formation blocks SOX activity. Both nuclear and cytoplasmic poly(A binding proteins are critical cellular cofactors for SOX function, the latter of which undergoes striking nuclear relocalization by SOX. SOX-induced mRNA turnover therefore represents both a novel mechanism of host shutoff as well as a new model system to probe the regulation of poly(A tail-stimulated mRNA turnover in mammalian cells.

  5. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail:


    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  6. Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection. (United States)

    Uchida, Hirokuni; Itaka, Keiji; Nomoto, Takahiro; Ishii, Takehiko; Suma, Tomoya; Ikegami, Masaru; Miyata, Kanjiro; Oba, Makoto; Nishiyama, Nobuhiro; Kataoka, Kazunori


    Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

  7. KSHV SOX mediated host shutoff: the molecular mechanism underlying mRNA transcript processing. (United States)

    Lee, Hyunah; Patschull, Anathe O M; Bagnéris, Claire; Ryan, Hannah; Sanderson, Christopher M; Ebrahimi, Bahram; Nobeli, Irene; Barrett, Tracey E


    Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Designing degradable hydrogels for orthogonal control of cell microenvironments (United States)

    Kharkar, Prathamesh M.


    Degradable and cell-compatible hydrogels can be designed to mimic the physical and biochemical characteristics of native extracellular matrices and provide tunability of degradation rates and related properties under physiological conditions. Hence, such hydrogels are finding widespread application in many bioengineering fields, including controlled bioactive molecule delivery, cell encapsulation for controlled three-dimensional culture, and tissue engineering. Cellular processes, such as adhesion, proliferation, spreading, migration, and differentiation, can be controlled within degradable, cell-compatible hydrogels with temporal tuning of biochemical or biophysical cues, such as growth factor presentation or hydrogel stiffness. However, thoughtful selection of hydrogel base materials, formation chemistries, and degradable moieties is necessary to achieve the appropriate level of property control and desired cellular response. In this review, hydrogel design considerations and materials for hydrogel preparation, ranging from natural polymers to synthetic polymers, are overviewed. Recent advances in chemical and physical methods to crosslink hydrogels are highlighted, as well as recent developments in controlling hydrogel degradation rates and modes of degradation. Special attention is given to spatial or temporal presentation of various biochemical and biophysical cues to modulate cell response in static (i.e., non-degradable) or dynamic (i.e., degradable) microenvironments. This review provides insight into the design of new cell-compatible, degradable hydrogels to understand and modulate cellular processes for various biomedical applications. PMID:23609001

  9. A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells. (United States)

    Jan, Eric; Mohr, Ian; Walsh, Derek


    Although viruses require cellular functions to replicate, their absolute dependence upon the host translation machinery to produce polypeptides indispensable for their reproduction is most conspicuous. Despite their incredible diversity, the mRNAs produced by all viruses must engage cellular ribosomes. This has proven to be anything but a passive process and has revealed a remarkable array of tactics for rapidly subverting control over and dominating cellular regulatory pathways that influence translation initiation, elongation, and termination. Besides enforcing viral mRNA translation, these processes profoundly impact host cell-intrinsic immune defenses at the ready to deny foreign mRNA access to ribosomes and block protein synthesis. Finally, genome size constraints have driven the evolution of resourceful strategies for maximizing viral coding capacity. Here, we review the amazing strategies that work to regulate translation in virus-infected cells, highlighting both virus-specific tactics and the tremendous insight they provide into fundamental translational control mechanisms in health and disease.

  10. Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export. (United States)

    Zhang, Liang; Das, Priyabrata; Schmolke, Mirco; Manicassamy, Balaji; Wang, Yaming; Deng, Xiaoyi; Cai, Ling; Tu, Benjamin P; Forst, Christian V; Roth, Michael G; Levy, David E; García-Sastre, Adolfo; de Brabander, Jef; Phillips, Margaret A; Fontoura, Beatriz M A


    The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors. © 2012 Zhang et al.

  11. Single-cell nanobiopsy reveals compartmentalization of mRNA in neuronal cells. (United States)

    Tóth, Eszter N; Lohith, Akshar; Mondal, Manas; Guo, Jia; Fukamizu, Akiyoshi; Pourmand, Nader


    In highly polarized cells such as neurons, compartmentalization of mRNA and of local protein synthesis enables remarkably fast, precise, and local responses to external stimuli. These responses are highly important for neuron growth cone guidance, synapse formation, and regeneration following injury. Because an altered spatial distribution of mRNA can result in mental retardation or neurodegenerative diseases, subcellular transcriptome analysis of neurons could be a useful tool for studying these conditions, but current techniques, such as in situ hybridization, bulk microarray, or RNA-Seq, impose tradeoffs between spatial resolution and multiplexing. To obtain a comprehensive analysis of the cell body versus neurite transcriptome from the same neuron, we have recently developed a label-free, single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM), that uses electrowetting within a quartz nanopipette to extract cellular material from living cells with minimal disruption of the cellular membrane and milieu. In this study, we used this platform to collect samples from the cell bodies and neurites of human neurons and analyzed the mRNA pool with multiplex RNA-Seq. The minute volume of a nanobiopsy sample allowed us to extract samples from several locations in the same cell and to map the various mRNA species to specific subcellular locations. In addition to previously identified transcripts, we discovered new sets of mRNAs localizing to neurites, including nuclear genes such as Eomes and Nap1l3. In summary, our single-neuron nanobiopsy analysis provides opportunities to improve our understanding of intracellular mRNA transport and local protein composition in neuronal growth, connectivity, and function. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

  12. Is central dogma a global property of cellular information flow? (United States)

    Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar


    The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

  13. Is central dogma a global property of cellular information flow?

    Directory of Open Access Journals (Sweden)

    Vincent ePiras


    Full Text Available The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcript to protein show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

  14. Protein Repair and Degradation during Aging

    Directory of Open Access Journals (Sweden)

    Bertrand Friguet


    Full Text Available Cellular aging is characterized by a build-up of oxidatively modified proteins. The steady-state level of oxidized proteins depends on the balance between the rate of protein oxidative damage and the rates of protein degradation and repair. Therefore, the accumulation of oxidized protein with age can be due to increased protein damage, decreased oxidized protein degradation and repair, or the combination of both mechanisms. The proteasomal system is the major intracellular proteolytic pathway implicated in the degradation of oxidized protein, and the peptide methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide (i.e., oxidized methionine to methionine within proteins. A short summary on protein oxidative damage and oxidized protein degradation is given, and evidence for a decline of proteasome function with age is presented. Arguments for the implication of peptide methionine sulfoxide reductase in the age-related accumulation of oxidized protein are also discussed.

  15. Epigenetics and Cellular Metabolism

    Directory of Open Access Journals (Sweden)

    Wenyi Xu


    Full Text Available Living eukaryotic systems evolve delicate cellular mechanisms for responding to various environmental signals. Among them, epigenetic machinery (DNA methylation, histone modifications, microRNAs, etc. is the hub in transducing external stimuli into transcriptional response. Emerging evidence reveals the concept that epigenetic signatures are essential for the proper maintenance of cellular metabolism. On the other hand, the metabolite, a main environmental input, can also influence the processing of epigenetic memory. Here, we summarize the recent research progress in the epigenetic regulation of cellular metabolism and discuss how the dysfunction of epigenetic machineries influences the development of metabolic disorders such as diabetes and obesity; then, we focus on discussing the notion that manipulating metabolites, the fuel of cell metabolism, can function as a strategy for interfering epigenetic machinery and its related disease progression as well.

  16. Cellular Basis of Antiproliferative and Antitumor Activity of the Novel Camptothecin Derivative, Gimatecan, in Bladder Carcinoma Models

    Directory of Open Access Journals (Sweden)

    Paola Ulivi


    Full Text Available To investigate the cellular/molecular basis of the activity of a novel lipophilic camptothecin, gimatecan (ST1481, against slowly proliferating cells, we performed a comparative study of topotecan and gimatecan in human bladder cancer models (HT1376 and MCR. Gimatecan was significantly more effective than topotecan in inhibiting the growth of HT1376 tumor, thus reflecting anti proliferative potency. In both HT1376 and MCR cells, gimatecan caused a persistent S-phase arrest, indicating an efficient DNA damage checkpoint. This response was consistent with a cytostatic effect, because no evidence of apoptosis was detected. In contrast to gimatecan, topotecan at equitoxic concentrations caused an early and persistent downregulation of topoisomerase I. Modulation of protein level could not be solely ascribed to the proteasome-mediated degradation of the enzyme because the proteasome inhibitor PS341 sensitized MCR but not HT1376 cells to camptothecins, suggesting alternative mechanisms of drug-induced topoisomerase I downregulation. Indeed, the two camptothecins caused a differential inhibition of topoisomerase I transcription, which is more marked in topotecan-treated cells. The HT1376 model was more sensitive to this immediate decrease of mRNA level. Our data document a marked antitumor activity of gimatecan against a bladder carcinoma model. A limited downregulation of topoisomerase I by gimatecan provides additional insights into the cellular basis of drug potency.

  17. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis


    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

  18. Wireless Cellular Mobile Communications

    Directory of Open Access Journals (Sweden)

    V. Zalud


    Full Text Available In this article is briefly reviewed the history of wireless cellularmobile communications, examined the progress in current secondgeneration (2G cellular standards and discussed their migration to thethird generation (3G. The European 2G cellular standard GSM and itsevolution phases GPRS and EDGE are described somewhat in detail. Thethird generation standard UMTS taking up on GSM/GPRS core network andequipped with a new advanced access network on the basis of codedivision multiple access (CDMA is investigated too. A sketch of theperspective of mobile communication beyond 3G concludes this article.

  19. Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability

    DEFF Research Database (Denmark)

    Vytvytska, O; Jakobsen, J S; Balcunaite, G


    The stability of the ompA mRNA depends on the bacterial growth rate. The 5' untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5' untranslated region......RNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has...

  20. N6-methyladenosine modification in mRNA: machinery, function and implications for health and diseases. (United States)

    Maity, Arpita; Das, Biswadip


    N6-methyladenosine (m(6) A) modification in mRNA is extremely widespread, and functionally modulates the eukaryotic transcriptome to influence mRNA splicing, export, localization, translation, and stability. Methylated adenines are present in a large subset of mRNAs and long noncoding RNAs (lncRNAs). Methylation is reversible, and this is accomplished by the orchestrated action of highly conserved methyltransferase (m(6) A writer) and demethylase (m(6) A eraser) enzymes to shape the cellular 'epitranscriptome'. The engraved 'methyl code' is subsequently decoded and executed by a group of m(6) A reader/effector components, which, in turn, govern the fate of the modified transcripts, thereby dictating their potential for translation. Reversible mRNA methylation thus adds another layer of regulation at the post-transcriptional level in the gene expression programme of eukaryotes that finely sculpts a highly dynamic proteome in order to respond to diverse cues during cellular differentiation, immune tolerance, and neuronal signalling. © 2015 FEBS.

  1. Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification. (United States)

    Zhang, Yaqi; Liu, Baonian; Shao, Chengchen; Xu, Hongmei; Xue, Aimin; Zhao, Ziqin; Shen, Yiwen; Tang, Qiqun; Xie, Jianhui


    The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

  2. The New Cellular Immunology (United States)

    Claman, Henry N.


    Discusses the nature of the immune response and traces many of the discoveries that have led to the present state of knowledge in immunology. The new cellular immunology is directing its efforts toward improving health by proper manipulation of the immune mechanisms of the body. (JR)

  3. m6A Facilitates eIF4F-Independent mRNA Translation. (United States)

    Coots, Ryan A; Liu, Xiao-Min; Mao, Yuanhui; Dong, Leiming; Zhou, Jun; Wan, Ji; Zhang, Xingqian; Qian, Shu-Bing


    In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M


    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  5. Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi. (United States)

    Tanaka, Mizuki; Tokuoka, Masafumi; Gomi, Katsuya


    Filamentous fungi, particularly Aspergillus species, have recently attracted attention as host organisms for recombinant protein production. Because the secretory yields of heterologous proteins are generally low compared with those of homologous proteins or proteins from closely related fungal species, several strategies to produce substantial amounts of recombinant proteins have been conducted. Codon optimization is a powerful tool for improving the production levels of heterologous proteins. Although codon optimization is generally believed to improve the translation efficiency of heterologous genes without affecting their mRNA levels, several studies have indicated that codon optimization causes an increase in the steady-state mRNA levels of heterologous genes in filamentous fungi. However, the mechanism that determines the low mRNA levels when native heterologous genes are expressed was poorly understood. We recently showed that the transcripts of heterologous genes are polyadenylated prematurely within the coding region and that the heterologous gene transcripts can be stabilized significantly by codon optimization, which is probably attributable to the prevention of premature polyadenylation in Aspergillus oryzae. In this review, we describe the detailed mechanism of premature polyadenylation and the rapid degradation of mRNA transcripts derived from heterologous genes in filamentous fungi.

  6. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A) Polymorphism. (United States)

    Inaoka, Yoshihiko; Osawa, Motoki; Mukasa, Nahoko; Miyashita, Keiko; Satoh, Fumiko; Kakimoto, Yu


    Alpha 2-HS glycoprotein (AHSG), also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG(∗) 1 and AHSG(∗) 2. The serum level in the AHSG(∗) 1 homozygote is significantly higher than that of the AHSG(∗) 2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG(∗) 1 to AHSG(∗) 2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  7. Allelic Imbalance of mRNA Associated with α2-HS Glycoprotein (Fetuin-A Polymorphism

    Directory of Open Access Journals (Sweden)

    Yoshihiko Inaoka


    Full Text Available Alpha 2-HS glycoprotein (AHSG, also designated as fetuin-A, exhibits polymorphism in population genetics consisting of two major alleles of AHSG∗1 and AHSG∗2. The serum level in the AHSG∗1 homozygote is significantly higher than that of the AHSG∗2 homozygote. This study examined the molecular mechanism for the cis-regulatory expression. To quantitate allele-specific mRNA in intra-assays of the heterozygote, RT-PCR method employing primers that were incorporated to the two closely located SNPs was developed. The respective magnitudes of AHSG∗1 to AHSG∗2 in the liver tissues and hepatic culture cells of PLC/PRF/5 were determined quantitatively as 2.5-fold and 6.2-fold. The mRNA expressional difference of two major alleles was observed, which is consistent with that in the serum level. The culture cells carried heterozygous genotypes in rs4917 and rs4918, but homozygous one in rs2248690. It was unlikely that the imbalance was derived from the SNP located in the promotor site. Furthermore, to investigate the effect of mRNA degradation, RNA synthesis in the cell culture was inhibited potently by the addition of actinomycin-D. No marked change was apparent between the two alleles. The results indicated that the cis-regulatory expressional difference is expected to occur at the level of transcription or splicing of mRNA.

  8. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)


    Jul 10, 2013 ... mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional .... Figure 1. mRNA life-cycle in eukaryotic cell: Schematic view of the nuclear and cytoplasmic phases of mRNA life cycle, namely ..... structure is characteristic and critical feature of an mRNA.

  9. Simple and inexpensive ribosome profiling analysis of mRNA translation. (United States)

    Reid, David W; Shenolikar, Shirish; Nicchitta, Christopher V


    The development and application of ribosome profiling has markedly advanced our understanding of ribosomes and mRNA translation. The experimental approach, which relies on deep sequencing of ribosome-protected mRNA fragments generated by treatment of polyribosomes with exogenous nucleases, provides a transcriptome-wide assessment of translation. The broad application of ribosome profiling has been slowed by the complexity and expense of the protocol. Here, we provide a simplified ribosome profiling method that uses micrococcal nuclease to generate ribosome footprints in crude cellular extracts, which are then purified simply by size selection via polyacrylamide gel electrophoresis. This simplification removes the laborious or expensive purification of ribosomes that has typically been used. This direct extraction method generates gene-level ribosome profiling data that are similar to a method that includes ribosome purification. This protocol should significantly ease the barrier to entry for research groups interested in employing ribosome profiling. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Molecular analysis of methanogens involved in methanogenic degradation of tetramethylammonium hydroxide in full-scale bioreactors. (United States)

    Whang, Liang-Ming; Hu, Tai-Ho; Liu, Pao-Wen Grace; Hung, Yu-Ching; Fukushima, Toshikazu; Wu, Yi-Ju; Chang, Shao-Hsiung


    This study investigated methanogenic communities involved in degradation of tetramethylammonium hydroxide (TMAH) in three full-scale bioreactors treating TMAH-containing wastewater. Based on the results of terminal-restriction fragment-length polymorphism (T-RFLP) and quantitative PCR analyses targeting the methyl-coenzyme M reductase alpha subunit (mcrA) genes retrieved from three bioreactors, Methanomethylovorans and Methanosarcina were the dominant methanogens involved in the methanogenic degradation of TMAH in the bioreactors. Furthermore, batch experiments were conducted to evaluate mcrA messenger RNA (mRNA) expression during methanogenic TMAH degradation, and the results indicated that a higher level of TMAH favored mcrA mRNA expression by Methansarcina, while Methanomethylovorans could only express considerable amount of mcrA mRNA at a lower level of TMAH. These results suggest that Methansarcina is responsible for methanogenic TMAH degradation at higher TMAH concentrations, while Methanomethylovorans may be important at a lower TMAH condition.

  11. PEM fuel cell degradation

    Energy Technology Data Exchange (ETDEWEB)

    Borup, Rodney L [Los Alamos National Laboratory; Mukundan, Rangachary [Los Alamos National Laboratory


    The durability of PEM fuel cells is a major barrier to the commercialization of these systems for stationary and transportation power applications. While significant progress has been made in understanding degradation mechanisms and improving materials, further improvements in durability are required to meet commercialization targets. Catalyst and electrode durability remains a primary degradation mode, with much work reported on understanding how the catalyst and electrode structure degrades. Accelerated Stress Tests (ASTs) are used to rapidly evaluate component degradation, however the results are sometimes easy, and other times difficult to correlate. Tests that were developed to accelerate degradation of single components are shown to also affect other component's degradation modes. Non-ideal examples of this include ASTs examining catalyst degradation performances losses due to catalyst degradation do not always well correlate with catalyst surface area and also lead to losses in mass transport.

  12. Identification of T-cell epitopes by a novel mRNA PCR-based epitope chase technique. (United States)

    Doucet, Jean-Daniel; Gauchat, Dominique; Lapointe, Réjean


    The identification of specific viral and tumor antigen T-cell epitopes remains a challenge. Indeed, epitope mapping methods are generally costly and time-consuming. Thus, few techniques allow for efficient CD4+ T-lymphocyte epitope identification. Here, we introduce a novel polymerase chain reaction-based mRNA epitope identification method, called mPEC, to rapidly and precisely identify relevant T-cell epitopes recognized by CD8+ or CD4+ T lymphocytes. This method is based on the use of mRNA fragments synthesized from polymerase chain reaction-amplified cDNA with a choice of 3'end deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to deduce an epitope's localization in a given protein antigen. Considering mRNA's sensitivity to degradation, we also inserted a defined epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and its capacity to be translated. Using this method, we rapidly and successfully identified the specific epitope of 2 CD8+ and 1 CD4+ T-lymphocyte clones derived from influenza model antigens. Hence, mPEC could be used to identify new, in vivo-relevant T-cell epitopes for cancer immunotherapy and vaccination in general.

  13. Reduced m6A mRNA methylation is correlated with the progression of human cervical cancer. (United States)

    Wang, Xiuli; Li, Zenghui; Kong, Beihua; Song, Chen; Cong, Jianglin; Hou, Jianqing; Wang, Shaoguang


    The m6A mRNA methylation involves in mRNA splicing, degradation and translation. Recent studies have revealed that reduced m6A mRNA methylation might promote cancer development. However, the role of m6A mRNA methylation in cervical cancer development remains unknown. Therefore, we investigated the role of m6A methylation in cervical cancer in the current study. We first evaluated the m6A mRNA methylation level in 286 pairs of cervical cancer samples and their adjacent normal tissues by dot blot assay. Then the role of m6A on patient survival rates and cervical cancer progression were assessed. The m6A level was significantly reduced in the cervical cancer when comparing with the adjacent normal tissue. The m6A level reduction was significantly correlated with the FIGO stage, tumor size, differentiation, lymph invasion and cancer recurrence. It was also shown to be an independent prognostic indicator of disease-free survival and overall survival for patients with cervical cancer. Reducing m6A level via manipulating the m6A regulators expression promoted cervical cancer cell proliferation. And increasing m6A level significantly suppressed tumor development both in vitro and in vivo. Our results showed that the reduced m6A level is tightly associated with cervical cancer development and m6A mRNA methylation might be a potential therapeutic target in cervical cancer.

  14. Molecular and Cellular Signaling

    CERN Document Server

    Beckerman, Martin


    A small number of signaling pathways, no more than a dozen or so, form a control layer that is responsible for all signaling in and between cells of the human body. The signaling proteins belonging to the control layer determine what kinds of cells are made during development and how they function during adult life. Malfunctions in the proteins belonging to the control layer are responsible for a host of human diseases ranging from neurological disorders to cancers. Most drugs target components in the control layer, and difficulties in drug design are intimately related to the architecture of the control layer. Molecular and Cellular Signaling provides an introduction to molecular and cellular signaling in biological systems with an emphasis on the underlying physical principles. The text is aimed at upper-level undergraduates, graduate students and individuals in medicine and pharmacology interested in broadening their understanding of how cells regulate and coordinate their core activities and how diseases ...

  15. Vitamin D and the RNA transcriptome: more than mRNA regulation

    Directory of Open Access Journals (Sweden)

    Moray J Campbell


    Full Text Available The GRCh37.p13 primary assembly of the human genome contains 20805 protein coding mRNA, and 37147 non-protein coding genes and pseudogenes that as a result of RNA processing and editing generate 196501 gene transcripts. Given the size and diversity of the human transcriptome, it is timely to revisit what is known of VDR function in the regulation and targeting of transcription.Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approxamte size, and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. With the discovery of microRNA, investigators also considered VDR regulation of these non-protein coding RNA. Again, cell and time dependency has emerged. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. As the awareness of the diversity of non-coding RNA increases, it is evident that VDR actions are mediated through these molecules also. Key knowledge gaps remain over the VDR transcriptome. The causes for the cell and type dependent transcriptional heterogenetiy remain enigmatic. ChIP-Seq approaches have confirmed that VDR binding choices differ very significantly by cell type, but as yet the underlying causes distilling VDR binding choices are unclear. Similarly, it is clear that many of the VDR binding sites are non-canonical in nature but again the mechanisms underlying these interactions are unclear. Finally, although alternative splicing is clearly a very significant process in cellular transcriptional control, the lack of RNA-Seq data centered on VDR function are currently limiting the global assessment of the VDR transcriptome. VDR focused research

  16. Localized IRES-dependent translation of ER chaperone protein mRNA in sensory axons.

    Directory of Open Access Journals (Sweden)

    Almudena Pacheco

    Full Text Available Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.

  17. Probabilistic cellular automata. (United States)

    Agapie, Alexandru; Andreica, Anca; Giuclea, Marius


    Cellular automata are binary lattices used for modeling complex dynamical systems. The automaton evolves iteratively from one configuration to another, using some local transition rule based on the number of ones in the neighborhood of each cell. With respect to the number of cells allowed to change per iteration, we speak of either synchronous or asynchronous automata. If randomness is involved to some degree in the transition rule, we speak of probabilistic automata, otherwise they are called deterministic. With either type of cellular automaton we are dealing with, the main theoretical challenge stays the same: starting from an arbitrary initial configuration, predict (with highest accuracy) the end configuration. If the automaton is deterministic, the outcome simplifies to one of two configurations, all zeros or all ones. If the automaton is probabilistic, the whole process is modeled by a finite homogeneous Markov chain, and the outcome is the corresponding stationary distribution. Based on our previous results for the asynchronous case-connecting the probability of a configuration in the stationary distribution to its number of zero-one borders-the article offers both numerical and theoretical insight into the long-term behavior of synchronous cellular automata.

  18. Predictability in cellular automata. (United States)

    Agapie, Alexandru; Andreica, Anca; Chira, Camelia; Giuclea, Marius


    Modelled as finite homogeneous Markov chains, probabilistic cellular automata with local transition probabilities in (0, 1) always posses a stationary distribution. This result alone is not very helpful when it comes to predicting the final configuration; one needs also a formula connecting the probabilities in the stationary distribution to some intrinsic feature of the lattice configuration. Previous results on the asynchronous cellular automata have showed that such feature really exists. It is the number of zero-one borders within the automaton's binary configuration. An exponential formula in the number of zero-one borders has been proved for the 1-D, 2-D and 3-D asynchronous automata with neighborhood three, five and seven, respectively. We perform computer experiments on a synchronous cellular automaton to check whether the empirical distribution obeys also that theoretical formula. The numerical results indicate a perfect fit for neighbourhood three and five, which opens the way for a rigorous proof of the formula in this new, synchronous case.

  19. Messenger RNA (mRNA) nanoparticle tumour vaccination (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.


    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  20. Environment Aware Cellular Networks

    KAUST Repository

    Ghazzai, Hakim


    The unprecedented rise of mobile user demand over the years have led to an enormous growth of the energy consumption of wireless networks as well as the greenhouse gas emissions which are estimated currently to be around 70 million tons per year. This significant growth of energy consumption impels network companies to pay huge bills which represent around half of their operating expenditures. Therefore, many service providers, including mobile operators, are looking for new and modern green solutions to help reduce their expenses as well as the level of their CO2 emissions. Base stations are the most power greedy element in cellular networks: they drain around 80% of the total network energy consumption even during low traffic periods. Thus, there is a growing need to develop more energy-efficient techniques to enhance the green performance of future 4G/5G cellular networks. Due to the problem of traffic load fluctuations in cellular networks during different periods of the day and between different areas (shopping or business districts and residential areas), the base station sleeping strategy has been one of the main popular research topics in green communications. In this presentation, we present several practical green techniques that provide significant gains for mobile operators. Indeed, combined with the base station sleeping strategy, these techniques achieve not only a minimization of the fossil fuel consumption but also an enhancement of mobile operator profits. We start with an optimized cell planning method that considers varying spatial and temporal user densities. We then use the optimal transport theory in order to define the cell boundaries such that the network total transmit power is reduced. Afterwards, we exploit the features of the modern electrical grid, the smart grid, as a new tool of power management for cellular networks and we optimize the energy procurement from multiple energy retailers characterized by different prices and pollutant

  1. Cellular contractility requires ubiquitin mediated proteolysis.

    Directory of Open Access Journals (Sweden)

    Yuval Cinnamon

    Full Text Available BACKGROUND: Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility. PRINCIPAL FINDINGS: We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation. CONCLUSIONS: Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.

  2. Processing bodies are not required for mammalian nonsense-mediated mRNA decay


    Stalder, L.; Muhlemann, O.


    Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-bo...

  3. Fibronectin 1 mRNA expression correlates with advanced disease in renal cancer

    Directory of Open Access Journals (Sweden)

    Stenzl Arnulf


    Full Text Available Abstract Background Fibronectin 1 (FN1 is a glycoprotein involved in cellular adhesion and migration processes. The aim of this study was to elucidate the role of FN1 in development of renal cell cancer (RCC and to determine a prognostic relevance for optimal clinical management. Methods 212 renal tissue samples (109 RCC, 86 corresponding tissues from adjacent normal renal tissue and 17 oncocytomas were collected from patients undergoing surgery for renal tumors and subjected to total RNA extraction. Detection of FN1 mRNA expression was performed using quantitative real time PCR, three endogenous controls, renal proximal tubular epithelial cells (RPTEC as biological control and the ΔΔCt method for calculation of relative quantities. Results Mean tissue specific FN1 mRNA expression was found to be increased approximately seven fold comparing RCC and corresponding kidney control tissues (p FN1 expression was increased approx. 11 fold in clear cell compared to papillary RCC (p = 9×10-5; Wilcoxon rank sum test. Patients with advanced disease had higher FN1 expression when compared to organ-confined disease (p FN1 mRNA expression between organ-confined and advanced disease in the papillary and not in the clear cell RCC group (p = 0.02 vs. p = 0.2; Wilcoxon rank sum test. There was an increased expression in RCC compared to oncocytoma (p = 0.016; ANOVA. Conclusions To our knowledge, this is the first study to show that FN1 mRNA expression is higher in RCC compared to normal renal tissue. FN1 mRNA expression might serve as a marker for RCC aggressiveness, indicating early systemic progression particularly for patients with papillary RCC.

  4. Cosserat modeling of cellular solids

    NARCIS (Netherlands)

    Onck, P.R.


    Cellular solids inherit their macroscopic mechanical properties directly from the cellular microstructure. However, the characteristic material length scale is often not small compared to macroscopic dimensions, which limits the applicability of classical continuum-type constitutive models. Cosserat

  5. In vitro selection of external guide sequences for directing human RNase P to cleave a target mRNA. (United States)

    Raj, Stephen; Liu, Fenyong


    External guide sequences (EGSs) are oligonucleotides that consist of a sequence that is complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. Recent studies indicate that increasing the targeting activity of EGSs in directing human RNase P to cleave an mRNA in vitro can lead to better efficacies of the EGSs in inducing RNase P-mediated inhibition of the expression of the target mRNA in cultured cells. This chapter will describe the procedure for the generation of highly functional EGSs by in vitro selection. We also describe protocols for in vitro evaluation of the activity of the EGSs. These methods should provide general guidelines for using in vitro selection for generating highly active EGSs for gene-targeting applications.

  6. Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display (United States)

    Brzostowicz, Patricia C.; Gibson, Katharine L.; Thomas, Stuart M.; Blasko, Mary Sue; Rouvière, Pierre E.


    The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes. PMID:10894733

  7. Cellular communication through light.

    Directory of Open Access Journals (Sweden)

    Daniel Fels

    Full Text Available Information transfer is a fundamental of life. A few studies have reported that cells use photons (from an endogenous source as information carriers. This study finds that cells can have an influence on other cells even when separated with a glass barrier, thereby disabling molecule diffusion through the cell-containing medium. As there is still very little known about the potential of photons for intercellular communication this study is designed to test for non-molecule-based triggering of two fundamental properties of life: cell division and energy uptake. The study was performed with a cellular organism, the ciliate Paramecium caudatum. Mutual exposure of cell populations occurred under conditions of darkness and separation with cuvettes (vials allowing photon but not molecule transfer. The cell populations were separated either with glass allowing photon transmission from 340 nm to longer waves, or quartz being transmittable from 150 nm, i.e. from UV-light to longer waves. Even through glass, the cells affected cell division and energy uptake in neighboring cell populations. Depending on the cuvette material and the number of cells involved, these effects were positive or negative. Also, while paired populations with lower growth rates grew uncorrelated, growth of the better growing populations was correlated. As there were significant differences when separating the populations with glass or quartz, it is suggested that the cell populations use two (or more frequencies for cellular information transfer, which influences at least energy uptake, cell division rate and growth correlation. Altogether the study strongly supports a cellular communication system, which is different from a molecule-receptor-based system and hints that photon-triggering is a fine tuning principle in cell chemistry.

  8. Engineering Cellular Metabolism

    DEFF Research Database (Denmark)

    Nielsen, Jens; Keasling, Jay


    Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds...... of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation....

  9. Review of cellular mechanotransduction (United States)

    Wang, Ning


    Living cells and tissues experience physical forces and chemical stimuli in the human body. The process of converting mechanical forces into biochemical activities and gene expression is mechanochemical transduction or mechanotransduction. Significant advances have been made in understanding mechanotransduction at the cellular and molecular levels over the last two decades. However, major challenges remain in elucidating how a living cell integrates signals from mechanotransduction with chemical signals to regulate gene expression and to generate coherent biological responses in living tissues in physiological conditions and diseases.

  10. Engineering Cellular Metabolism

    DEFF Research Database (Denmark)

    Nielsen, Jens; Keasling, Jay


    of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.......Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds...

  11. Role of cysteine cathepsins in matrix degradation and cell signalling. (United States)

    Obermajer, Natasa; Jevnikar, Zala; Doljak, Bojan; Kos, Janko


    Cysteine cathepsins participate in extracellular matrix (ECM) degradation and remodelling and thus influence important cellular processes such as cell transformation and differentiation, motility, adhesion, invasion, angiogenesis, and metastasis. Also, cathepsins are involved in cell signalling and are capable of activating specific cell receptors and growth factors or liberating them from the ECM. In this review we emphasize recent studies on cathepsins in regard to ECM degradation and cell signalling.

  12. Integrated cellular systems (United States)

    Harper, Jason C.

    The generation of new three-dimensional (3D) matrices that enable integration of biomolecular components and whole cells into device architectures, without adversely altering their morphology or activity, continues to be an expanding and challenging field of research. This research is driven by the promise that encapsulated biomolecules and cells can significantly impact areas as diverse as biocatalysis, controlled delivery of therapeutics, environmental and industrial process monitoring, early warning of warfare agents, bioelectronics, photonics, smart prosthetics, advanced physiological sensors, portable medical diagnostic devices, and tissue/organ replacement. This work focuses on the development of a fundamental understanding of the biochemical and nanomaterial mechanisms that govern the cell directed assembly and integration process. It was shown that this integration process relies on the ability of cells to actively develop a pH gradient in response to evaporation induced osmotic stress, which catalyzes silica condensation within a thin 3D volume surrounding the cells, creating a functional bio/nano interface. The mechanism responsible for introducing functional foreign membrane-bound proteins via proteoliposome addition to the silica-lipid-cell matrix was also determined. Utilizing this new understanding, 3D cellular immobilization capabilities were extended using sol-gel matrices endowed with glycerol, trehalose, and media components. The effects of these additives, and the metabolic phase of encapsulated S. cerivisiase cells, on long-term viability and the rate of inducible gene expression was studied. This enabled the entrapment of cells within a novel microfluidic platform capable of simultaneous colorimetric, fluorescent, and electrochemical detection of a single analyte, significantly improving confidence in the biosensor output. As a complementary approach, multiphoton protein lithography was utilized to engineer 3D protein matrices in which to

  13. mRNA vaccines - a new era in vaccinology. (United States)

    Pardi, Norbert; Hogan, Michael J; Porter, Frederick W; Weissman, Drew


    mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

  14. Bacterial Degradation of Pesticides

    DEFF Research Database (Denmark)

    Knudsen, Berith Elkær

    . Bioaugmentation i.e. addition of specific degrader organisms, has been suggested as an environmentally friendly and economically competitive strategy for cleaning polluted sites. Several organisms have been isolated, capable of degrading different compounds. However the capacity to degrade the desired compound...... SRS2, Variovorax SRS16 and Arthrobacter globiformis D47. The degradation capacity of each strain individually as well as two- and three-member consortia was studied in a sand column set up. Glass beads were added to the set up to create a dry patch, separating the organisms and the diuron-spiked sand...

  15. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for NOS mRNA expression

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Dijkstra, G; Moshage, H; Elsinga, PH; Jansen, PLM; Vaalburg, W

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[F-18]fluorobenzyl)-2-bromoacetamide. Cellular

  16. New markers for old stains: Stable mRNA markers for blood and saliva identification from up to 16-year-old stains

    NARCIS (Netherlands)

    Zubakov, D.; Kokshoorn, M.; Kloosterman, A.; Kayser, M.


    In forensic science, the unequivocal identification of the cellular origin of crime scene samples used for DNA profiling can provide crucial information for crime scene reconstruction. We have previously shown that various mRNA markers from genes with expression patterns specific for blood and

  17. Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells. (United States)

    Choi, Hye Yeon; Lee, Tae-Jin; Yang, Gwang-Mo; Oh, Jaesur; Won, Jihye; Han, Jihae; Jeong, Gun-Jae; Kim, Jongpil; Kim, Jin-Hoi; Kim, Byung-Soo; Cho, Ssang-Goo


    Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings. Copyright © 2016. Published by Elsevier B.V.

  18. [Senescence and cellular immortality]. (United States)

    Trentesaux, C; Riou, J-F


    Senescence was originally described from the observation of the limited ability of normal cells to grow in culture, and may be generated by telomere erosion, accumulation of DNA damages, oxidative stress and modulation of oncogenes or tumor suppressor genes. Senescence corresponds to a cellular response aiming to control tumor progression by limiting cell proliferation and thus constitutes an anticancer barrier. Senescence is observed in pre-malignant tumor stages and disappears from malignant tumors. Agents used in standard chemotherapy also have the potential to induce senescence, which may partly explain their therapeutic activities. It is possible to restore senescence in tumors using targeted therapies that triggers telomere dysfunction or reactivates suppressor genes functions, which are essential for the onset of senescence.

  19. Cellular image classification

    CERN Document Server

    Xu, Xiang; Lin, Feng


    This book introduces new techniques for cellular image feature extraction, pattern recognition and classification. The authors use the antinuclear antibodies (ANAs) in patient serum as the subjects and the Indirect Immunofluorescence (IIF) technique as the imaging protocol to illustrate the applications of the described methods. Throughout the book, the authors provide evaluations for the proposed methods on two publicly available human epithelial (HEp-2) cell datasets: ICPR2012 dataset from the ICPR'12 HEp-2 cell classification contest and ICIP2013 training dataset from the ICIP'13 Competition on cells classification by fluorescent image analysis. First, the reading of imaging results is significantly influenced by one’s qualification and reading systems, causing high intra- and inter-laboratory variance. The authors present a low-order LP21 fiber mode for optical single cell manipulation and imaging staining patterns of HEp-2 cells. A focused four-lobed mode distribution is stable and effective in optical...

  20. Making Myelin Basic Protein -from mRNA transport to localized translation

    Directory of Open Access Journals (Sweden)

    Christina eMüller


    Full Text Available In the central nervous system (CNS of most vertebrates, oligodendrocytes enwrap neuronal axons with extensions of their plasma membrane to form the myelin sheath. Several proteins are characteristically found in myelin of which Myelin Basic Protein (MBP is the second most abundant one after Proteolipid Protein (PLP. The lack of functional MBP in rodents results in a severe hypomyelinated phenotype in the CNS demonstrating its importance for myelin synthesis. Mbp mRNA is transported from the nucleus to the plasma membrane and is translated locally at the axon-glial contact site. Axonal properties such as diameter or electrical activity influence the degree of myelination. As oligodendrocytes can myelinate many axonal segments with varying properties, localized MBP translation represents an important part of a rapid and axon-tailored synthesis machinery. MBP’s ability to compact cellular membranes may be problematic for the integrity of intracellular membranous organelles and can also explain why MBP is transported in oligodendrocytes in the form of an mRNA rather than as a protein. Here we review the recent findings regarding intracellular transport and signalling mechanisms leading to localized translation of Mbp mRNA in oligodendrocytes. More detailed insights into the MBP synthesis pathway are important for a better understanding of the myelination process and may foster the development of remyelination therapies for demyelinating diseases.

  1. Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA

    Directory of Open Access Journals (Sweden)

    Raúl Ortiz


    Full Text Available Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs.

  2. A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes.

    Directory of Open Access Journals (Sweden)

    Wanjun Gu


    Full Text Available Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.

  3. Recruitment of Staufen2 Enhances Dendritic Localization of an Intron-Containing CaMKIIα mRNA. (United States)

    Ortiz, Raúl; Georgieva, Maya V; Gutiérrez, Sara; Pedraza, Neus; Fernández-Moya, Sandra M; Gallego, Carme


    Regulation of mRNA localization is a conserved cellular process observed in many types of cells and organisms. Asymmetrical mRNA distribution plays a particularly important role in the nervous system, where local translation of localized mRNA represents a key mechanism in synaptic plasticity. CaMKIIα is a very abundant mRNA detected in neurites, consistent with its crucial role at glutamatergic synapses. Here, we report the presence of CaMKIIα mRNA isoforms that contain intron i16 in dendrites, RNA granules, and synaptoneurosomes from primary neurons and brain. This subpopulation of unspliced mRNA preferentially localizes to distal dendrites in a synaptic-activity-dependent manner. Staufen2, a well-established marker of RNA transport in dendrites, interacts with intron i16 sequences and enhances its distal dendritic localization, pointing to the existence of intron-mediated mechanisms in the molecular pathways that modulate dendritic transport and localization of synaptic mRNAs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Rapid identification of mRNA processing defects with a novel single-cell yeast reporter. (United States)

    Sorenson, Matthew R; Stevens, Scott W


    It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter using multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 nonessential yeast genes revealed striking phenotypic overlaps between chromatin remodeling, histone modification, and pre-mRNA splicing. Additionally, we developed a copper-inducible reporter, with which we demonstrate that 5-fluorouracil mimics the mRNA decay phenotype of cells lacking the 3'-5' exonuclease Rrp6p. Our reporter is capable of performing high-throughput, rapid, and large-scale screens to identify and characterize genetic and chemical perturbations of the major eukaryotic gene expression processes.

  5. Chromatoid Body Protein TDRD6 Supports Long 3' UTR Triggered Nonsense Mediated mRNA Decay.

    Directory of Open Access Journals (Sweden)

    Grigorios Fanourgakis


    Full Text Available Chromatoid bodies (CBs are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3' UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3' UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3' UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs.

  6. Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design

    Directory of Open Access Journals (Sweden)

    Chapman Elinor A


    Full Text Available Abstract Background RNA interference (RNAi has been one of the most rapidly expanding areas of biological research in the past decade, revolutionizing the ability to analyze gene function. Thorough validation of siRNA duplexes is required prior to use in experimental systems, ideally by western blotting to show a reduction in protein levels. However, in many cases good antibodies are not available, and researchers must rely on RT-qPCR to detect knockdown of the mRNA species. Findings We have observed a phenomenon that gives a disparity between analyzing small interfering RNA (siRNA efficacy by western blotting of the protein levels and real-time quantitative PCR (RT-qPCR measurement of mRNA levels. Detection of this phenomenon was dependent upon the location of the target amplicon for PCR primers within the mRNA. Conclusions Our data suggests that for certain mRNAs, degradation of the 3' mRNA fragment resulting from siRNA mediated cleavage is blocked, leaving an mRNA fragment that can act as a template for cDNA synthesis, giving rise to false negative results and the rejection of a valid siRNA duplex. We show that this phenomenon may be avoided by the careful design of RT-qPCR primers for each individual siRNA experiment.

  7. Anticancer agent CHS-828 inhibits cellular synthesis of NAD

    DEFF Research Database (Denmark)

    Olesen, U.H.; Christensen, M.K.; Bjorkling, F.


    Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD...... different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting...

  8. Degradable polymeric nano-films and particles as delivery platforms for vaccines and immunotherapeutics (United States)

    Su, Xingfang

    Degradable polymeric materials provide opportunities for the development of improved vaccines and immunotherapies by acting as platforms that facilitate the delivery of molecules to appropriate tissue and cellular locations to achieve therapeutic outcomes. To this end, we have designed and characterized nano-films and particles employing a hydrolytically degradable polymer for the delivery of vaccine antigens and immunotherapeutics. We first describe protein- and oligonucleotide-loaded layer-by-layer (LbL)-assembled multilayer thin films constructed based on electrostatic interactions between a cationic poly(beta-amino ester) (PBAE, denoted Poly-1) with a model protein antigen, ovalbumin (OVA), and/or immunostimulatory CpG oligonucleotides for transcutaneous delivery. Linear growth of nanoscale Poly-I/OVA bilayers was observed. Dried OVA protein-loaded films rapidly deconstructed when rehydrated in saline solutions, releasing OVA as non-aggregated/non-degraded protein, suggesting that the structure of biomolecules integrated into these multilayer films are preserved during release. Using confocal fluorescence microscopy and an in vivo murine ear skin model, we demonstrated delivery of OVA from LbL films into barrier-disrupted skin, uptake of the protein by skin-resident antigen-presenting cells (Langerhans cells), and transport of the antigen to the skin-draining lymph nodes. Dual incorporation of OVA and CpG oligonucleotides into the nanolayers of LbL films enabled dual release of the antigen and adjuvant with distinct kinetics for each component; OVA was rapidly released while CpG was released in a relatively sustained manner. Applied as skin patches, these films delivered OVA and CpG to Langerhans Cells in the skin. To our knowledge, this is the first demonstration of LbL films applied for the delivery of biomolecules into skin. This approach provides a new route for storage of vaccines and other immunotherapeutics in a solid-state thin film for subsequent

  9. Autophagy mediates degradation of nuclear lamina. (United States)

    Dou, Zhixun; Xu, Caiyue; Donahue, Greg; Shimi, Takeshi; Pan, Ji-An; Zhu, Jiajun; Ivanov, Andrejs; Capell, Brian C; Drake, Adam M; Shah, Parisha P; Catanzaro, Joseph M; Ricketts, M Daniel; Lamark, Trond; Adam, Stephen A; Marmorstein, Ronen; Zong, Wei-Xing; Johansen, Terje; Goldman, Robert D; Adams, Peter D; Berger, Shelley L


    Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.

  10. Enzymatically degradable mussel-inspired adhesive hydrogel. (United States)

    Brubaker, Carrie E; Messersmith, Phillip B


    Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (G') achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material's adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials.

  11. Integrin beta 4 MRNA expression levels in bronchial asthma patients ...

    African Journals Online (AJOL)

    Serum total IgE was measured by ELISA and mRNA expression of ITGβ4 was assessed by reverse transcriptase PCR (RT-PCR) using real time PCR.. ITGβ4 mRNA expression was significantly down regulated with increased serum total IgE in patients with asthma compared to controls. Moreover, ITGβ4 expression was ...

  12. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol


    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  13. Phytochrome B mRNA expression enhances biomass yield and ...

    African Journals Online (AJOL)

    The present study shows successful transformation and mRNA expression in Phytochrome B transformed CIM 482 cotton plants. Transgenic cotton plants expressing Phytochrome B mRNA have showed more than two times increase in relative leaf growth rate (RLGR) and photosynthetic rate, more than one time increase in ...

  14. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad


    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots...

  15. Regulation of pesticide degradation in the detritusphere (United States)

    Pagel, Holger; Poll, Christian; Ingwersen, Joachim; Ditterich, Franziska; Gebala, Aurelia; Kandeler, Ellen; Streck, Thilo


    The detritusphere is a microbial hot spot of C turnover and degradation of pesticides in soils. We aimed at an improved understanding of the regulation mechanisms, which are responsible for stimulated degradation of the herbicide MCPA (2-Methyl-4-chlorophenoxyacetic acid) in response to increased C availability in the detritusphere. We combined a microcosm experiment with biogeochemical modeling and linked genetic information on abundances of total bacteria, fungi and specific pesticide degraders in soil to the coupled biogeochemical dynamics of C and MCPA. As a result of diffusive and convective C transport from litter into the adjacent soil we found increased dissolved organic C (DOC) in soil up to a 6 mm distance to litter (detritusphere). In the detritusphere, we observed increased microbial C and accelerated MCPA degradation. These dynamics were accurately reproduced by the model. Whereas the observed increase of bacteria and pesticide degrader populations in the detritusphere was simulated satisfactorily, the model could not reproduce the steep increase of fungi indicated by the fungal marker gene. Our simulations suggest that bacterial MCPA degraders mostly benefited from high-quality DOC, whereas fungal activity and growth were specifically stimulated by low-quality DOC. According to the simulations, MCPA was predominantly degraded via fungal co-metabolism. Our study demonstrates that biogeochemical processes in soil hotspots are regulated by the interaction of transport processes and microbial dynamics. It further reveals that mathematical modelling is as powerful tool to gain comprehensive insight into the microbial regulation of matter cycling in soil. Genetic information has a high potential to parameterize and evaluate complex mechanistic models, but model approaches must be improved based on extended information on gene dynamics at the cellular level.

  16. atz gene expressions during atrazine degradation in the soil drilosphere. (United States)

    Monard, C; Martin-Laurent, F; Devers-Lamrani, M; Lima, O; Vandenkoornhuyse, P; Binet, F


    One of the various ecosystemic services sustained by soil is pollutant degradation mediated by adapted soil bacteria. The pathways of atrazine biodegradation have been elucidated but in situ expression of the genes involved in atrazine degradation has yet to be demonstrated in soil. Expression of the atzA and atzD genes involved in atrazine dechlorination and s-triazine ring cleavage, respectively, was investigated during in situ degradation of atrazine in the soil drilosphere and bulked samples from two agricultural soils that differed in their ability to mineralize atrazine. Interestingly, expression of the atzA gene, although present in both soils, was not detected. Atrazine mineralization was greatest in Epoisses soil, where a larger pool of atzD mRNA was consistently measured 7 days after atrazine treatment, compared with Vezin soil (146 vs. 49 mRNA per 10(6)16S rRNA, respectively). Expression of the atzD gene varied along the degradation time course and was profoundly modified in soil bioturbated by earthworms. The atzD mRNA pool was the highest in the soil drilosphere (casts and burrow-linings) and it was significantly different in burrow-linings compared with bulk soil (e.g. 363 vs. 146 mRNA per 10(6)16S rRNA, 7 days after atrazine treatment in Epoisses soil). Thus, consistent differences in atrazine mineralization were demonstrated between the soil drilosphere and bulk soil. However, the impact of bioturbation on atrazine mineralization depended on soil type. Mineralization was enhanced in casts, compared with bulk soil, from Epoisses soil but in burrow-linings from Vezin soil. This study is the first to report the effects of soil bioturbation by earthworms on s-triazine ring cleavage and its spatial variability in soil.

  17. Engineering intranasal mRNA vaccines to enhance lymph node trafficking and immune responses. (United States)

    Li, Man; Li, You; Peng, Ke; Wang, Ying; Gong, Tao; Zhang, Zhirong; He, Qin; Sun, Xun


    Intranasal mRNA vaccination provides immediate immune protection against pandemic diseases. Recent studies have shown that diverse forms of polyethyleneimine (PEI) have potent mucosal adjuvant activity, which could significantly facilitate the delivery of intranasal mRNA vaccines. Nevertheless, optimizing the chemical structure of PEI to maximize its adjuvanticity and decrease its toxicity remains a challenge. Here we show that the chemical structure of PEI strongly influences how well nanocomplexes of PEI and mRNA migrate to the lymph nodes and elicit immune responses. Conjugating cyclodextrin (CD) with PEI600 or PEI2k yielded CP (CD-PEI) polymers with different CD/PEI ratios. We analyzed the delivery efficacy of CP600, CP2k, and PEI25k as intranasal mRNA vaccine carriers by evaluating the lymph nodes migration and immune responses. Among these polymers, CP2k/mRNA showed significantly higher in vitro transfection efficiency, stronger abilities to migrate to lymph nodes and stimulate dendritic cells maturation in vivo, which further led to potent humoral and cellular immune responses, and showed lower local and systemic toxicity than PEI25k/mRNA. These results demonstrate the potential of CD-PEI2k/mRNA nanocomplex as a self-adjuvanting vaccine delivery vehicle that traffics to lymph nodes with high efficiency. As we face outbreaks of pandemic diseases such as Zika virus, intranasal mRNA vaccination provides instant massive protection against highly variant viruses. Various polymer-based delivery systems have been successfully applied in intranasal vaccine delivery. However, the influence of molecular structure of the polymeric carriers on the lymph node trafficking and dendritic cell maturation is seldom studied for intranasal vaccination. Therefore, engineering polymer-based vaccine delivery system and elucidating the relationship between molecular structure and the intranasal delivery efficiency are essential for maximizing the immune responses. We hereby

  18. Cometabolic degradation of chlorinated solvents: Bacterial inhibition, inactivation, and recovery

    Energy Technology Data Exchange (ETDEWEB)

    Ely, R.L.; Williamson, K.J.; Hyman, M.R.; Arp, D.J. [Oregon State Univ., Corvallis, OR (United States). Dept. of Civil Engineering


    This paper summarizes an approach for quantifying degradation kinetics and bacterial activity changes during cometabolic degradation of chlorinated solvents, results from trichloroethylene (TCE) degradation experiments, and a mathematical model addressing fluctuations in activity caused by enzyme inhibition, inactivation, and respondent enzyme synthesis. Using Nitrosomonas duropaea as a slow-growing exemplar capable of effecting cometabolic transformations, quasi-steady-state ammonia oxidation was established in a small bioreactor. A chlorinated solvent was injected to perturb the system, and bacterial activity and solvent degradation were monitored. At TCE concentrations to about 3.5 mg/L, from slight to nearly complete ammonia monooxygenase (AMO) inactivation occurred without causing immediate cell death. Results suggested cellular injury was limited primarily to AMO, most metabolic systems remained functional, and bacterial recovery processes, independent of cell growth, were initiated while degrading TCE.

  19. ECM degradation assays for analyzing local cell invasion. (United States)

    Artym, Vira V; Yamada, Kenneth M; Mueller, Susette C


    Proteolytic degradation of extracellular matrix (ECM) is a critical step during cell invasion and tissue transmigration that is required for many physiological and pathological processes. Cellular structures that mediate cell adhesion to, degradation of, and invasion into ECM are invadopodia of transformed and tumor cells and podosomes of normal monocytic, endothelial, and smooth muscle cells. Detecting the ability of the cell to form invadopodia and podosomes and to degrade ECM is required for studying the invasive capability of the cell. We have developed approximately 50 nm thick fluorescent gelatin matrices that provide a rapid, sensitive, and reliable in vitro system for detection of invadopodia and podosomes, and measurements of the extent of ECM degradation. In this chapter, we provide a detailed protocol for preparation of thin fluorescent gelatin matrices and for evaluation of the results from this degradation assay.

  20. Intermittent degradation and schizotypy

    Directory of Open Access Journals (Sweden)

    Matthew W. Roché


    Full Text Available Intermittent degradation refers to transient detrimental disruptions in task performance. This phenomenon has been repeatedly observed in the performance data of patients with schizophrenia. Whether intermittent degradation is a feature of the liability for schizophrenia (i.e., schizotypy is an open question. Further, the specificity of intermittent degradation to schizotypy has yet to be investigated. To address these questions, 92 undergraduate participants completed a battery of self-report questionnaires assessing schizotypy and psychological state variables (e.g., anxiety, depression, and their reaction times were recorded as they did so. Intermittent degradation was defined as the number of times a subject’s reaction time for questionnaire items met or exceeded three standard deviations from his or her mean reaction time after controlling for each item’s information processing load. Intermittent degradation scores were correlated with questionnaire scores. Our results indicate that intermittent degradation is associated with total scores on measures of positive and disorganized schizotypy, but unrelated to total scores on measures of negative schizotypy and psychological state variables. Intermittent degradation is interpreted as potentially derivative of schizotypy and a candidate endophenotypic marker worthy of continued research.

  1. Cellular phenotype and extracellular vesicles: basic and clinical considerations. (United States)

    Quesenberry, Peter J; Goldberg, Laura R; Aliotta, Jason M; Dooner, Mark S; Pereira, Mandy G; Wen, Sicheng; Camussi, Giovanni


    Early work on platelet and erythrocyte vesicles interpreted the phenomena as a discard of material from cells. Subsequently, vesicles were studied as possible vaccines and, most recently, there has been a focus on the effects of vesicles on cell fate. Recent studies have indicated that extracellular vesicles, previously referred to as microvesicles or exosomes, have the capacity to change the phenotype of neighboring cells. Extensive work has shown that vesicles derived from either the lung or liver can enter bone marrow cells (this is a prerequisite) and alter their fate toward that of the originating liver and lung tissue. Lung vesicles interacted with bone marrow cells result in the bone marrow cells expressing surfactants A-D, Clara cell protein, and aquaporin-5 mRNA. In a similar vein, liver-derived vesicles induce albumin mRNA in target marrow cells. The vesicles contain protein, mRNA, microRNA, and noncoding RNA and variably some DNA. This genetic package is delivered to cells and alters the phenotype. Further studies have shown that initially the altered phenotype is due to the transfer of mRNA and a transcriptional modulator, but long-term epigenetic changes are induced through transfer of a transcriptional factor, and the mRNA is rapidly degraded in the cell. Studies on the capacity of vesicles to restore injured tissue have been quite informative. Mesenchymal stem cell-derived vesicles are able to reverse the injury to the damaged liver and kidney. Other studies have shown that mesenchymal stem cell-derived vesicles can reverse radiation toxicity of bone marrow stem cells. Extracellular vesicles offer an intriguing strategy for treating a number of diseases characterized by tissue injury.

  2. XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail:


    Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediated cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.

  3. Sequence-based analysis of protein degradation rates

    NARCIS (Netherlands)

    Correa Marrero, Miguel; Dijk, van Aalt-Jan; Ridder, de Dick


    Protein turnover is a key aspect of cellular homeostasis and proteome dynamics. However, there is little consensus on which properties of a protein determine its lifetime in the cell. In this work, we exploit two reliable datasets of experimental protein degradation rates to learn models and uncover


    NARCIS (Netherlands)



    High molecular weight as-polymerized poly(l-lactide) (PLLA) has been successfully used for fracture fixation and orbital floor reconstruction in animals and humans. As this PLLA takes more than 3 years to resorb, a method was developed to obtain insight into the final cellular degradation process of

  5. Sterile Inflammation and Degradation Systems in Heart Failure. (United States)

    Nishida, Kazuhiko; Otsu, Kinya


    In most patients with chronic heart failure (HF), levels of circulating cytokines are elevated and the elevated cytokine levels correlate with the severity of HF and prognosis. Various stresses induce subcellular component abnormalities, such as mitochondrial damage. Damaged mitochondria induce accumulation of reactive oxygen species and apoptogenic proteins, and subcellular inflammation. The vicious cycle of subcellular component abnormalities, inflammatory cell infiltration and neurohumoral activation induces cardiomyocyte injury and death, and cardiac fibrosis, resulting in cardiac dysfunction and HF. Quality control mechanisms at both the protein and organelle levels, such as elimination of apoptogenic proteins and damaged mitochondria, maintain cellular homeostasis. An imbalance between protein synthesis and degradation is likely to result in cellular dysfunction and disease. Three major protein degradation systems have been identified, namely the cysteine protease system, autophagy, and the ubiquitin proteasome system. Autophagy was initially believed to be a non-selective process. However, recent studies have described the process of selective mitochondrial autophagy, known as mitophagy. Elimination of damaged mitochondria by autophagy is important for maintenance of cellular homeostasis. DNA and RNA degradation systems also play a critical role in regulating inflammation and maintaining cellular homeostasis mediated by damaged DNA clearance and post-transcriptional regulation, respectively. This review discusses some recent advances in understanding the role of sterile inflammation and degradation systems in HF.

  6. Free fall and cellular automata

    Directory of Open Access Journals (Sweden)

    Pablo Arrighi


    Full Text Available Three reasonable hypotheses lead to the thesis that physical phenomena can be described and simulated with cellular automata. In this work, we attempt to describe the motion of a particle upon which a constant force is applied, with a cellular automaton, in Newtonian physics, in Special Relativity, and in General Relativity. The results are very different for these three theories.

  7. Cellular automata analysis and applications

    CERN Document Server

    Hadeler, Karl-Peter


    This book focuses on a coherent representation of the main approaches to analyze the dynamics of cellular automata. Cellular automata are an inevitable tool in mathematical modeling. In contrast to classical modeling approaches as partial differential equations, cellular automata are straightforward to simulate but hard to analyze. In this book we present a review of approaches and theories that allow the reader to understand the behavior of cellular automata beyond simulations. The first part consists of an introduction of cellular automata on Cayley graphs, and their characterization via the fundamental Cutis-Hedlund-Lyndon theorems in the context of different topological concepts (Cantor, Besicovitch and Weyl topology). The second part focuses on classification results: What classification follows from topological concepts (Hurley classification), Lyapunov stability (Gilman classification), and the theory of formal languages and grammars (Kůrka classification). These classifications suggest to cluster cel...

  8. MIMO Communication for Cellular Networks

    CERN Document Server

    Huang, Howard; Venkatesan, Sivarama


    As the theoretical foundations of multiple-antenna techniques evolve and as these multiple-input multiple-output (MIMO) techniques become essential for providing high data rates in wireless systems, there is a growing need to understand the performance limits of MIMO in practical networks. To address this need, MIMO Communication for Cellular Networks presents a systematic description of MIMO technology classes and a framework for MIMO system design that takes into account the essential physical-layer features of practical cellular networks. In contrast to works that focus on the theoretical performance of abstract MIMO channels, MIMO Communication for Cellular Networks emphasizes the practical performance of realistic MIMO systems. A unified set of system simulation results highlights relative performance gains of different MIMO techniques and provides insights into how best to use multiple antennas in cellular networks under various conditions. MIMO Communication for Cellular Networks describes single-user,...

  9. MSAT and cellular hybrid networking (United States)

    Baranowsky, Patrick W., II

    Westinghouse Electric Corporation is developing both the Communications Ground Segment and the Series 1000 Mobile Phone for American Mobile Satellite Corporation's (AMSC's) Mobile Satellite (MSAT) system. The success of the voice services portion of this system depends, to some extent, upon the interoperability of the cellular network and the satellite communication circuit switched communication channels. This paper will describe the set of user-selectable cellular interoperable modes (cellular first/satellite second, etc.) provided by the Mobile Phone and described how they are implemented with the ground segment. Topics including roaming registration and cellular-to-satellite 'seamless' call handoff will be discussed, along with the relevant Interim Standard IS-41 Revision B Cellular Radiotelecommunications Intersystem Operations and IOS-553 Mobile Station - Land Station Compatibility Specification.

  10. Degradation of implant materials

    CERN Document Server

    Eliaz, Noam


    This book surveys the degradation of implant materials, reviewing in detail such failure mechanisms as corrosion, fatigue and wear, along with monitoring techniques. Surveys common implant biomaterials, as well as procedures for implant retrieval and analysis.

  11. Bacterial Degradation of Pesticides

    DEFF Research Database (Denmark)

    Knudsen, Berith Elkær

    This PhD project was carried out as part of the Microbial Remediation of Contaminated Soil and Water Resources (MIRESOWA) project, funded by the Danish Council for Strategic Research (grant number 2104-08-0012). The environment is contaminated with various xenobiotic compounds e.g. pesticides....... Bioaugmentation i.e. addition of specific degrader organisms, has been suggested as an environmentally friendly and economically competitive strategy for cleaning polluted sites. Several organisms have been isolated, capable of degrading different compounds. However the capacity to degrade the desired compound...... is just one requirement for successful bioaugmentation. There are several challenges that need to be overcome in order for bioaugmentation to be sufficiently efficient. The purpose of this PhD project was to study the degradative abilities of different bacteria, and, in collaboration with a fellow Ph...

  12. Pathogenesis of pulmonary emphysema – cellular and molecular events

    Directory of Open Access Journals (Sweden)

    Antonio Di Petta


    Full Text Available Pulmonary emphysema is a chronic obstructive disease, resulting fromimportant alterations in the whole distal structure of terminal bronchioles, either by enlargement of air spaces or by destruction of the alveolar wall, leading to loss of respiratory surface, decreased elastic recoil and lung hyperinflation. For many years, the hypothesis of protease-antiprotease unbalance prevailed as the central theme in the pathogenesis of pulmonary emphysema. According to this hypothesis, the release of active proteolytic enzymes, produced mainly by neutrophils and macrophages, degrades the extracellular matrix, affecting the integrity of its components, especially collagen and elastic fibers. However, new concepts involving cellular and molecular events were proposed, including oxidative stress, cell apoptosis, cellular senescence and failed lung tissue repair. The aim of this review paper was to evaluate the cellular and molecular mechanisms seen in the pathogenesis of pulmonary emphysema.

  13. Molecular and cellular constraints on proteins (United States)

    Kortemme, Tanja

    Engineering proteins with new sequences, structures and functions has many exciting practical applications, and provides new ways to dissect design principles for function. Recent successes in computational protein design provide a cause for optimism. Yet many functions are currently too complex to engineer predictively, and successful design of new biological activities also requires an understanding of the functional pressures acting on proteins in the context of cells and organisms. I will present two vignettes describing our progress with dissecting both molecular and cellular constraints on protein function. In the first, we characterized the cost and benefit of protein production upon sequence perturbations in a classic system for gene regulation, the lac operon. Our results were unexpected in light of the common assumption that the dominant fitness costs are due to protein expression. Instead, we discovered a direct linear relationship between cost and lacpermease activity, not protein or mRNA production. The magnitude of the cost of permease activity, relative to protein production, has consequences for regulation. Our model predicts an advantage of direct regulation of protein activity (not just expression), providing a new explanation for the long-known mechanism of ``inducer exclusion'' that inhibits transport through the permease. Similar pressures and cost/benefit tradeoffs may be key to engineering synthetic systems with improved fitness. In the second vignette, I will describe our recent efforts to develop computational approaches that predict protein sequences consistent with multiple functional conformations. We expect such ``multi-constraint'' models to improve predictions of functional sequences determined by deep mutational scanning in bacteria, to provide insights into how the balance between functional conformations shapes sequence space, and to highlight molecular and cellular constraints that cannot be captured by the model.

  14. Cellular Chaperones As Therapeutic Targets in ALS to Restore Protein Homeostasis and Improve Cellular Function

    Directory of Open Access Journals (Sweden)

    Bernadett Kalmar


    Full Text Available Heat shock proteins (Hsps are ubiquitously expressed chaperone proteins that enable cells to cope with environmental stresses that cause misfolding and denaturation of proteins. With aging this protein quality control machinery becomes less effective, reducing the ability of cells to cope with damaging environmental stresses and disease-causing mutations. In neurodegenerative disorders such as Amyotrophic Lateral Sclerosis (ALS, such mutations are known to result in protein misfolding, which in turn results in the formation of intracellular aggregates cellular dysfunction and eventual neuronal death. The exact cellular pathology of ALS and other neurodegenerative diseases has been elusive and thus, hindering the development of effective therapies. However, a common scheme has emerged across these “protein misfolding” disorders, in that the mechanism of disease involves one or more aspects of proteostasis; from DNA transcription, RNA translation, to protein folding, transport and degradation via proteosomal and autophagic pathways. Interestingly, members of the Hsp family are involved in each of these steps facilitating normal protein folding, regulating the rate of protein synthesis and degradation. In this short review we summarize the evidence that suggests that ALS is a disease of protein dyshomeostasis in which Hsps may play a key role. Overwhelming evidence now indicates that enabling protein homeostasis to cope with disease-causing mutations might be a successful therapeutic strategy in ALS, as well as other neurodegenerative diseases. Novel small molecule co-inducers of Hsps appear to be able to achieve this aim. Arimoclomol, a hydroxylamine derivative, has shown promising results in cellular and animal models of ALS, as well as other protein misfolding diseases such as Inclusion Body Myositis (IBM. Initial clinical investigations of Arimoclomol have shown promising results. Therefore, it is possible that the long series of

  15. Thraustochytrid protists degrade hydrocarbons

    Digital Repository Service at National Institute of Oceanography (India)

    Raikar, M.T.; Raghukumar, S.; Vani, V.; David, J.J.; Chandramohan, D.

    that thraustochytrids have the capability to utilize a wide range of organic nitrogen and carbon compounds for their nutrition. However, the capability of these protists to degrade hydrocarbons has not been examined so far. Hydrocarbons occur in seawater either... chromatography. (1) Gravimetry: Tarballs were extracted from experimental flasks with 10 ml of carbon tetrachloride, the extract transferred to pre- weighed Petri dish and the solvent allowed to RAIKAR et al.: THRAUSTOCHYTRID PROTISTS DEGRADE HYDROCARBONS...

  16. Role of mRNA Methylation in Prostate Cancer (United States)


    regulation of mRNA. Recent technological advances have made it possible to detect mRNA methylation . The m6A was found near regulatory regions and...TERMS mRNA methylation , FTO, MeRIP-seq, RNA-seq, m6A 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...10 9. Appendices……………………………………………………………10 1. INTRODUCTION: Methylation at the N6 position of adenosine ( m6A ) is a post-transcriptional modification of

  17. The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate. (United States)

    Rao, T R; Slobin, L I


    The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.

  18. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA.

    Directory of Open Access Journals (Sweden)

    David G Hendrickson


    start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.

  19. Glucocorticoid receptor mRNA and protein isoform alterations in the orbitofrontal cortex in schizophrenia and bipolar disorder (United States)


    Background The orbitofrontal cortex (OFC) may play a role in the pathogenesis of psychiatric illnesses such as bipolar disorder and schizophrenia, in which hypothalamic-pituitary-adrenal (HPA) axis abnormalities are observed and stress has been implicated. A critical component of the HPA axis which mediates cellular stress responses in the OFC, and has been implicated in psychiatric illness, is the glucocorticoid receptor (GR). Methods In the lateral OFC, we employed quantitative real-time PCR and western blotting to investigate GR mRNA and protein expression in 34 bipolar disorder cases, 35 schizophrenia cases and 35 controls. Genotype data for eleven GR gene (NR3C1) polymorphisms was also used to explore possible effects of NR3C1 sequence variation on GR mRNA and protein expression in the lateral OFC. Results We found no diagnostic differences in pan GR, GR-1C or GR-1F mRNA expression. However, the GR-1B mRNA transcript variant was decreased (14.3%) in bipolar disorder cases relative to controls (p schizophrenia cases relative to controls (p disorder (56.1%, p schizophrenia (31.5% p < 0.05). Using genotype data for eleven NR3C1 polymorphisms, we found no evidence of effects of NR3C1 genotype on GR mRNA or GRα protein expression in the OFC. Conclusions These findings reveal selective abnormalities of GR mRNA expression in the lateral OFC in psychiatric illness, which are more specific and may be less influenced by NR3C1 genotype than those of the dorsolateral prefrontal cortex reported previously. Our results suggest that the GRα-D1 protein isoform may be up-regulated widely across the frontal cortex in psychiatric illness. PMID:22812453

  20. DDE remediation and degradation. (United States)

    Thomas, John E; Ou, Li-Tse; All-Agely, Abid


    DDT and its metabolites, DDD and DDE, have been shown to be recalcitrant to degradation. The parent compound, DDT, was used extensively worldwide starting in 1939 and was banned in the United States in 1973. The daughter compound, DDE, may result from aerobic degradation, abiotic dehydrochlorination, or photochemical decomposition. DDE has also occurred as a contaminant in commercial-grade DDT. The p,p'-DDE isomer is more biologically active than the o,p-DDE, with a reported half-life of -5.7 years. However, when DDT was repeatedly applied to the soil, the DDE concentration may remain unchanged for more than 20 yr. Remediation of DDE-contaminated soil and water may be done by several techniques. Phytoremediation involves translocating DDT, DDD, and DDE from the soil into the plant, although some aquatic species (duckweed > elodea > parrot feather) can transform DDT into predominantly DDD with some DDE being formed. Of all the plants that can uptake DDE, Cucurbita pepo has been the most extensively studied, with translocation values approaching "hyperaccumulation" levels. Soil moisture, temperature, and plant density have all been documented as important factors in the uptake of DDE by Cucurbita pepo. Uptake may also be influenced positively by amendments such as biosurfactants, mycorrhizal inoculants, and low molecular weight organic acids (e.g., citric and oxalic acids). DDE microbial degradation by dehalogenases, dioxygenases, and hydrolases occurs under the proper conditions. Although several aerobic degradation pathways have been proposed, none has been fully verified. Very few aerobic pure cultures are capable of fully degrading DDE to CO2. Cometabolism of DDE by Pseudomonas sp., Alicaligens sp., and Terrabacter sp. grown on biphenyl has been reported; however, not all bacterial species that produce biphenyl dioxygenase degraded DDE. Arsenic and copper inhibit DDE degradation by aerobic microorganisms. Similarly, metal chelates such as EDTA inhibit the

  1. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement* (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh


    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

  2. Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5'-to-3' directional decay and 5'-end protection of mRNA in Crenarchaeota. (United States)

    Hasenöhrl, David; Konrat, Robert; Bläsi, Udo


    In both Bacteria and Eukaryotes, degradation is known to start at the 5' and at the 3' extremities of mRNAs. Until the recent discovery of 5'-to-3' exoribonucleases in hyperthermophilic Euryarchaeota, the exosome was assumed to be the key enzyme in mRNA degradation in Archaea. By means of zymogram assays and bioinformatics, we have identified a 5'-to-3' exoribonuclease activity in the crenarchaeum Sulfolobus solfataricus (Sso), which is affected by the phosphorylation state of the 5'-end of the mRNA. The protein comprises typical signature motifs of the β-CASP family of metallo-β-lactamases and was termed Sso-RNAse J. Thus, our study provides the first evidence for a 5'-to-3' directional mRNA decay pathway in the crenarchaeal clade of Archaea. In Bacteria the 5'-end of mRNAs is often protected by a tri-phosphorylated 5'-terminus and/or by stem-loop structures, while in Eukaryotes the cap-binding complex is responsible for this task. Here, we show that binding of translation initiation factor a/eIF2(γ) to the 5'-end of mRNA counteracts the 5'-to-3' exoribonucleolytic activity of Sso-RNase J in vitro. Hence, 5'-to-3' directional decay and 5'-end protection appear to be conserved features of mRNA turnover in all kingdoms of life.

  3. The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

    Directory of Open Access Journals (Sweden)

    Tachibana Taro


    Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

  4. [6]-Gingerol attenuates β-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system. (United States)

    Lee, Chan; Park, Gyu Hwan; Kim, Chang-Yul; Jang, Jung-Hee


    β-Amyloid (Aβ) is involved in the formation of senile plaques, the typical neuropathological marker for Alzheimer's disease (AD) and has been reported to cause apoptosis in neurons via oxidative and/or nitrosative stress. In this study, we have investigated the neuroprotective effect and molecular mechanism of [6]-gingerol, a pungent ingredient of ginger against Αβ(25-35)-induced oxidative and/or nitrosative cell death in SH-SY5Y cells. [6]-Gingerol pretreatment protected against Aβ(25-35)-induced cytotoxicity and apoptotic cell death such as DNA fragmentation, disruption of mitochondrial membrane potential, elevated Bax/Bcl-2 ratio, and activation of caspase-3. To elucidate the neuroprotective mechanism of [6]-gingerol, we have examined Aβ(25-35)-induced oxidative and/or nitrosative stress and cellular antioxidant defense system against them. [6]-Gingerol effectively suppressed Aβ(25-35)-induced intracellular accumulation of reactive oxygen and/or nitrogen species and restored Aβ(25-35)-depleted endogenous antioxidant glutathione levels. Furthermore, [6]-gingerol treatment up-regulated the mRNA and protein expression of antioxidant enzymes such as γ-glutamylcysteine ligase (GCL) and heme oxygenase-1 (HO-1), the rate limiting enzymes in the glutathione biosynthesis and the degradation of heme, respectively. The expression of aforementioned antioxidant enzymes seemed to be mediated by activation of NF-E2-related factor 2 (Nrf2). These results suggest that [6]-gingerol exhibits preventive and/or therapeutic potential for the management of AD via augmentation of antioxidant capacity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Degradable Polymersomes for Targeted Drug Delivery (United States)

    Petersen, Matthew Alan

    Chemotherapy today is often accompanied by major side effects due to delivery of toxic drugs to healthy tissue in addition to diseased cells. Targeted drug delivery offers the possibility of minimizing these side effects by specific delivery to cancer cells using targeted nanocarriers that enhance drug accumulation in tumors and facilitate target-specific cellular uptake. Polymersomes, vesicles self-assembled from polymeric amphiphiles, are an attractive targeted vehicle, as they are capable of encapsulating both hydrophobic and hydrophilic drugs, have lengthy circulation times in vivo, and can employ degradable functionality for triggered release of payload and clearance from the body. This thesis reports on efforts to enhance the capabilities of degradable polymersomes for targeted delivery. First, targeting functionality is incorporated into polymersomes of the block copolymer poly(ethylene oxide)-b-poly(gamma-methyl-epsilon-caprolactone) by incorporating the reactive vinyl sulfone group into the amphiphile's hydrophilic terminus, allowing site-selective reaction with cysteine-functionalized targeting peptides following self-assembly. The performance of targeted delivery using this polymersome is then evaluated in vitro. Binding and delivery to model cell lines for targeted and bystander cells is tracked using nontargeted polymersomes and compared to that for polymersomes using a high- or low-affinity ligand. Polymer degradation is also tracked both in simple media and during cellular delivery. Finally, a new monomer is developed incorporating acid-labile acetal functionality into a cyclic polyester. The polymerization of this monomer to two distinct polymers is also characterized and the degradation behavior of both polymers evaluated.

  6. PI3Kδ activates E2F1 synthesis in response to mRNA translation stress. (United States)

    Gnanasundram, Sivakumar Vadivel; Pyndiah, Slovénie; Daskalogianni, Chrysoula; Armfield, Kate; Nylander, Karin; Wilson, Joanna B; Fåhraeus, Robin


    The c-myc oncogene stimulates ribosomal biogenesis and protein synthesis to promote cellular growth. However, the pathway by which cells sense and restore dysfunctional mRNA translation and how this is linked to cell proliferation and growth is not known. We here show that mRNA translation stress in cis triggered by the gly-ala repeat sequence of Epstein-Barr virus (EBV)-encoded EBNA1, results in PI3Kδ-dependent induction of E2F1 mRNA translation with the consequent activation of c-Myc and cell proliferation. Treatment with a specific PI3Kδ inhibitor Idelalisib (CAL-101) suppresses E2F1 and c-Myc levels and causes cell death in EBNA1-induced B cell lymphomas. Suppression of PI3Kδ prevents E2F1 activation also in non-EBV-infected cells. These data illustrate an mRNA translation stress-response pathway for E2F1 activation that is exploited by EBV to promote cell growth and proliferation, offering new strategies to treat EBV-carrying cancers.

  7. Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells (United States)

    Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.


    Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

  8. Nature-inspired DNA nanosensor for real-time in situ detection of mRNA in living cells. (United States)

    Tay, Chor Yong; Yuan, Liang; Leong, David Tai


    Rapid and precise in situ detection of gene expressions within a single cell is highly informative and offers valuable insights into its state. Detecting mRNA within single cells in real time and nondestructively remains an important challenge. Using DNA nanotechnology and inspired by nature's many examples of "protective-yet-accessible" exoskeletons, we designed our mRNA nanosensor, nano-snail-inspired nucleic acid locator (nano-SNEL), to illustrate these elements. The design of the nano-SNEL is composed of a sensory molecular beacon module to detect mRNA and a DNA nanoshell component, mimicking the functional anatomy of a snail. Accurate and sensitive visualization of mRNA is achieved by the exceptional protection conferred by the nanoshell to the sensory component from nucleases-mediated degradation by approximately 9-25-fold compared to its unprotected counterpart. Our nano-SNEL design strategy improved cell internalization is a demonstration of accurate, dynamic spatiotemporal resolved detection of RNA transcripts in living cells.

  9. Drift Degradation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    D. Kicker


    Degradation of underground openings as a function of time is a natural and expected occurrence for any subsurface excavation. Over time, changes occur to both the stress condition and the strength of the rock mass due to several interacting factors. Once the factors contributing to degradation are characterized, the effects of drift degradation can typically be mitigated through appropriate design and maintenance of the ground support system. However, for the emplacement drifts of the geologic repository at Yucca Mountain, it is necessary to characterize drift degradation over a 10,000-year period, which is well beyond the functional period of the ground support system. This document provides an analysis of the amount of drift degradation anticipated in repository emplacement drifts for discrete events and time increments extending throughout the 10,000-year regulatory period for postclosure performance. This revision of the drift degradation analysis was developed to support the license application and fulfill specific agreement items between the U.S. Nuclear Regulatory Commission (NRC) and the U.S. Department of Energy (DOE). The earlier versions of ''Drift Degradation Analysis'' (BSC 2001 [DIRS 156304]) relied primarily on the DRKBA numerical code, which provides for a probabilistic key-block assessment based on realistic fracture patterns determined from field mapping in the Exploratory Studies Facility (ESF) at Yucca Mountain. A key block is defined as a critical block in the surrounding rock mass of an excavation, which is removable and oriented in an unsafe manner such that it is likely to move into an opening unless support is provided. However, the use of the DRKBA code to determine potential rockfall data at the repository horizon during the postclosure period has several limitations: (1) The DRKBA code cannot explicitly apply dynamic loads due to seismic ground motion. (2) The DRKBA code cannot explicitly apply loads due to thermal

  10. CeFra-seq reveals broad asymmetric mRNA and noncoding RNA distribution profiles in Drosophila and human cells. (United States)

    Benoit Bouvrette, Louis Philip; Cody, Neal A L; Bergalet, Julie; Lefebvre, Fabio Alexis; Diot, Cédric; Wang, Xiaofeng; Blanchette, Mathieu; Lécuyer, Eric


    Cells are highly asymmetrical, a feature that relies on the sorting of molecular constituents, including proteins, lipids, and nucleic acids, to distinct subcellular locales. The localization of RNA molecules is an important layer of gene regulation required to modulate localized cellular activities, although its global prevalence remains unclear. We combine biochemical cell fractionation with RNA-sequencing (CeFra-seq) analysis to assess the prevalence and conservation of RNA asymmetric distribution on a transcriptome-wide scale in Drosophila and human cells. This approach reveals that the majority (∼80%) of cellular RNA species are asymmetrically distributed, whether considering coding or noncoding transcript populations, in patterns that are broadly conserved evolutionarily. Notably, a large number of Drosophila and human long noncoding RNAs and circular RNAs display enriched levels within specific cytoplasmic compartments, suggesting that these RNAs fulfill extra-nuclear functions. Moreover, fraction-specific mRNA populations exhibit distinctive sequence characteristics. Comparative analysis of mRNA fractionation profiles with that of their encoded proteins reveals a general lack of correlation in subcellular distribution, marked by strong cases of asymmetry. However, coincident distribution profiles are observed for mRNA/protein pairs related to a variety of functional protein modules, suggesting complex regulatory inputs of RNA localization to cellular organization. © 2018 Benoit Bouvrette et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase. (United States)

    Alam, Md Badrul; Bajpai, Vivek K; Lee, JungIn; Zhao, Peijun; Byeon, Jung-Hee; Ra, Jeong-Sic; Majumder, Rajib; Lee, Jong Sung; Yoon, Jung-In; Rather, Irfan A; Park, Yong-Ha; Kim, Kangmin; Na, MinKyun; Lee, Sang-Han


    In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

  12. Demonstration that a mRNA Binding Protein is Responsible for GADD45 mRNA Destabilization

    National Research Council Canada - National Science Library

    Abcouwer, Steve


    ...) Using regions of the GADD45 mRNA 3'-untranslated region (UTR) in KNA gel shift assays, we have observed that glutamine causes distinct changes in RBP activities in cytoplasmic and nuclear protein extracts...

  13. Systems biology of cellular rhythms. (United States)

    Goldbeter, A; Gérard, C; Gonze, D; Leloup, J-C; Dupont, G


    Rhythms abound in biological systems, particularly at the cellular level where they originate from the feedback loops present in regulatory networks. Cellular rhythms can be investigated both by experimental and modeling approaches, and thus represent a prototypic field of research for systems biology. They have also become a major topic in synthetic biology. We review advances in the study of cellular rhythms of biochemical rather than electrical origin by considering a variety of oscillatory processes such as Ca++ oscillations, circadian rhythms, the segmentation clock, oscillations in p53 and NF-κB, synthetic oscillators, and the oscillatory dynamics of cyclin-dependent kinases driving the cell cycle. Finally we discuss the coupling between cellular rhythms and their robustness with respect to molecular noise.

  14. A Course in Cellular Bioengineering. (United States)

    Lauffenburger, Douglas A.


    Gives an overview of a course in chemical engineering entitled "Cellular Bioengineering," dealing with how chemical engineering principles can be applied to molecular cell biology. Topics used are listed and some key references are discussed. Listed are 85 references. (YP)

  15. When are cellular automata random? (United States)

    Coe, J. B.; Ahnert, S. E.; Fink, T. M. A.


    A random cellular automaton is one in which a cell's behaviour is independent of its previous states. We derive analytical conditions which must be satisfied by random cellular automata and find deterministic and probabilistic cellular automata that satisfy these conditions. Many random cellular automata are seen to have a flow as they are updated through time. We define a correlation current that describes this flow and develop an analytical expression for its size. We compare results from this analytical expression with those from simulation. The randomness in a cell comes from randomness in adjacent cells or from the stochastic nature of update rules. We give an expression for how much randomness comes from each of these two sources.

  16. Procefual Non Uniform Cellular Noise


    Jonchier, Théo; Salvati, Marc; Derouet-Jourdan, Alexandre


    Procedural cellular textures have been widely used in movie production to reproduce various natural and organic looks. The advantage of procedural texture is to trade memory for computer power and obtain potentially unlimited resolution. In this paper, we propose to compute non-uniform density cellular noise by using a procedural quad-tree. We will explain how to efficiently traverse the tree recursively (CPU) and iteratively (CPU and GPU).

  17. Snipper, an Eri1 homologue, affects histone mRNA abundance and is crucial for normal Drosophila melanogaster development. (United States)

    Alexiadis, Anastasios; Delidakis, Christos; Kalantidis, Kriton


    The conserved 3'-5' RNA exonuclease ERI1 is implicated in RNA interference inhibition, 5.8S rRNA maturation and histone mRNA maturation and turnover. The single ERI1 homologue in Drosophila melanogaster Snipper (Snp) is a 3'-5' exonuclease, but its in vivo function remains elusive. Here, we report Snp requirement for normal Drosophila development, since its perturbation leads to larval arrest and tissue-specific downregulation results in abnormal tissue development. Additionally, Snp directly interacts with histone mRNA, and its depletion results in drastic reduction in histone transcript levels. We propose that Snp protects the 3'-ends of histone mRNAs and upon its absence, histone transcripts are readily degraded. This in turn may lead to cell cycle delay or arrest, causing growth arrest and developmental perturbations. © 2017 Federation of European Biochemical Societies.

  18. Integrating iron and oxygen/antioxidant signals via a combinatorial array of DNA - (antioxidant response elements) and mRNA (iron responsive elements) sequences. (United States)

    Theil, Elizabeth C


    Fe (cellular iron), O (dioxygen, antioxidant inducers, hydrogen peroxide), and P (protein phosphorylation) signals combine to regulate DNA activity (transcription/mRNA synthesis) for antioxidant/Phase II response proteins (e.g., ferritin H, ferritin L, thioredoxin reductase I, NAD(P)H quinone oxido-reductase, heme oxygenase1 and beta-globin) and mRNA activity for proteins of iron transport, storage or oxygen metabolism (e.g., ferritin H, ferritin L, transferrin receptor1, ferroportin, mt-aconitase-TCA cycle and aminolevulinate synthase - heme biosynthesis). Ferritin regulation links the two groups of genetic controls via DNA (ARE-antioxidant response element) and mRNA (IRE-iron responsive element) structures. More is known about the IRE-mRNA and protein repressors, IRPs (iron regulatory proteins/aconitase homologues), than the DNA-ARE and protein repressors, e.g., Bach1. Iron responsive elements are very similar (65-80% sequence identity), but each mRNA has sufficient IRE specificity (>90% phylogenetic sequence conservation), that IRP binding and signal responses vary quantitatively. The structural specificity of each IRE-RNA provides an opportunity for finding small molecule regulators in vitro, and possibly in vivo. The potential of manipulating mRNA function with small molecules targeted to specific RNA regulatory structures, e.g., ferritin mRNA in iron overload, or viral mRNA control structures for replication, is high.

  19. Update: Mechanisms underlying N6-methyladenosine modification of eukaryotic mRNA (United States)

    Wang, Yang; Zhao, Jing Crystal


    Summary Eukaryotic messenger RNA (mRNA) undergoes chemical modification both at the 5′cap [1, 2] and internally [3–14]. Among internal modifications, m6A, by far the most abundant, is present in all eukaryotes examined, including mammals [3–6], flies [15], plants [16, 17] and yeast [18, 19]. m6A modification plays an essential role in diverse biological processes. Over the past few years, our knowledge relevant to establishment and function of this modification has grown rapidly. This review focuses on technologies that have facilitated m6A detection in mRNAs, identification of m6A methylation enzymes and binding proteins, and potential functions of the modification at the molecular level. Regarding m6A function at cellular or organismal levels or in disease, please refer to other recent reviews [20–23]. PMID:27793360

  20. Motor degradation prediction methods

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, J.R.; Kelly, J.F.; Delzingaro, M.J.


    Motor Operated Valve (MOV) squirrel cage AC motor rotors are susceptible to degradation under certain conditions. Premature failure can result due to high humidity/temperature environments, high running load conditions, extended periods at locked rotor conditions (i.e. > 15 seconds) or exceeding the motor`s duty cycle by frequent starts or multiple valve stroking. Exposure to high heat and moisture due to packing leaks, pressure seal ring leakage or other causes can significantly accelerate the degradation. ComEd and Liberty Technologies have worked together to provide and validate a non-intrusive method using motor power diagnostics to evaluate MOV rotor condition and predict failure. These techniques have provided a quick, low radiation dose method to evaluate inaccessible motors, identify degradation and allow scheduled replacement of motors prior to catastrophic failures.

  1. Hemagglutinin of Influenza A Virus Antagonizes Type I Interferon (IFN) Responses by Inducing Degradation of Type I IFN Receptor 1. (United States)

    Xia, Chuan; Vijayan, Madhuvanthi; Pritzl, Curtis J; Fuchs, Serge Y; McDermott, Adrian B; Hahm, Bumsuk


    Influenza A virus (IAV) employs diverse strategies to circumvent type I interferon (IFN) responses, particularly by inhibiting the synthesis of type I IFNs. However, it is poorly understood if and how IAV regulates the type I IFN receptor (IFNAR)-mediated signaling mode. In this study, we demonstrate that IAV induces the degradation of IFNAR subunit 1 (IFNAR1) to attenuate the type I IFN-induced antiviral signaling pathway. Following infection, the level of IFNAR1 protein, but not mRNA, decreased. Indeed, IFNAR1 was phosphorylated and ubiquitinated by IAV infection, which resulted in IFNAR1 elimination. The transiently overexpressed IFNAR1 displayed antiviral activity by inhibiting virus replication. Importantly, the hemagglutinin (HA) protein of IAV was proved to trigger the ubiquitination of IFNAR1, diminishing the levels of IFNAR1. Further, influenza A viral HA1 subunit, but not HA2 subunit, downregulated IFNAR1. However, viral HA-mediated degradation of IFNAR1 was not caused by the endoplasmic reticulum (ER) stress response. IAV HA robustly reduced cellular sensitivity to type I IFNs, suppressing the activation of STAT1/STAT2 and induction of IFN-stimulated antiviral proteins. Taken together, our findings suggest that IAV HA causes IFNAR1 degradation, which in turn helps the virus escape the powerful innate immune system. Thus, the research elucidated an influenza viral mechanism for eluding the IFNAR signaling pathway, which could provide new insights into the interplay between influenza virus and host innate immunity. Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide and remains a major health concern. When triggered by influenza viral infection, host cells produce type I interferon (IFN) to block viral replication. Although IAV was shown to have diverse strategies to evade this powerful, IFN-mediated antiviral response, it is not well-defined if IAV manipulates the IFN receptor-mediated signaling pathway. Here, we

  2. Analysis of interferon-beta mRNA stability control after poly(I:C) stimulation using RNA metabolic labeling by ethynyluridine. (United States)

    Abe, Kaito; Ishigami, Tomoaki; Shyu, Ann-Bin; Ohno, Shigeo; Umemura, Satoshi; Yamashita, Akio


    Interferon-beta (IFN-β) is a critical antiviral cytokine and is essential for innate and acquired immune responses to pathogens. Treatment with polyinosinic:polycytidylic acid (poly(I:C)) induces transient accumulation of IFN-β mRNA, which involves an increase and a decrease of IFN-β mRNA. This phenomenon has been extensively analyzed as a model for understanding the mechanisms of transient gene induction in response to external stimuli. Using a new RNA metabolic labeling method with ethynyluridine to directly measure de novo RNA synthesis and RNA stability, we reassessed both de novo synthesis and degradation of IFN-β mRNA. We found that transcriptional activity is maintained after the maximum accumulation of IFN-β mRNA following poly(I:C) treatment on immortalized human bronchial epithelial cells. We also observed an unexpected change in the stability of IFN-β mRNA before and after the maximum accumulation. The results indicate that this method of RNA metabolic labeling provides a general approach for the simultaneous analysis of transcriptional activity and mRNA stability coupled with transcriptional timing. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay

    DEFF Research Database (Denmark)

    Colak, Dilek; Ji, Sheng-Jian; Porse, Bo T


    Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we...... (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.......2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding....

  4. Photovoltaic Degradation Risk: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Jordan, D. C.; Kurtz, S. R.


    The ability to accurately predict power delivery over the course of time is of vital importance to the growth of the photovoltaic (PV) industry. Important cost drivers include the efficiency with which sunlight is converted into power, how this relationship changes over time, and the uncertainty in this prediction. An accurate quantification of power decline over time, also known as degradation rate, is essential to all stakeholders - utility companies, integrators, investors, and researchers alike. In this paper we use a statistical approach based on historical data to quantify degradation rates, discern trends and quantify risks related to measurement uncertainties, number of measurements and methodologies.

  5. Antifoam degradation testing

    Energy Technology Data Exchange (ETDEWEB)

    Lambert, D. P. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Zamecnik, J. R. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Newell, D. D. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL); Williams, M. S. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River Ecology Lab. (SREL)


    This report describes the results of testing to quantify the degradation products resulting from the dilution and storage of Antifoam 747. Antifoam degradation is of concern to the Defense Waste Processing Facility (DWPF) due to flammable decomposition products in the vapor phase of the Chemical Process Cell vessels, as well as the collection of flammable and organic species in the offgas condensate. The discovery that hexamethyldisiloxane is formed from the antifoam decomposition was the basis for a Potential Inadequacy in the Safety Analysis declaration by the DWPF.

  6. Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors. (United States)

    Piecyk, Karolina; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena


    Synthetic analogs of the 5' end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC50 similar to dinucleotide triphosphate m(7)GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m(7)GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity. © 2014 Piecyk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways. (United States)

    Li, Hua-Bing; Tong, Jiyu; Zhu, Shu; Batista, Pedro J; Duffy, Erin E; Zhao, Jun; Bailis, Will; Cao, Guangchao; Kroehling, Lina; Chen, Yuanyuan; Wang, Geng; Broughton, James P; Chen, Y Grace; Kluger, Yuval; Simon, Matthew D; Chang, Howard Y; Yin, Zhinan; Flavell, Richard A


    N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

  8. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    Directory of Open Access Journals (Sweden)

    Cathal Seoighe

    Full Text Available BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  9. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal


    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  10. The Substrates of Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Virginia S. Muir


    Full Text Available Nonsense-mediated mRNA decay (NMD is a conserved pathway that strongly influences eukaryotic gene expression. Inactivating or inhibiting NMD affects the abundance of a substantial fraction of the transcriptome in numerous species. Transcripts whose abundance is altered in NMD-deficient cells may represent either direct substrates of NMD or indirect effects of inhibiting NMD. We present a genome-wide investigation of the direct substrates of NMD in Caenorhabditis elegans. Our goals were (i to identify mRNA substrates of NMD and (ii to distinguish those mRNAs from others whose abundance is indirectly influenced by the absence of NMD. We previously demonstrated that Upf1p/SMG-2, the central effector of NMD in all studied eukaryotes, preferentially associates with mRNAs that contain premature translation termination codons. We used this preferential association to distinguish direct from indirect effects by coupling immunopurification of Upf1/SMG-2 with high-throughput mRNA sequencing of NMD-deficient mutants and NMD-proficient controls. We identify 680 substrates of NMD, 171 of which contain novel spliced forms that (i include sequences of annotated introns and (ii have not been previously documented in the C. elegans transcriptome. NMD degrades unproductively spliced mRNAs with sufficient efficiency in NMD-proficient strains that such mRNAs were not previously known. Two classes of genes are enriched among the identified NMD substrates: (i mRNAs of expressed pseudogenes and (ii mRNAs of gene families whose gene number has recently expanded in the C. elegans genome. Our results identify novel NMD substrates and provide a context for understanding NMD’s role in normal gene expression and genome evolution.

  11. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal


    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  12. Inflammation-regulated mRNA stability and the progression of vascular inflammatory diseases. (United States)

    Herman, Allison B; Autieri, Michael V


    Cardiovascular disease remains a major medical and socioeconomic burden in developed and developing societies, and will increase with an aging and increasingly sedentary society. Vascular disease and atherosclerotic vascular syndromes are essentially inflammatory disorders, and transcriptional and post-transcriptional processes play essential roles in the ability of resident vascular and inflammatory cells to adapt to environmental stimuli. The regulation of mRNA translocation, stability, and translation are key processes of post-transcriptional regulation that permit these cells to rapidly respond to inflammatory stimuli. For the most part, these processes are controlled by elements in the 3'-UTR of labile, proinflammatory transcripts. Since proinflammatory transcripts almost exclusively contain AU-rich elements (AREs), this represents a tightly regulated and specific mechanism for initiation and maintenance of the proinflammatory phenotype. RNA-binding proteins (RBPs) recognize cis elements in 3'-UTR, and regulate each of these processes, but there is little literature exploring the concept that RBPs themselves can be directly regulated by inflammatory stimuli. Conceptually, inflammation-responsive RBPs represent an attractive target of rational therapies to combat vascular inflammatory syndromes. Herein we briefly describe the cellular and molecular etiology of atherosclerosis, and summarize our current understanding of RBPs and their specific roles in regulation of inflammatory mRNA stability. We also detail RBPs as targets of current anti-inflammatory modalities and how this may translate into better treatment for vascular inflammatory diseases. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  13. Characterization of gene organization and generation of heterogeneous mRNA species of rat ISK protein. (United States)

    Iwai, M; Masu, M; Tsuchida, K; Mori, T; Ohkubo, H; Nakanishi, S


    The ISK protein is a novel, probably epithelial potassium channel which differs from conventional potassium channels in its structure, electrophysiology, and tissue distribution. In this investigation, we isolated and analyzed genomic and cDNA clones coding for the rat ISK protein to characterize the structural organization and expression pattern of the ISK protein gene. This analysis, together with primer extension and RNase protection experiments, indicated that the ISK protein mRNA is initiated from two different upstream exons and then encoded by an uninterrupted downstream exon covering the protein-coding and the 3'-untranslated regions of the mRNA. RNA blot hybridization analysis showed additional generation of several large species of mRNAs which result from inclusion of a part of the intron sequence and the 3'-flanking region of the ISK protein gene. Thus, the single ISK protein gene is involved in the production of multiple species of mRNAs through a variety of cellular mechanisms including transcription initiation at different sites, alternative RNA splicing, and polyadenylation at different sites. The heterogeneity of the ISK protein mRNAs may be associated with the emergence of the functional and regulatory diversity observed for potassium ion permeation in epithelial cells.

  14. Anaerobic degradation of benzoate by sulfate-reducing bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Silva, S.P.; Adorno, M.A.T.; Moraes, E.M.; Varesche, M.B.A. [Sao Paulo Univ., Sao Carlos, SP (Brazil). Biological Processes Laboratory


    Anaerobic processes are an efficient way to degrade aromatic compounds in industrial wastewater, such as phenol, cresol and benzoate. This study characterized the bacteria that degrades benzoate, an anaerobic degradation intermediate of several complex aromatic compounds. In particular, the study assessed the capacity to use benzoate with sulfate reducing bacteria in mesophilic conditions. Biofilm from polyurethane foam matrices of a fixed bed reactor was used as the cellular inoculum to treat industrial wastewater containing organic peroxide. Dilution techniques were used to purify the material and obtain cultures of cocci. The benzoate consumption capacity in sulfidogenic conditions was observed when the purified inoculum was applied to batch reactors with different benzoate/sulfate relations. Results indicate that purification was positive to bacteria that can degrade aromatic compounds. Desulfococcus multivorans bacteria was identified following the physiologic and kinetic experiments. The 0.6 benzoate/sulfate relation was considered ideal for complete consumption of carbon and total use of sulfur. 10 refs., 3 figs.

  15. Processing bodies are not required for mammalian nonsense-mediated mRNA decay. (United States)

    Stalder, Lukas; Mühlemann, Oliver


    Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.

  16. Tis11 mediated mRNA decay promotes the reacquisition of Drosophila intestinal stem cell quiescence. (United States)

    McClelland, Lindy; Jasper, Heinrich; Biteau, Benoît


    Adult stem cell proliferation rates are precisely regulated to maintain long-term tissue homeostasis. Defects in the mechanisms controlling stem cell proliferation result in impaired regeneration and hyperproliferative diseases. Many stem cell populations increase proliferation in response to tissue damage and reacquire basal proliferation rates after tissue repair is completed. Although proliferative signals have been extensively studied, much less is known about the molecular mechanisms that restore stem cell quiescence. Here we show that Tis11, an Adenine-uridine Rich Element (ARE) binding protein that promotes mRNA degradation, is required to re-establish basal proliferation rates of adult Drosophila intestinal stem cells (ISC) after a regenerative episode. We find that Tis11 limits ISC proliferation specifically after proliferation has been stimulated in response to heat stress or infection, and show that Tis11 expression and activity are increased in ISCs during tissue repair. Based on stem cell transcriptome analysis and RNA immunoprecipitation, we propose that Tis11 activation represents an integral part of a negative feedback mechanism that limits the expression of key components of several signaling pathways that control ISC function and proliferation. Our results identify Tis11 mediated mRNA decay as an evolutionarily conserved mechanism of re-establishing basal proliferation rates of stem cells in regenerating tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Inhibition of cellular DNA synthesis by vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    McGowan, J.J.; Wagner, R.R.


    DNA synthesis in mouse myeloma (MPC-11) cells and L cells was rapidly and progressively inhibited by infection with vesicular stomatitis virus (VSV). No significant difference in cellular DNA synthesis inhibition was noted between synchronized and unsynchronized cells, nor did synchronized cells vary in their susceptibility to VSV infection after release from successive thymidine and hydroxyurea blocks. Cellular RNA synthesis was inhibited to about the same extent as DNA synthesis, but cellular protein synthesis was less affected by VSV at the same multiplicity of infection. The effect of VSV on cellular DNA synthesis could not be attributed to degradation of existing DNA or to decreased uptake of deoxynucleoside triphosphates, nor were DNA polymerase and thymidine kinase activities significantly different in VSV-infected and uninfected cell extracts. Analysis by alkaline sucrose gradients of DNA in pulse-labeled uninfected and VSV-infected cells indicated that VSV infection did not appear to influence DNA chain elongation. Cellular DNA synthesis was not significantly inhibited by infection with the VSV polymerase mutant tsG114(I) at the restrictive temperature or by infection with defective-interfering VSV DI-011 (5' end of the genome), but DI-HR-LT (3' end of genome) exhibited initially rapid but not prolonged inhibition of MPC-11 cell DNA synthesis. DNA synthesis inhibitory activity of wild-type VSV was only slowly and partially inactivated by very large doses of UV irradiation. These data suggest that, as in the effect of VSV on cellular RNA synthesis inhibition of cellular DNA synthesis by VSV requires transcription of a small segment of the viral genome.

  18. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C


    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  19. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

    Directory of Open Access Journals (Sweden)

    Sarah M Johler

    Full Text Available During the last years the potential role of in vitro transcribed (IVT mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE. A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000 in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages

  20. Endocytic collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H.; Jürgensen, Henrik J.; Ingvarsen, Signe Ziir


    it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked...

  1. Degradation of fluorotelomer alcohols

    DEFF Research Database (Denmark)

    Ellis, David A; Martin, Jonathan W; De Silva, Amila O


    . The significance of the gas-phase peroxy radical cross reactions that produce PFCAs has not been recognized previously. Such reactions are expected to occur during the atmospheric degradation of all polyfluorinated materials, necessitating a reexamination of the environmental fate and impact of this important...... class of industrial chemicals....

  2. [The influence of fibronectin on the formation of multi-cellular spheroid of ovarian cancer]. (United States)

    Xie, Xiao-Yan; Liu, Shan-Ling; Wang, He; Lin, Lin; Zhu, Hong-Mei; Zhang, Jian-Jun; Zheng, Ying


    To investigate the role of Fibronectin in the formation of multi-cellular spheroid of ovarian cancer and the integrin receptor involved in the process. In vitro model of multi-cellular spheroid of SKOV3 was constructed by liguid overlay technique. The influence of fibronectin on the formation of the spheroid was observed. The gene expressions of potential integrin receptors were examined from the levels of mRNA and protein using real time reverse transcription PCR and Western-blot. Fibronctin stimulated the formation of multi-cellular spheroid of ovarian cancer larger than 250 microm. fibronectin suppressed the expression of subtype of integrin receptor ITGA5. Fibronectin can enhance the formation of multi-cellular spheroid of ovarian cancer. The subtype of integrin receptor ITGA5 is probably involved in the process.

  3. EGF receptor inhibitors increase ErbB3 mRNA and protein levels in breast cancer cells

    DEFF Research Database (Denmark)

    Grøvdal, Lene Melsæther; Kim, Jiyoung; Holst, Mikkel Roland


    gene expression of FAS which is involved in apoptotic signaling. Together, our data strongly suggest that resistance to EGFR inhibitors may result from the compensation of other family members and that combinations of anti-cancer drugs are required to increase the sensitivity of these treatments....... induced by gefitinib treatment on mRNA levels of the most common genes known to be involved in breast cancer. As expected, we found that gefitinib downregulated genes whose functions were linked to cellular proliferation, such as Ki-67, topoisomerase II alpha and cyclins, and surprisingly downregulated...

  4. Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

    Directory of Open Access Journals (Sweden)

    Valentina Iadevaia


    Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

  5. Protein targeting to subcellular organelles via MRNA localization. (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William


    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  6. Mechanism of Regulation of bcl-2 mRNA by Nucleolin and A+U-rich Element-binding Factor 1 (AUF1)* (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K.; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J.; Spicer, Eleanor K.


    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3′-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3′-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5′ end of the 136-nucleotide bcl-2 AU-rich element (AREbcl-2). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to AREbcl-2. In RNA decay assays, AREbcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of AREbcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability. PMID:20571027

  7. Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1). (United States)

    Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar; Sengupta, Tapas K; Bandyopadhyay, Sumita; Reuben, Adrian; Fernandes, Daniel J; Spicer, Eleanor K


    The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3'-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its half-life. Here, we examined the site on the bcl-2 3'-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5' end of the 136-nucleotide bcl-2 AU-rich element (ARE(bcl-2)). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE(bcl-2). In RNA decay assays, ARE(bcl-2) transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE(bcl-2) transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.

  8. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han


    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  9. Detection of pump degradation

    Energy Technology Data Exchange (ETDEWEB)

    Greene, R.H.; Casada, D.A.; Ayers, C.W. [and others


    This Phase II Nuclear Plant Aging Research study examines the methods of detecting pump degradation that are currently employed in domestic and overseas nuclear facilities. This report evaluates the criteria mandated by required pump testing at U.S. nuclear power plants and compares them to those features characteristic of state-of-the-art diagnostic programs and practices currently implemented by other major industries. Since the working condition of the pump driver is crucial to pump operability, a brief review of new applications of motor diagnostics is provided that highlights recent developments in this technology. The routine collection and analysis of spectral data is superior to all other technologies in its ability to accurately detect numerous types and causes of pump degradation. Existing ASME Code testing criteria do not require the evaluation of pump vibration spectra but instead overall vibration amplitude. The mechanical information discernible from vibration amplitude analysis is limited, and several cases of pump failure were not detected in their early stages by vibration monitoring. Since spectral analysis can provide a wealth of pertinent information concerning the mechanical condition of rotating machinery, its incorporation into ASME testing criteria could merit a relaxation in the monthly-to-quarterly testing schedules that seek to verify and assure pump operability. Pump drivers are not included in the current battery of testing. Operational problems thought to be caused by pump degradation were found to be the result of motor degradation. Recent advances in nonintrusive monitoring techniques have made motor diagnostics a viable technology for assessing motor operability. Motor current/power analysis can detect rotor bar degradation and ascertain ranges of hydraulically unstable operation for a particular pump and motor set. The concept of using motor current or power fluctuations as an indicator of pump hydraulic load stability is presented.

  10. The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression

    Directory of Open Access Journals (Sweden)

    Kirkpatrick Katharine L


    Full Text Available Abstract Background Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal stability leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase is the rate-limiting determinant of telomerase reactivation. Telomerase has been associated with negative prognostic indicators in some studies. The present study aims to detect any correlation between hTERT and the negative prognostic indicators VEGF and PCNA by quantitatively measuring the mRNA expression of these genes in human breast cancer and in adjacent non-cancerous tissue (ANCT. Materials and methods RNA was extracted from 38 breast carcinomas and 40 ANCT. hTERT and VEGF165, VEGF189 and PCNA mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR and Taqman methodology. Results The level of expression of VEGF-165 and PCNA was significantly higher in carcinoma tissue than ANCT (p = 0.02. The ratio of VEGF165/189 expression was significantly higher in breast carcinoma than ANCT (p = 0.025. hTERT mRNA expression correlated with VEGF-189 mRNA (p = 0.008 and VEGF165 (p = 0.07. Conclusions hTERT mRNA expression is associated with the expression of the VEGF189 and 165 isoforms. This could explain the poorer prognosis reported in breast tumours expressing high levels of hTERT. The relative expression of the VEGF isoforms is significantly different in breast tumour to ANCT, and this may be important in breast carcinogenesis.

  11. The origins of cellular life. (United States)

    Koonin, Eugene V


    All life on earth can be naturally classified into cellular life forms and virus-like selfish elements, the latter being fully dependent on the former for their reproduction. Cells are reproducers that not only replicate their genome but also reproduce the cellular organization that depends on semipermeable, energy-transforming membranes and cannot be recovered from the genome alone, under the famous dictum of Rudolf Virchow, Omnis cellula e cellula. In contrast, simple selfish elements are replicators that can complete their life cycles within the host cell starting from genomic RNA or DNA alone. The origin of the cellular organization is the central and perhaps the hardest problem of evolutionary biology. I argue that the origin of cells can be understood only in conjunction with the origin and evolution of selfish genetic elements. A scenario of precellular evolution is presented that involves cohesion of the genomes of the emerging cellular life forms from primordial pools of small genetic elements that eventually segregated into hosts and parasites. I further present a model of the coevolution of primordial membranes and membrane proteins, discuss protocellular and non-cellular models of early evolution, and examine the habitats on the primordial earth that could have been conducive to precellular evolution and the origin of cells.

  12. Continuum representations of cellular solids

    Energy Technology Data Exchange (ETDEWEB)

    Neilsen, M.K.


    Cellular materials consist of interconnected struts or plates which form cells. The struts or plates are constructed from a variety of metals, polymers, ceramics and wood products. Cellular materials are often used in impact limiters for shipping containers to protect the contents from accidental impact events. These materials exhibit a variety of complex behavior when subjected to crushing loads. This research focuses on the development of continuum representations of cellular solids that can be used in the finite element analysis of shipping container accidents. A significant portion of this work is the development of a new methodology to relate localized deformations to appropriate constitutive descriptions. This methodology provides the insight needed to select constitutive descriptions for cellular solids that capture the localized deformations that are observed experimentally. Constitutive relations are developed for two different cellular materials, aluminum honeycomb and polyurethane foam. These constitutive relations are based on plasticity and continuum damage theories. Plasticity is used to describe the permanent deformation exhibited by both aluminum honeycomb and polyurethane foam. Continuum damage is needed to capture the change in elastic parameters due to cracking of the polyurethane cell wall materials. The new constitutive description of polyurethane foam is implemented in both static and dynamic finite element codes, and analytical and numerical predictions are compared with available experimental data.

  13. Antiinflammatory Effect of Rosiglitazone via Modulation of mRNA Stability of Interleukin 10 and Cyclooxygenase 2 in Astrocytes. (United States)

    Pankevich, E V; Astakhova, A A; Chistyakov, D V; Sergeeva, M G


    Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E 2 , PGE 2 ) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.

  14. Nickel and cadmium-induced SLBP depletion: A potential pathway to metal mediated cellular transformation.

    Directory of Open Access Journals (Sweden)

    Ashley Jordan

    Full Text Available Both nickel and cadmium compounds have been established as group I carcinogens for several decades. Despite over-whelming evidence of these compounds' carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal induced cellular transformation remain unclear. In this study, we found that there were slightly different effects on decreased SLBP mRNA and protein as well as increased polyA H3.1 in our nickel exposed cells. This suggested that nickel and arsenic have similar effects on canonical histone mRNA transcription and translation. We also saw that the depletion of SLBP protein was reversed by inhibiting the proteosome. Finally, we showed that inhibiting the SLBP mRNA and protein levels were rescued by epigenetic modifiers suggesting that nickel's effects on SLBP may be mediated via epigenetic mechanisms. Taken together these results suggest a similar mechanism by which both arsenic and nickel may exert their carcinogenic effects.

  15. Tissue-specific induction of Hsp90 mRNA and plasma cortisol response in chinook salmon following heat shock, seawater challenge, and handling challenge (United States)

    Palmisano, Aldo N.; Winton, J.R.; Dickhoff, Walton W.


    In studying the whole-body response of chinook salmon (Oncorhynchus tshawytscha) to various stressors, we found that 5-hour exposure to elevated temperature (mean 21.6??C; + 10.6??C over ambient) induced a marked increase in Hsp90 messenger RNA accumulation in heart, brain, gill, muscle, liver, kidney, and tail fin tissues. The most vital tissues (heart, brain, gill, and muscle) showed the greatest Hsp90-mRNA response, with heart tissue increasing approximately 35-fold, Heat shock induced no increase in plasma cortisol. In contrast, a standard handling challenge induced high plasma cortisol levels, but no elevation in Hsp90 mRNA in any tissue, clearly separating the physiological and cellular stress responses. We saw no increase either in tissue Hsp90 mRNA levels or in plasma cortisol concentrations after exposing the fish to seawater overnight.

  16. Cues for cellular assembly of vascular elastin networks (United States)

    Kothapalli, Chandrasekhar R.

    Elastin, a structural protein distributed in the extracellular matrix of vascular tissues is critical to the maintenance of vascular mechanics, besides regulation of cell-signaling pathways involved in injury response and morphogenesis. Thus, congenital absence or disease-mediated degradation of vascular elastin and its malformation within native vessels due to innately poor elastin synthesis by adult vascular cells compromise vascular homeostasis. Current elastin regenerative strategies using tissue engineering principles are limited by the progressive destabilization of tropoelastin mRNA expression in adult vascular cells and the unavailability of scaffolds that can provide cellular cues necessary to up-regulate elastin synthesis and regenerate faithful mimics of native elastin. Since our earlier studies demonstrated the elastogenic utility of hyaluronan (HA)-based cues, we have currently sought to identify a unique set of culture conditions based on HA fragments (0.756-2000 kDa), growth factors (TGF-beta1, IGF-1) and other biomolecules (Cu2+ ions, LOX), which will together enhance synthesis, crosslinking, maturation and fibrous elastin matrix formation by adult SMCs, under both healthy and inflammatory conditions. It was observed that TGF-beta1 (1 ng/mL) together with HA oligomers (0.2 microg/mL) synergistically suppressed SMC proliferation, enhanced tropoelastin (8-fold) and matrix elastin synthesis (5.5-fold), besides improving matrix yield (4.5-fold), possibly by increasing production and activity of lysyl oxidase (LOX). Though addition of IGF-1 alone did not offer any advantage, HA fragments (20-200 kDa) in the presence of IGF-1 stimulated tropoelastin and soluble elastin synthesis more than 2.2-fold, with HMW HA contributing for ˜5-fold increase in crosslinked matrix elastin synthesis. Similarly, 0.1 M of Cu2+ ions, alone or together with HA fragments stimulated synthesis of tropoelastin (4-fold) and crosslinked matrix elastin (4.5-fold), via increases in

  17. Increased cellular proliferation in rat skeletal muscle and tendon in response to exercise

    DEFF Research Database (Denmark)

    Skovgaard, Dorthe; Bayer, Monika L; Mackey, Abigail


    PURPOSE: The purpose of this study is to investigate exercise-induced cellular proliferation in rat skeletal muscle/tendon with the use of 3'-[F-18]fluoro-3'deoxythymidine (FLT) and to quantitatively study concomitant changes in the proliferation-associated factor, Ki67. PROCEDURES: Wistar rats (......-derived results were supported by a correlation in calf muscle to Ki67 (protein and mRNA level), while this coherence was not found in tendon. CONCLUSION: FLT-PET seems to be a promising tool for imaging of exercise-induced cellular proliferation in musculo-tendinous tissue....

  18. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B


    . In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV......). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p alpha 6(IV) mRNA coincides...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6...

  19. Post-transcriptional gene regulation by mRNA modifications (United States)

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan


    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  20. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.


    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  1. BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation. (United States)

    Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro


    Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Rapid degradation of an active formylglycine generating enzyme variant leads to a late infantile severe form of multiple sulfatase deficiency. (United States)

    Schlotawa, Lars; Radhakrishnan, Karthikeyan; Baumgartner, Matthias; Schmid, Regula; Schmidt, Bernhard; Dierks, Thomas; Gärtner, Jutta


    Multiple sulfatase deficiency (MSD) is a rare inborn error of metabolism affecting posttranslational activation of sulfatases by the formylglycine generating enzyme (FGE). Due to mutations in the encoding SUMF1 gene, FGE's catalytic capacity is impaired resulting in reduced cellular sulfatase activities. Both, FGE protein stability and residual activity determine disease severity and have previously been correlated with the clinical MSD phenotype. Here, we report a patient with a late infantile severe course of disease. The patient is compound heterozygous for two so far undescribed SUMF1 mutations, c.156delC (p.C52fsX57) and c.390A>T (p.E130D). In patient fibroblasts, mRNA of the frameshift allele is undetectable. In contrast, the allele encoding FGE-E130D is expressed. FGE-E130D correctly localizes to the endoplasmic reticulum and has a very high residual molecular activity in vitro (55% of wildtype FGE); however, it is rapidly degraded. Thus, despite substantial residual enzyme activity, protein instability determines disease severity, which highlights that potential MSD treatment approaches should target protein folding and stabilization mechanisms.

  3. Aging, Cellular Senescence, and Cancer (United States)

    Campisi, Judith


    For most species, aging promotes a host of degenerative pathologies that are characterized by debilitating losses of tissue or cellular function. However, especially among vertebrates, aging also promotes hyperplastic pathologies, the most deadly of which is cancer. In contrast to the loss of function that characterizes degenerating cells and tissues, malignant (cancerous) cells must acquire new (albeit aberrant) functions that allow them to develop into a lethal tumor. This review discusses the idea that, despite seemingly opposite characteristics, the degenerative and hyperplastic pathologies of aging are at least partly linked by a common biological phenomenon: a cellular stress response known as cellular senescence. The senescence response is widely recognized as a potent tumor suppressive mechanism. However, recent evidence strengthens the idea that it also drives both degenerative and hyper-plastic pathologies, most likely by promoting chronic inflammation. Thus, the senescence response may be the result of antagonistically pleiotropic gene action. PMID:23140366

  4. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani


    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  5. Analysis of the Tumor Microenvironment Transcriptome via NanoString mRNA and miRNA Expression Profiling. (United States)

    M'Boutchou, Marie-Noël; van Kempen, Léon C


    Gene expression analysis in the tumor microenvironment using archived clinical samples is challenging because of formalin fixation, RNA degradation, and limiting sample volume. NanoString gene expression profiling is a RNA-DNA hybrid capture technology that does not require PCR and can accurately quantify the expression of to 800 transcripts in a single reaction. The technology requires 50-100 ng of RNA, which can be degraded ( is this correct?) to a 200 bp fragment size. In contrast to amplification technologies, nanoString counts the actual numbers of transcripts that are captured with transcript-specific and fluorescently-barcoded probes. This chapter describes protocols for RNA extraction, quantification, mRNA and miRNA profiling and data analysis.

  6. Cellular structures with interconnected microchannels

    Energy Technology Data Exchange (ETDEWEB)

    Shaefer, Robert Shahram; Ghoniem, Nasr M.; Williams, Brian


    A method for fabricating a cellular tritium breeder component includes obtaining a reticulated carbon foam skeleton comprising a network of interconnected ligaments. The foam skeleton is then melt-infiltrated with a tritium breeder material, for example, lithium zirconate or lithium titanate. The foam skeleton is then removed to define a cellular breeder component having a network of interconnected tritium purge channels. In an embodiment the ligaments of the foam skeleton are enlarged by adding carbon using chemical vapor infiltration (CVI) prior to melt-infiltration. In an embodiment the foam skeleton is coated with a refractory material, for example, tungsten, prior to melt infiltration.

  7. Cellular uptake of metallated cobalamins

    DEFF Research Database (Denmark)

    Tran, Mai Thanh Quynh; Stürup, Stefan; Lambert, Ian Henry


    Cellular uptake of vitamin B12-cisplatin conjugates was estimated via detection of their metal constituents (Co, Pt, and Re) by inductively coupled plasma mass spectrometry (ICP-MS). Vitamin B12 (cyano-cob(iii)alamin) and aquo-cob(iii)alamin [Cbl-OH2](+), which differ in the β-axial ligands (CN......(-) and H2O, respectively), were included as control samples. The results indicated that B12 derivatives delivered cisplatin to both cellular cytosol and nuclei with an efficiency of one third compared to the uptake of free cisplatin cis-[Pt(II)Cl2(NH3)2]. In addition, uptake of charged B12 derivatives...

  8. Interleukin-29 Enhances Synovial Inflammation and Cartilage Degradation in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Lingxiao Xu


    Full Text Available We have recently shown that IL-29 was an important proinflammatory cytokine in pathogenesis of rheumatoid arthritis (RA. Inflammation also contributes to the pathogenesis of osteoarthritis (OA. The aim of this study was to investigate the effect and mechanism of IL-29 on cytokine production and cartilage degradation in OA. The mRNA levels of IL-29 and its specific receptor IL-28Ra in peripheral blood mononuclear cells (PBMCs were significantly increased in OA patients when compared to healthy controls (HC. In the serum, IL-29 protein levels were higher in OA patients than those in HC. Immunohistochemistry revealed that both IL-29 and IL-28Ra were dramatically elevated in OA synovium compared to HC; synovial fibroblasts (FLS and macrophages were the main IL-29-producing cells in OA synovium. Furthermore, recombinant IL-29 augmented the mRNA expression of IL-1β, IL-6, IL-8, and matrix-metalloproteinase-3 (MMP-3 in OA FLS and increased cartilage degradation when ex vivo OA cartilage explant was coincubated with OA FLS. Finally, in OA FLS, IL-29 dominantly activated MAPK and nuclear factor-κB (NF-κB, but not Jak-STAT and AKT signaling pathway as examined by western blot. In conclusion, IL-29 stimulates inflammation and cartilage degradation by OA FLS, indicating that this cytokine is likely involved in the pathogenesis of OA.

  9. TALSPEAK Solvent Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Leigh R. Martin; Bruce J. Mincher


    Understanding the radiolytic degradation behavior of organic molecules involved in new or existing schemes for the recycle of used nuclear fuels is of significant interest for sustaining a closed nuclear fuel cycle. Here we have conducted several lines of investigation to begin understanding the effects of radiolysis on the aqueous phase of the TALSPEAK process for the separation of the trivalent lanthanides from the trivalent actinides. Using the 60-Co irradiator at the INL, we have begun to quantify the effects of radiation on the aqueous phase complexants used in this separation technique, and how this will affect the actinide lanthanide separation factor. In addition we have started to develop methodologies for stable product identification, a key element in determining the degradation pathways. We have also introduced a methodology to investigate the effects of alpha radiolysis that has previously received limited attention.

  10. Nylon separators. [thermal degradation (United States)

    Lim, H. S.


    A nylon separator was placed in a flooded condition in K0H solution and heated at various high temperatures ranging from 60 C to 110 C. The weight decrease was measured and the molecular weight and decomposition product were analyzed to determine: (1) the effect of K0H concentration on the hydrolysis rate; (2) the effect of K0H concentration on nylon degradation; (3) the activation energy at different K0H concentrations; and (4) the effect of oxygen on nylon degradation. The nylon hydrolysis rate is shown to increase as K0H concentration is decreased 34%, giving a maximum rate at about 16%. Separator hydrolysis is confirmed by molecular weight decrease in age of the batteries, and the reaction of nylon with molecular oxygen is probably negligible, compared to hydrolysis. The extrapolated rate value from the high temperature experiment correlates well with experimental values at 35 degrees.

  11. Role of RNase Y in Clostridium perfringens mRNA Decay and Processing. (United States)

    Obana, Nozomu; Nakamura, Kouji; Nomura, Nobuhiko


    RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5' untranslated region (5'UTR), and we observe that RNase Y depletion diminishes colA 5'UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation. RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results

  12. Cellular Automata and the Humanities. (United States)

    Gallo, Ernest


    The use of cellular automata to analyze several pre-Socratic hypotheses about the evolution of the physical world is discussed. These hypotheses combine characteristics of both rigorous and metaphoric language. Since the computer demands explicit instructions for each step in the evolution of the automaton, such models can reveal conceptual…

  13. Auxin and Cellular Elongation1 (United States)

    Velasquez, Silvia Melina; Barbez, Elke


    Auxin is a crucial growth regulator in plants. However, a comprehensive understanding of how auxin induces cell expansion is perplexing, because auxin acts in a concentration- and cell type-dependent manner. Consequently, it is desirable to focus on certain cell types to exemplify the underlying growth mechanisms. On the other hand, plant tissues display supracellular growth (beyond the level of single cells); hence, other cell types might compromise the growth of a certain tissue. Tip-growing cells do not display neighbor-induced growth constraints and, therefore, are a valuable source of information for growth-controlling mechanisms. Here, we focus on auxin-induced cellular elongation in root hairs, exposing a mechanistic view of plant growth regulation. We highlight a complex interplay between auxin metabolism and transport, steering root hair development in response to internal and external triggers. Auxin signaling modules and downstream cascades of transcription factors define a developmental program that appears rate limiting for cellular growth. With this knowledge in mind, the root hair cell is a very suitable model system in which to dissect cellular effectors required for cellular expansion. PMID:26787325

  14. Analysis of cellular manufacturing systems

    NARCIS (Netherlands)

    Heragu, Sunderesh; Zijm, Willem H.M.; Meng, Gang; Heragu, S.S.; van Ommeren, Jan C.W.; van Houtum, Geert-Jan


    In this paper, we present an open queuing network modeling approach to estimate performance measures of a cellular manufacturing layout. It is assumed a layout and production data for a planning period of specified length are available. The production data takes into account, processing and handling

  15. Conformational flexibility may explain multiple cellular roles of PEST motifs. (United States)

    Sandhu, Kuljeet Singh; Dash, Debasis


    PEST sequences are one of the major motifs that serve as signal for the protein degradation and are also involved in various cellular processes such as phosphorylation and protein-protein interaction. In our earlier study, we found that these motifs contribute largely to eukaryotic protein disorder. This observation led us to evaluate their conformational variability in the nonredundant Protein Data Bank (PDB) structures. For this purpose, crystallographic temperature factors, structural alignment of multiple NMR models, and dihedral angle order parameters have been used in this study. The study has revealed the hypermobility of PEST motifs as compared to other regions of the protein. Conformational flexibility may allow them to participate in number of molecular interactions under different conditions. This analysis may explain the role of protein backbone flexibility in bringing about multiple cellular roles of PEST motifs. 2006 Wiley-Liss, Inc.

  16. Biogeochemical Cycles in Degraded Lands (United States)

    Davidson, Eric A.; Vieira, Ima Celia G.; ReisdeCarvalho, Claudio Jose; DeanedeAbreuSa, Tatiana; deSouzaMoutinho, Paulo R.; Figueiredo, Ricardo O.; Stone, Thomas A.


    The objectives of this project were to define and describe the types of landscapes that fall under the broad category of "degraded lands" and to study biogeochemical cycles across this range of degradation found in secondary forests. We define degraded land as that which has lost part of its capacity of renovation of a productive ecosystem, either in the context of agroecosystems or as native communities of vegetation. This definition of degradation permits evaluation of biogeochemical constraints to future land uses.

  17. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages

    National Research Council Canada - National Science Library

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D


    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA...

  18. Analysis of mRNA recognition by human thymidylate synthase. (United States)

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas


    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

  19. Association between VDAC1 mRNA expression and intracellular ...

    African Journals Online (AJOL)

    One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium ...

  20. Cytokine mRNA expression during experimental corneal allograft rejection

    NARCIS (Netherlands)

    Torres, P. F.; de Vos, A. F.; van der Gaag, R.; Martins, B.; Kijlstra, A.


    Allograft rejection is the main cause of corneal graft failure. T lymphocytes and macrophages have been implied to be involved in corneal rejection, but little is known about the molecular mechanism in this process. In this study, cytokine mRNA expression in the cornea was analysed during

  1. Human mRNA response to exercise and temperature. (United States)

    Slivka, D R; Dumke, C L; Tucker, T J; Cuddy, J S; Ruby, B


    The purpose of this research was to determine the mRNA response to exercise in different environmental temperatures. 9 recreationally active males (27±1 years, 77.4±2.7  kg, 13.5±1.5% fat, 4.49±0.15  L · min (-1) VO2 max) completed 3 trials consisting of 1 h cycling exercise at 60% Wmax followed by a 3 h recovery in the cold (7°C), room temperature (20°C), and hot (33°C) environments. Muscle biopsies were obtained pre, post, and 3 h post exercise for the analysis of glycogen and mRNA. Expired gases were collected to calculate substrate use. PGC-1α increased to a greater degree in the cold trial than in the room temperature trial (p=0.036) and the hot trial (p=0.006). PGC1-α mRNA was also higher after the room temperature trial than the hot trial (p=0.050). UCP3 and MFN2 mRNA increased with exercise (pcold than exercise in the heat. However, VO2 was higher during recovery in the cold trial than in the room temperature and hot trials (p<0.05). This study presents evidence of PGC-1α temperature sensitivity in human skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

  2. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

  3. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)



    Jul 13, 2011 ... This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye. (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results.

  4. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)


    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  5. Proteoglycan degradation by the ADAMTS family of proteinases. (United States)

    Stanton, Heather; Melrose, James; Little, Christopher B; Fosang, Amanda J


    Proteoglycans are key components of extracellular matrices, providing structural support as well as influencing cellular behaviour in physiological and pathological processes. The diversity of proteoglycan function reported in the literature is equally matched by diversity in proteoglycan structure. Members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family of enzymes degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular function. In this review, we focus on ADAMTS enzymes that degrade the lectican and small leucine-rich repeat families of proteoglycans. We discuss the known ADAMTS cleavage sites and the consequences of cleavage at these sites. We illustrate our discussion with examples from the literature in which ADAMTS proteolysis of proteoglycans makes profound changes to tissue function. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Endoplasmic reticulum-associated degradation of glycoproteins in plants

    Directory of Open Access Journals (Sweden)

    Silvia eHüttner


    Full Text Available In all eukaryotes the endoplasmic reticulum (ER has a central role in protein folding and maturation of secretory and membrane proteins. Upon translocation into the ER polypeptides are immediately subjected to folding and modifications involving the formation of disulfide bridges, assembly of subunits to multi-protein complexes and glycosylation. During these processes incompletely folded, terminally misfolded and unassembled proteins can accumulate which endanger the cellular homeostasis and subsequently the survival of cells and tissues. Consequently, organisms have developed a quality control system to cope with this problem and remove the unwanted protein load from the ER by a process collectively referred to as endoplasmic reticulum-associated degradation (ERAD pathway. Recent studies in Arabidopsis have identified plant ERAD components involved in the degradation of aberrant proteins and evidence was provided for a specific role in abiotic stress tolerance. In this short review we discuss our current knowledge about this important cellular pathway.

  7. 'Biomoleculas': cellular metabolism didactic software

    Energy Technology Data Exchange (ETDEWEB)

    Menghi, M L [Chair of Physiology and Biophysics, Facultad de Ingenieria, Universidad Nacional de Entre Rios, CC 57 Suc 3, Parana 3100, Entre Rios (Argentina); Novella, L P [Chair of Physiology and Biophysics, Facultad de Ingenieria, Universidad Nacional de Entre Rios, CC 57 Suc 3, Parana 3100, Entre Rios (Argentina); Siebenlist, M R [Chair of Physiology and Biophysics, Facultad de Ingenieria, Universidad Nacional de Entre Rios, CC 57 Suc 3, Parana 3100, Entre Rios (Argentina)


    'Biomoleculas' is a software that deals with topics such as the digestion, cellular metabolism and excretion of nutrients. It is a pleasant, simple and didactic guide, made by and for students. In this program, each biomolecule (carbohydrates, lipids and proteins) is accompanied until its degradation and assimilation by crossing and interrelating the different metabolic channels to finally show the destination of the different metabolites formed and the way in which these are excreted. It is used at present as a teaching-learning process tool by the chair of Physiology and Biophysics at the Facultad de Ingenieria - Universidad Nacional de Entre Rios.

  8. "Biomoléculas": cellular metabolism didactic software (United States)

    Menghi, M. L.; Novella, L. P.; Siebenlist, M. R.


    "Biomoléculas" is a software that deals with topics such as the digestion, cellular metabolism and excretion of nutrients. It is a pleasant, simple and didactic guide, made by and for students. In this program, each biomolecule (carbohydrates, lipids and proteins) is accompanied until its degradation and assimilation by crossing and interrelating the different metabolic channels to finally show the destination of the different metabolites formed and the way in which these are excreted. It is used at present as a teaching-learning process tool by the chair of Physiology and Biophysics at the Facultad de Ingeniería - Universidad Nacional de Entre Ríos.

  9. Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation. (United States)

    Ouyang, Weiming; Guo, Pengfei; Fang, Hui; Frucht, David M


    Anthrax is a life-threatening disease caused by infection with Bacillus anthracis , which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2-Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2-Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4-JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.

  10. Regulation of mRNA Levels by Decay-Promoting Introns that Recruit the Exosome Specificity Factor Mmi1

    Directory of Open Access Journals (Sweden)

    Cornelia Kilchert


    Full Text Available In eukaryotic cells, inefficient splicing is surprisingly common and leads to the degradation of transcripts with retained introns. How pre-mRNAs are committed to nuclear decay is unknown. Here, we uncover a mechanism by which specific intron-containing transcripts are targeted for nuclear degradation in fission yeast. Sequence elements within these “decay-promoting” introns co-transcriptionally recruit the exosome specificity factor Mmi1, which induces degradation of the unspliced precursor and leads to a reduction in the levels of the spliced mRNA. This mechanism negatively regulates levels of the RNA helicase DDX5/Dbp2 to promote cell survival in response to stress. In contrast, fast removal of decay-promoting introns by co-transcriptional splicing precludes Mmi1 recruitment and relieves negative expression regulation. We propose that decay-promoting introns facilitate the regulation of gene expression. Based on the identification of multiple additional Mmi1 targets, including mRNAs, long non-coding RNAs, and sn/snoRNAs, we suggest a general role in RNA regulation for Mmi1 through transcript degradation.

  11. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling. (United States)

    Reid, David W; Nicchitta, Christopher V


    In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a more substantial role for the ER cellular protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression.

  12. Phase Space Invertible Asynchronous Cellular Automata

    Directory of Open Access Journals (Sweden)

    Simon Wacker


    Full Text Available While for synchronous deterministic cellular automata there is an accepted definition of reversibility, the situation is less clear for asynchronous cellular automata. We first discuss a few possibilities and then investigate what we call phase space invertible asynchronous cellular automata in more detail. We will show that for each Turing machine there is such a cellular automaton simulating it, and that it is decidable whether an asynchronous cellular automaton has this property or not, even in higher dimensions.

  13. Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. (United States)

    Ma, M; Benimetskaya, L; Lebedeva, I; Dignam, J; Takle, G; Stein, C A


    Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.

  14. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    Zhen Li


    Full Text Available The reproductive function of G-protein subunit Galphaq (GNAQ, a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR. The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01 and testis (p<0.05. Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.

  15. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli. (United States)

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel


    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  16. A case of cervical cancer expressed three mRNA variant of Hyaluronan-mediated motility receptor (United States)

    Villegas-Ruíz, Vanessa; Salcedo, Mauricio; Zentella-Dehesa, Alejandro; de Oca, Edén V Montes; Román-Basaure, Edgar; Mantilla-Morales, Alejandra; Dávila-Borja, Víctor M; Juárez-Méndez, Sergio


    Cervical cancer is the second malignancy in Mexico, little is known about the prognostic factors associated with this disease. Several cellular components are important in their transformation and progression. Alternative mRNA splice is an important mechanism for generating protein diversity, nevertheless, in cancer unknown mRNA diversity is expressed. Hyaluronan-mediated motility receptor (HMMR, RHAMM, CD168) is a family member of proteins, hyaluronan acid dependent, and has been associated with different malignant processes such as: angiogenesis, cell invasiveness, proliferation, metastasis and poor outcome in some tumors. In the present study we identified expression of HMMR in cervical cancer by means of RT-PCR and sequencing. Our results indicate co-expression of two HMMR variants in all samples, and one case expressed three alternative HMMR splice transcripts. These results showed the heterogeneity of mRNA transcripts of HMMR that could express in cancer and the expression of HMMR could be marker of malignancy in CC. PMID:24966934

  17. Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay (United States)

    Fanourgakis, Grigorios; Akpinar, Müge; Dahl, Andreas; Jessberger, Rolf


    Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. PMID:27149095

  18. Coexpression index of estrogen receptor alpha mRNA isoforms in simple, complex hyperplasia without atypia, complex atypical hyperplasia and adenocarcinoma. (United States)

    Witek, Andrzej; Paul-Samojedny, Monika; Stojko, Rafał; Seifert, Bohdan; Mazurek, Urszula


    Estrogen receptor isoforms are postulated to play an important role in modulating the estrogen response. To clarify the molecular mechanisms through which malignant changes are activated in endometrium, this study aims to examine the expression profiles of wild-type ER-alpha and their splice variants and to assess the number of coexisting mRNA isoforms of ER-alpha in normal endometrium as well as in endometrial hyperplasia and endometrial endometrioid adenocarcinoma. Human endometrium and specimens including endometrial hyperplasia and endometrial cancer were obtained during surgery. Endometrial data were classified into four groups: simple hyperplasia (n=24), complex hyperplasia (n=15), atypical hyperplasia (n=11), endometrial endometrioid adenocarcinoma (n=19) (grade 1, grade 2 morphological degree) and proliferative endometrium (n=24) as a control group. Total cellular RNA was extracted from endometrial tissues using Total RNA Prep Plus. A real-time quantitative RT-PCR assay was developed to quantify the wild-type ER-alpha and ER-alpha mRNA isoforms copy numbers. We have evaluated the variation in ERs mRNA level between normal endometrium and endometrial hyperplasia and adenocarcinoma. We also evaluated the "sharing indicator". It is a factor of mRNA ER-alpha holding shares in whole mRNA it assume quotient of ER-alpha slicing variant to all variants of mRNA ER-alpha. It was found that the number of coexisting mRNA isoforms was significantly higher in adenocarcinoma endometrium than that evaluated for various degrees of hyperplasia endometrium and normal proliferative endometrium (palpha.

  19. Sodium arsenite-induced inhibition of cell proliferation is related to inhibition of IL-2 mRNA expression in mouse activated T cells

    Energy Technology Data Exchange (ETDEWEB)

    Conde, Patricia; Acosta-Saavedra, Leonor C.; Calderon-Aranda, Emma S. [Centro de Investigacion y de Estudios Avanzados, CINVESTAV, Seccion Toxicologia, P.O. Box 14-740, Mexico, D.F. (Mexico); Goytia-Acevedo, Raquel C. [Universidad Juarez del Estado de Durango, Facultad de Medicina, Gomez Palacio, Durango (Mexico)


    A proposed mechanism for the As-induced inhibition of cell proliferation is the inhibition of IL-2 secretion. However, the effects of arsenite on IL-2 mRNA expression or on the ERK pathway in activated-T cells have not yet been described. We examined the effect of arsenite on IL-2 mRNA expression, cell activation and proliferation in PHA-stimulated murine lymphocytes. Arsenite (1 and 10 {mu}M) decreased IL-2 mRNA expression, IL-2 secretion and cell proliferation. Arsenite (10 {mu}M) strongly inhibited ERK-phosphorylation. However, the partial inhibition (50%) of IL-2 mRNA produced by 1 {mu}M, consistent with the effects on IL-2 secretion and cell proliferation, could not be explained by the inhibition of ERK-phosphorylation, which was not affected at this concentration. The inhibition of IL-2 mRNA expression caused by 1 {mu}M could be associated to effects on pathways located downstream or parallel to ERK. Arsenite also decreased early activation (surface CD69{sup +} expression) in both CD4{sup +} and CD8{sup +}, and decreased total CD8{sup +} count without significantly affecting CD4{sup +}, supporting that the cellular immune response mediated by cytotoxic T cells is an arsenic target. Thus, our results suggest that arsenite decreases IL-2 mRNA levels and T-cell activation and proliferation. However, further studies on the effects of arsenite on IL-2 gene transcription and IL-2 mRNA stability are needed. (orig.)

  20. Role of a redox-based methylation switch in mRNA life cycle ( pre- & post- transcriptional maturation and protein turnover : Implications in neurological disorders

    Directory of Open Access Journals (Sweden)



    Full Text Available Homeostatic synaptic scaling in response to neuronal stimulus or activation, as well as due to changes in cellular niche, is an important phenomenon for memory consolidation, retrieval, and other similar cognitive functions. Neurological disorders and cognitive disabilities in autism, Rett syndrome, schizophrenia, dementia etc., are strongly correlated to alterations in protein expression (both synaptic and cytoplasmic. This correlation suggests that efficient temporal regulation of synaptic protein expression is important for synaptic plasticity. In addition, equilibrium between mRNA processing, protein translation and protein turnover is a critical sensor/trigger for recording synaptic information, normal cognition and behavior. Thus a regulatory switch, controlling the lifespan, maturation and processing of mRNA, might influence cognition and adaptive behavior. Here, we propose a two part novel hypothesis that methylation might act as this suggested coordinating switch to critically regulate mRNA maturation at 1.The pre-transcription level, by regulating precursor-RNA (pre-RNA processing into mRNA, via other non-coding RNAs and their influence on splicing phenomenon, and 2. the post-transcription level by modulating the regulatory functions of ribonucleoproteins (RNP and RNA binding proteins (RNABP in mRNA translation, dendritic translocation as well as protein synthesis and synaptic turnover. DNA methylation changes are well recognized and highly correlated to gene expression levels as well as, learning and memory; however, RNA methylation changes are recently characterized and yet their functional implications are not established. This review article provides some insight on the intriguing consequences of changes in methylation levels on mRNA life-cycle. We also suggest that, since methylation is under the control of glutathione antioxidant levels, the redox status of neurons might be the central regulatory switch for methylation

  1. Statistical modeling for degradation data

    CERN Document Server

    Lio, Yuhlong; Ng, Hon; Tsai, Tzong-Ru


    This book focuses on the statistical aspects of the analysis of degradation data. In recent years, degradation data analysis has come to play an increasingly important role in different disciplines such as reliability, public health sciences, and finance. For example, information on products’ reliability can be obtained by analyzing degradation data. In addition, statistical modeling and inference techniques have been developed on the basis of different degradation measures. The book brings together experts engaged in statistical modeling and inference, presenting and discussing important recent advances in degradation data analysis and related applications. The topics covered are timely and have considerable potential to impact both statistics and reliability engineering.

  2. Intracellular collagen degradation mediated by uPARAP/Endo180 is a major pathway of extracellular matrix turnover during malignancy

    DEFF Research Database (Denmark)

    Curino, Alejandro C; Engelholm, Lars H; Yamada, Susan S


    We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/...

  3. eIF3d is an mRNA cap-binding protein required for specialized translation initiation (United States)

    Lee, Amy S.Y.; Kranzusch, Philip J.; Doudna, Jennifer A.; Cate, Jamie H.D.


    Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation. PMID:27462815

  4. Tebuconazole photocatalytic degradation kinetics


    Prestes, Thiago de Hermann; Gibbon, Danielle de Oliveira; Lansarin, Marla Azário; Moro, Celso Camilo


    The tebuconazole photocatalytic degradation kinetics was studied in a batch reactor using TiO2 (P25-Degussa) as catalyst and a high pressure mercury lamp. The photolysis, adsorption and irradiation effects in the reaction rate were evaluated. Afterward, the suspension catalyst concentration and initial pH to the maximum reaction rate was determined. It was observed that the reaction rate can be approached by a pseudo-first order, with a maximum kinetics constant at 260 mg L-1catalyst concentr...

  5. Outdoor PV Degradation Comparison

    Energy Technology Data Exchange (ETDEWEB)

    Jordan, D. C.; Smith, R. M.; Osterwald, C. R.; Gelak, E.; Kurtz, S. R.


    As photovoltaic (PV) penetration of the power grid increases, it becomes vital to know how decreased power output; may affect cost over time. In order to predict power delivery, the decline or degradation rates must be determined; accurately. At the Performance and Energy Rating Testbed (PERT) at the Outdoor Test Facility (OTF) at the; National Renewable Energy Laboratory (NREL) more than 40 modules from more than 10 different manufacturers; were compared for their long-term outdoor stability. Because it can accommodate a large variety of modules in a; limited footprint the PERT system is ideally suited to compare modules side-by-side under the same conditions.

  6. Cellular and tissue expression of DAPIT, a phylogenetically conserved peptide

    Directory of Open Access Journals (Sweden)

    H. Kontro


    Full Text Available DAPIT (Diabetes Associated Protein in Insulin-sensitive Tissues is a small, phylogenetically conserved, 58 amino acid peptide that was previously shown to be down-regulated at mRNA level in insulin-sensitive tissues of type 1 diabetes rats. In this study we characterize a custom made antibody against DAPIT and confirm the mitochondrial presence of DAPIT on cellular level. We also show that DAPIT is localized in lysosomes of HUVEC and HEK 293T cells. In addition, we describe the histological expression of DAPIT in several tissues of rat and man and show that it is highly expressed especially in cells with high aerobic metabolism and epithelial cells related to active transport of nutrients and ions. We propose that DAPIT, in addition to indicated subunit of mitochondrial F-ATPase, is also a subunit of lysosomal V-ATPase suggesting that it is a common component in different proton pumps.

  7. Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent


    Full Text Available Abstract Background A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source, and mRNA inactivation and protein degradation (the sink. Results Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc and of the enhanced green fluorescent protein (eGFP. Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. Conclusions The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in

  8. Reversibly assembled cellular composite materials. (United States)

    Cheung, Kenneth C; Gershenfeld, Neil


    We introduce composite materials made by reversibly assembling a three-dimensional lattice of mass-produced carbon fiber-reinforced polymer composite parts with integrated mechanical interlocking connections. The resulting cellular composite materials can respond as an elastic solid with an extremely large measured modulus for an ultralight material (12.3 megapascals at a density of 7.2 milligrams per cubic centimeter). These materials offer a hierarchical decomposition in modeling, with bulk properties that can be predicted from component measurements and deformation modes that can be determined by the placement of part types. Because site locations are locally constrained, structures can be produced in a relative assembly process that merges desirable features of fiber composites, cellular materials, and additive manufacturing.

  9. Cellular multiplets in directional solidification

    Energy Technology Data Exchange (ETDEWEB)

    Kopczynski, P.; Rappel, W.; Karma, A. [Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115 (United States)


    We report the existence of new branches of steady state cellular structures in directional solidification. These structures consist of repeating cellular subunits, or multiplets, each containing a set of distinct cells separated by unequal grooves. A detailed numerical study of the symmetric model of directional solidification reveals that all multiplets bifurcate off the main singlet solution branch in two sets. Two points on the main branch, one corresponding to the onset of the Eckhaus instability at small cell spacing and the other to a fold of this branch at large spacing, are argued to be separate accumulation points for each set of multiplets. The set of structures bifurcating near the fold are morphologically similar to experimentally observed multiplets. In contrast, those bifurcating near the Eckhaus instability do not resemble experimental shapes. Furthermore, they are argued to be generically unstable. {copyright} {ital 1997} {ital The American Physical Society}

  10. Cellular IRES-mediated translation (United States)


    Translation of cellular mRNAs via initiation at internal ribosome entry sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death. PMID:21220943

  11. Xtoys: Cellular automata on xwindows

    Energy Technology Data Exchange (ETDEWEB)

    Creutz, M. [Brookhaven National Lab., Upton, NY (United States). Physics Dept.


    Xtoys is a collection of xwindow programs for demonstrating simulations of various statistical models. Included are xising, for the two dimensional Ising model, xpotts, for the q-state Potts model, xautomalab, for a fairly general class of totalistic cellular automata, xsand, for the Bak-Tang-Wiesenfield model of self organized criticality, and xfires, a simple forest fire simulation. The programs should compile on any machine supporting xwindows.

  12. Melanoma Screening with Cellular Phones


    Massone, Cesare; Hofmann-Wellenhof, Rainer; Ahlgrimm-Siess, Verena; Gabler, Gerald; Ebner, Christoph; Peter Soyer, H.


    BACKGROUND: Mobile teledermatology has recently been shown to be suitable for teledermatology despite limitations in image definition in preliminary studies. The unique aspect of mobile teledermatology is that this system represents a filtering or triage system, allowing a sensitive approach for the management of patients with emergent skin diseases. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated the feasibility of teleconsultation using a new generation of cellular phones in p...

  13. Aging, Cellular Senescence, and Cancer


    Campisi, Judith


    For most species, aging promotes a host of degenerative pathologies that are characterized by debilitating losses of tissue or cellular function. However, especially among vertebrates, aging also promotes hyperplastic pathologies, the most deadly of which is cancer. In contrast to the loss of function that characterizes degenerating cells and tissues, malignant (cancerous) cells must acquire new (albeit aberrant) functions that allow them to develop into a lethal tumor. This review discusses ...

  14. Cellular Senescence: A Translational Perspective


    Kirkland, James L.; Tamara Tchkonia


    Cellular senescence entails essentially irreversible replicative arrest, apoptosis resistance, and frequently acquisition of a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). Senescent cells accumulate in various tissues with aging and at sites of pathogenesis in many chronic diseases and conditions. The SASP can contribute to senescence-related inflammation, metabolic dysregulation, stem cell dysfunction, aging phenotypes, chronic diseases, geriatric sy...

  15. Cellular automata : dynamics, simulations, traces


    Guillon, Pierre


    A cellular automaton is a discrete dynamical system which can model objects that evolve parallelly and asynchronously : the space is divided into cells, each of which has a state evolving according to some single local rule and a finite number of neighboring cells. Though this system can easily be formalized, very complex behaviors can appear ; it turns out to be a powerful computational model. That complexity can be studied with respect to various theories : topology, measure, decidability, ...

  16. Cellular Adhesion and Adhesion Molecules


    SELLER, Zerrin


    In recent years, cell adhesion and cell adhesion molecules have been shown to be important for many normal biological processes, including embryonic cell migration, immune system functions and wound healing. It has also been shown that they contribute to the pathogenesis of a large number of common human disorders, such as rheumatoid arthritis and tumor cell metastasis in cancer. In this review, the basic mechanisms of cellular adhesion and the structural and functional features of adhes...

  17. 3'-5' RNA degradation pathways in human cells

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon

    RNA synthesis and degradation are key steps in the regulation of gene expression in all living organisms. During the course of his PhD studies, Michal Lubas centred his research on the nuclear and cytoplasmic RNA turnover of both noncoding and coding RNAs in human cells. His proteomic studies...... revealed the interaction network of the main 3'-5' RNA degradation machinery – the RNA exosome complex. One of the key findings was the identification and characterisation of the Nuclear Exosome Targeting (NEXT) complex, important for nuclear functions of the exosome. Michal Lubas also studied the role...... of the cytoplasmic 3'-5' exoribonuclease hDIS3L2. Using low throughout and high throughput techniques, both in vivo and in vitro, he characterised the nuclease and disclosed the role of hDIS3L2 in cytoplasmic mRNA metabolism....

  18. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E


    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  19. Sexual phenotype differences in zic2 mRNA abundance in the preoptic area of a protogynous teleost, Thalassoma bifasciatum.

    Directory of Open Access Journals (Sweden)

    Katherine McCaffrey

    Full Text Available The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP males and territorial, terminal phase (TP males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.

  20. Dynamic properties of cellular neural networks

    Directory of Open Access Journals (Sweden)

    Angela Slavova


    Full Text Available Dynamic behavior of a new class of information-processing systems called Cellular Neural Networks is investigated. In this paper we introduce a small parameter in the state equation of a cellular neural network and we seek for periodic phenomena. New approach is used for proving stability of a cellular neural network by constructing Lyapunov's majorizing equations. This algorithm is helpful for finding a map from initial continuous state space of a cellular neural network into discrete output. A comparison between cellular neural networks and cellular automata is made.

  1. Cellular communications a comprehensive and practical guide

    CERN Document Server

    Tripathi, Nishith


    Even as newer cellular technologies and standards emerge, many of the fundamental principles and the components of the cellular network remain the same. Presenting a simple yet comprehensive view of cellular communications technologies, Cellular Communications provides an end-to-end perspective of cellular operations, ranging from physical layer details to call set-up and from the radio network to the core network. This self-contained source forpractitioners and students represents a comprehensive survey of the fundamentals of cellular communications and the landscape of commercially deployed

  2. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis


    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  3. Translatability of rat kidney mRNA after mercury administration. (United States)

    Samji, S; Kuliszewski, M J; Girgis, G R; Nicholls, D M


    Young male rats received an intraperitoneal injection of 0.5 mg HgCl2/kg body weight and 16 h later the kidneys were removed and homogenized to prepare the polysomal fraction from which the poly(A)+ RNA was obtained. The activity of this fraction was assessed by translating the poly(A)+ RNA in a mRNA-dependent rabbit reticulocyte lysate and the activity was markedly elevated relative to preparations from control rat kidneys. The incorporation of labelled leucine and cysteine, but not phenylalanine, into a low molecular weight protein (approximately 10 000 as judged by denaturing polyacrylamide gel electrophoresis) accounted for the increased mRNA activity. The mobility during electrophoresis of the denatured labelled product and carboxymethylated product, as well as their acidic isoelectric points, provided evidence that it is metallothionein mRNA which exhibits increased translatability in preparations derived from mercury-treated rats.

  4. Cup regulates oskar mRNA stability during oogenesis. (United States)

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E


    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  5. Detection of cellular retinol-binding protein messenger RNA in the somatic cells of the rat seminiferous tubules. (United States)

    Galdieri, M; Faraonio, R; Colantuoni, V


    A cDNA clone coding for Cellular Retinol-Binding Protein (CRBP) was used as a probe to study the expression of the gene in the somatic cells of the seminiferous tubules (Sertoli and peritubular cells). In this paper we demonstrate that these cells are actively involved in the synthesis of the specific mRNA. In Sertoli cells the gene is modulated by the hormones effective in spermatogenesis, such as FSH and testosterone. Moreover, peritubular cells revealed an approximately two times higher concentration of CRBP steady-state mRNA levels when compared with Sertoli cells.

  6. Degradation of amyloid beta protein by purified myelin basic protein. (United States)

    Liao, Mei-Chen; Ahmed, Mahiuddin; Smith, Steven O; Van Nostrand, William E


    The progressive accumulation of beta-amyloid (Abeta) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer disease and related disorders. Impaired clearance of Abeta from the brain likely contributes to the prevalent sporadic form of Alzheimer disease. Several major pathways for Abeta clearance include receptor-mediated cellular uptake, blood-brain barrier transport, and direct proteolytic degradation. Myelin basic protein (MBP) is the major structural protein component of myelin and plays a functional role in the formation and maintenance of the myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autocatalytic cleavage liberating distinct fragments. Recently, we showed that MBP binds Abeta and inhibits Abeta fibril formation (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). Here we show that Abeta40 and Abeta42 peptides are degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment that lacks autolytic activity. MBP-mediated Abeta degradation is inhibited by serine proteinase inhibitors. Similarly, Cos-1 cells expressing MBP degrade exogenous Abeta40 and Abeta42. In addition, we demonstrate that purified MBP also degrades assembled fibrillar Abeta in vitro. Mass spectrometry analysis identified distinct degradation products generated from Abeta digestion by MBP. Lastly, we demonstrate in situ that purified MBP can degrade parenchymal amyloid plaques as well as cerebral vascular amyloid that form in brain tissue of Abeta precursor protein transgenic mice. Together, these findings indicate that purified MBP possesses Abeta degrading activity in vitro.

  7. Design of multimodal degradable hydrogels for controlled therapeutic delivery (United States)

    Kharkar, Prathamesh Madhav

    thiol exchange reaction facilitated rapid and responsive protein release in the presence of GSH. A photolabile o-nitrobenzyl ether group (o-NB) was subsequently incorporated within the PEG-based, gel-forming monomers to demonstrate cargo release triggered by exogenous stimuli for patient-specific therapies. Upon the application of cytocompatible doses of light, the photolabile o-NB linkage underwent irreversible cleavage yielding ketone and carboxylic acid-based cleavage products. Hydrogel degradation kinetics was characterized in response to externally applied cytocompatible light or GSH in aqueous microenvironments. By incorporating a photodegradable o-nitrobenzyl ether group, a thiol-sensitive succinimide thioether linkage, and ester linkages within the hydrogels, we demonstrated unique control over degradation via surface erosion or bulk degradation mechanisms, respectively, with degradation rate constants ranging from 10-1 min-1 to 10-4 min-1. As a proof of concept, the controlled release of nanobeads from the hydrogel was demonstrated in a preprogrammed and stimuli-responsive fashion. The multimodal degradable hydrogels were then investigated for the local controlled release of small molecular weight proteins, which are of interest for regulating various cellular functions and fates in vivo. Low molecular weight heparin, a highly sulfated polysaccharide was incorporated within the hydrogel network by Michael-type reaction due to its affinity with biologics such as growth factors and immunomodulatory proteins. Incorporation of reduction-sensitive linkages resulted in 2.3 fold differences in the release profile of fibroblast growth factor-2 (FGF-2) in the presence of GSH compared to non-reducing microenvironment. Bioactivity of released FGF-2 was comparable to pristine FGF-2, indicating the ability of the hydrogel to retain bioactivity of cargo molecules during encapsulation and release. Further, preliminary in vivo studies demonstrated control over hydrogel

  8. A Novel Cellular Senescence Gene, SENEX, Is Involved in Peripheral Regulatory T Cells Accumulation in Aged Urinary Bladder Cancer (United States)

    Chen, Tianping; Wang, Huiping; Zhang, Zhiqiang; Li, Qing; Yan, Kaili; Tao, Qianshan; Ye, Qianling; Xiong, Shudao; Wang, Yiping; Zhai, Zhimin


    Regulatory T cells (Tregs) play an essential role in sustaining self-tolerance and immune homeostasis. Despite many studies on the correlation between Tregs accumulation and age, or malignancies, the related mechanism hasn’t been well explored. To find out the mechanism of Tregs accumulation in aged urinary bladder cancer, we examined the novel cellular senesence gene SENEX and relevant apoptosis gene mRNA expression in sorted CD4+CD25hi Tregs from aged UBC donors, evaluated serum cytokine profiles related to tumor immunopathology, and further explored the relationship between SENEX expression, apoptosis gene expression and cytokine secretion. After having silenced down SENEX gene expression with RNA interference, we also evaluated the cellular apoptosis of Tregs sorted from aged UBC patients in response to H2O2-mediated stress. Our data indicated that upregulated SENEX mRNA expression in Tregs of aged UBC patients was correlated with pro-apoptotic gene expression and cytokine concentration. Silencing SENEX gene expression increased cellular apoptosis and pro-apoptotic gene expression of Tregs, in response to H2O2-mediated stress. Upregulated SENEX mRNA expression together with decreased pro-apoptotic gene expression and disturbances in cytokines synthesis may contribute to the Tregs proliferation and promote tumorigenesis and metastasis. Overall, upregulation of cellular senescence gene SENEX, was associated to regulatory T cells accumulation in aged urinary bladder cancer. Our study provides a new insight into understanding of peripheral Tregs accumulation in aged malignancies. PMID:24505313

  9. Thermal battery degradation mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Missert, Nancy A. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Brunke, Lyle Brent [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)


    Diffuse reflectance IR spectroscopy (DRIFTS) was used to investigate the effect of accelerated aging on LiSi based anodes in simulated MC3816 batteries. DRIFTS spectra showed that the oxygen, carbonate, hydroxide and sulfur content of the anodes changes with aging times and temperatures, but not in a monotonic fashion that could be correlated to phase evolution. Bands associated with sulfur species were only observed in anodes taken from batteries aged in wet environments, providing further evidence for a reaction pathway facilitated by H2S transport from the cathode, through the separator, to the anode. Loss of battery capacity with accelerated aging in wet environments was correlated to loss of FeS2 in the catholyte pellets, suggesting that the major contribution to battery performance degradation results from loss of active cathode material.

  10. Environmental degradation in biocomposites

    CSIR Research Space (South Africa)

    John, Maya J


    Full Text Available properties which occurs due to degradation of fibres and matrix. In the case of biocomposites, both natural fibres and the polymer matrix absorb the ultraviolet rays from the sun- light. This leads to changes in the chemical structure of the polymers via... a S ta ge : P ro of C ha pt er N o. : 7 T itl e N am e: R ay P ag e N um be r: 1 D at e: 0 2/ 02 /2 01 7 T im e: 0 1: 14 :0 4 B978-0-08-100793-8.00007-7, 00007 Ray, 978-0-08-100793-8 AUTHOR QUERY FORM Book: Biocomposites for High...

  11. Method for Analysis of Matrix Degradation by CCN2 Through the MMP/TIMP System. (United States)

    McLennan, Susan V; Min, Danqing; Wang, Xiaoyu; Twigg, Stephen M


    Many studies have shown effects of members of the CCN family on matrix synthesis and accumulation but few have examined degradative pathways. This scarcity of information is in part due to the lack of suitable model systems. Here we describe methods for making rhCCN2 and also for the preparation of a biosynthetically labeled matrix substrate that can be used to measure the effect of CCN on cellular or secreted degradative pathways.

  12. Autophagic degradation of peroxisomes in mammals. (United States)

    Zientara-Rytter, Katarzyna; Subramani, Suresh


    Peroxisomes are essential organelles required for proper cell function in all eukaryotic organisms. They participate in a wide range of cellular processes including the metabolism of lipids and generation, as well as detoxification, of hydrogen peroxide (H2O2). Therefore, peroxisome homoeostasis, manifested by the precise and efficient control of peroxisome number and functionality, must be tightly regulated in response to environmental changes. Due to the existence of many physiological disorders and diseases associated with peroxisome homoeostasis imbalance, the dynamics of peroxisomes have been widely examined. The increasing volume of reports demonstrating significant involvement of the autophagy machinery in peroxisome removal leads us to summarize current knowledge of peroxisome degradation in mammalian cells. In this review we present current models of peroxisome degradation. We particularly focus on pexophagy-the selective clearance of peroxisomes through autophagy. We also critically discuss concepts of peroxisome recognition for pexophagy, including signalling and selectivity factors. Finally, we present examples of the pathological effects of pexophagy dysfunction and suggest promising future directions. © 2016 Authors; published by Portland Press Limited.

  13. Cellular host responses to gliomas.

    Directory of Open Access Journals (Sweden)

    Joseph Najbauer

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites. METHODOLOGY/PRINCIPAL FINDINGS: Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a 'network' with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a 'pair-wise' manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a low-generation tumors (first in vivo passage in rats were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b high-generation xenografts (fifth passage had pronounced cellularity, were angiogenic with 'glomerulus-like' microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which

  14. Symmetry analysis of cellular automata

    Energy Technology Data Exchange (ETDEWEB)

    García-Morales, V., E-mail: [Institute for Advanced Study – Technische Universität München, Lichtenbergstr. 2a, D-85748 Garching (Germany)


    By means of B-calculus [V. García-Morales, Phys. Lett. A 376 (2012) 2645] a universal map for deterministic cellular automata (CAs) has been derived. The latter is shown here to be invariant upon certain transformations (global complementation, reflection and shift). When constructing CA rules in terms of rules of lower range a new symmetry, “invariance under construction” is uncovered. Modular arithmetic is also reformulated within B-calculus and a new symmetry of certain totalistic CA rules, which calculate the Pascal simplices modulo an integer number p, is then also uncovered.

  15. Repaglinide at a cellular level

    DEFF Research Database (Denmark)

    Krogsgaard Thomsen, M; Bokvist, K; Høy, M


    To investigate the hormonal and cellular selectivity of the prandial glucose regulators, we have undertaken a series of experiments, in which we characterised the effects of repaglinide and nateglinide on ATP-sensitive potassium ion (KATP) channel activity, membrane potential and exocytosis in rat...... pancreatic alpha-cells and somatotrophs. We found a pharmacological dissociation between the actions on KATP channels and exocytosis and suggest that compounds that, unlike repaglinide, have direct stimulatory effects on exocytosis in somatotrophs and alpha- and beta-cells, such as sulphonylureas...

  16. Game of Life Cellular Automata

    CERN Document Server

    Adamatzky, Andrew


    In the late 1960s, British mathematician John Conway invented a virtual mathematical machine that operates on a two-dimensional array of square cell. Each cell takes two states, live and dead. The cells' states are updated simultaneously and in discrete time. A dead cell comes to life if it has exactly three live neighbours. A live cell remains alive if two or three of its neighbours are alive, otherwise the cell dies. Conway's Game of Life became the most programmed solitary game and the most known cellular automaton. The book brings together results of forty years of study into computational

  17. Cellular automata a parallel model

    CERN Document Server

    Mazoyer, J


    Cellular automata can be viewed both as computational models and modelling systems of real processes. This volume emphasises the first aspect. In articles written by leading researchers, sophisticated massive parallel algorithms (firing squad, life, Fischer's primes recognition) are treated. Their computational power and the specific complexity classes they determine are surveyed, while some recent results in relation to chaos from a new dynamic systems point of view are also presented. Audience: This book will be of interest to specialists of theoretical computer science and the parallelism challenge.

  18. Cellular recycling of proteins in seed dormancy alleviation and germination

    Directory of Open Access Journals (Sweden)

    Krystyna Oracz


    Full Text Available Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway (UPP is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant’s photosynthetic tissues have been well characterized since many years, but in nonphotosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is

  19. Rapid detection of biothreat agents based on cellular machinery.

    Energy Technology Data Exchange (ETDEWEB)

    Lane, Todd W.; Gantt, Richard W.


    This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid, for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.

  20. Protein accounting in the cellular economy. (United States)

    Vázquez-Laslop, Nora; Mankin, Alexander S


    Knowing the copy number of cellular proteins is critical for understanding cell physiology. By being able to measure the absolute synthesis rates of the majority of cellular proteins, Li et al. gain insights into key aspects of translation regulation and fundamental principles of cellular strategies to adjust protein synthesis according to the functional needs. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Mapping the global mRNA transcriptome during development of the murine first molar

    Directory of Open Access Journals (Sweden)

    Maria A. eLandin


    Full Text Available The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the eleventh embryonic day (E11.5 up to the seventh post-natal day (P7. The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p<0.05 were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the elleventh embryonic day (E11.5 up to seven days post-natal (P7, 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure.Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5 studied were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0- P7 were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3, metallothionein 1(Mt1, cyclin-dependent kinase 4 (Cdk4, cathepsin D (Ctsd, keratin complex 2, basic, gene 6a (Krt2-6a, cofilin 1, non-muscle (Cfl1, cyclin 2 (Ccnd2, were verified by real-time RT-PCR.

  2. Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements.

    Directory of Open Access Journals (Sweden)

    Elina eWelchen


    Full Text Available Plant mitochondria are complex organelles that carry out numerous metabolic processes related with the generation of energy for cellular functions and the synthesis and degradation of several compounds. Mitochondria are semiautonomous and dynamic organelles changing in shape, number and composition depending on tissue or developmental stage. The biogenesis of functional mitochondria requires the coordination of genes present both in the nucleus and the organelle. In addition, due to their central role, all processes held inside mitochondria must be finely coordinated with those in other organelles according to cellular demands. Coordination is achieved by transcriptional control of nuclear genes encoding mitochondrial proteins by specific transcription factors that recognize conserved elements in their promoter regions. In turn, the expression of most of these transcription factors is linked to developmental and environmental cues, according to the availability of nutrients, light-dark cycles and warning signals generated in response to stress conditions. Among the signals impacting in the expression of nuclear genes, retrograde signals that originate inside mitochondria help to adjust mitochondrial biogenesis to organelle demands. Adding more complexity, several nuclear encoded proteins are dual localized to mitochondria and either chloroplasts or the nucleus. Dual targeting might establish a crosstalk between the nucleus and cell organelles to ensure a fine coordination of cellular activities. In this article, we discuss how the different levels of coordination of mitochondrial biogenesis interconnect to optimize the function of the organelle according to both internal and external demands.

  3. Fabrication of Biocompatible, Vibrational Magnetoelastic Materials for Controlling Cellular Adhesion

    Directory of Open Access Journals (Sweden)

    Rupak M. Rajachar


    Full Text Available This paper describes the functionalization of magnetoelastic (ME materials with Parylene-C coating to improve the surface reactivity to cellular response. Previous study has demonstrated that vibrating ME materials were capable of modulating cellular adhesion when activated by an externally applied AC magnetic field. However, since ME materials are not inherently biocompatible, surface modifications are needed for their implementation in biological settings. Here, the long-term stability of the ME material in an aqueous and biological environment is achieved by chemical-vapor deposition of a conformal Parylene-C layer, and further functionalized by methods of oxygen plasma etching and protein adsorption. In vitro cytotoxicity measurement and characterization of the vibrational behavior of the ME materials showed that Parylene-C coatings of 10 µm or greater could prevent hydrolytic degradation without sacrificing the vibrational behavior of the ME material. This work allows for long-term durability and functionality of ME materials in an aqueous and biological environment and makes the potential use of this technology in monitoring and modulating cellular behavior at the surface of implantable devices feasible.

  4. Expression and cellular localization of the Mas receptor in the adult and developing mouse retina. (United States)

    Prasad, Tuhina; Verma, Amrisha; Li, Qiuhong


    Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor mRNA

  5. Comparison of nonsense-mediated mRNA decay efficiency in various murine tissues

    Directory of Open Access Journals (Sweden)

    Mazoyer Sylvie


    Full Text Available Abstract Background The Nonsense-Mediated mRNA Decay (NMD pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type Men1 transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed Men1 gene. Results Significant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type Men1 transcripts, identical to the value measured in murine embryonic fibroblasts. The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%. Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the Men1 gene. Conclusion Mouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.

  6. Universal map for cellular automata

    Energy Technology Data Exchange (ETDEWEB)

    García-Morales, V., E-mail: [Institute for Advanced Study – Technische Universität München, Lichtenbergstr. 2a, D-85748 Garching (Germany)


    A universal map is derived for all deterministic 1D cellular automata (CAs) containing no freely adjustable parameters and valid for any alphabet size and any neighborhood range (including non-symmetrical neighborhoods). The map can be extended to an arbitrary number of dimensions and topologies and to arbitrary order in time. Specific CA maps for the famous Conway's Game of Life and Wolfram's 256 elementary CAs are given. An induction method for CAs, based in the universal map, allows mathematical expressions for the orbits of a wide variety of elementary CAs to be systematically derived. -- Highlights: ► A universal map is derived for all deterministic 1D cellular automata (CA). ► The map is generalized to 2D for Von Neumann, Moore and hexagonal neighborhoods. ► A map for all Wolfram's 256 elementary CAs is derived. ► A map for Conway's “Game of Life” is obtained.

  7. Melanoma screening with cellular phones.

    Directory of Open Access Journals (Sweden)

    Cesare Massone

    Full Text Available BACKGROUND: Mobile teledermatology has recently been shown to be suitable for teledermatology despite limitations in image definition in preliminary studies. The unique aspect of mobile teledermatology is that this system represents a filtering or triage system, allowing a sensitive approach for the management of patients with emergent skin diseases. METHODOLOGY/PRINCIPAL FINDINGS: In this study we investigated the feasibility of teleconsultation using a new generation of cellular phones in pigmented skin lesions. 18 patients were selected consecutively in the Pigmented Skin Lesions Clinic of the Department of Dermatology, Medical University of Graz, Graz (Austria. Clinical and dermoscopic images were acquired using a Sony Ericsson with a built-in two-megapixel camera. Two teleconsultants reviewed the images on a specific web application ( where images had been uploaded in JPEG format. Compared to the face-to-face diagnoses, the two teleconsultants obtained a score of correct telediagnoses of 89% and of 91.5% reporting the clinical and dermoscopic images, respectively. CONCLUSIONS/SIGNIFICANCE: The present work is the first study performing mobile teledermoscopy using cellular phones. Mobile teledermatology has the potential to become an easy applicable tool for everyone and a new approach for enhanced self-monitoring for skin cancer screening in the spirit of the eHealth program of the European Commission Information for Society and Media.

  8. Melanoma screening with cellular phones. (United States)

    Massone, Cesare; Hofmann-Wellenhof, Rainer; Ahlgrimm-Siess, Verena; Gabler, Gerald; Ebner, Christoph; Soyer, H Peter


    Mobile teledermatology has recently been shown to be suitable for teledermatology despite limitations in image definition in preliminary studies. The unique aspect of mobile teledermatology is that this system represents a filtering or triage system, allowing a sensitive approach for the management of patients with emergent skin diseases. In this study we investigated the feasibility of teleconsultation using a new generation of cellular phones in pigmented skin lesions. 18 patients were selected consecutively in the Pigmented Skin Lesions Clinic of the Department of Dermatology, Medical University of Graz, Graz (Austria). Clinical and dermoscopic images were acquired using a Sony Ericsson with a built-in two-megapixel camera. Two teleconsultants reviewed the images on a specific web application ( where images had been uploaded in JPEG format. Compared to the face-to-face diagnoses, the two teleconsultants obtained a score of correct telediagnoses of 89% and of 91.5% reporting the clinical and dermoscopic images, respectively. The present work is the first study performing mobile teledermoscopy using cellular phones. Mobile teledermatology has the potential to become an easy applicable tool for everyone and a new approach for enhanced self-monitoring for skin cancer screening in the spirit of the eHealth program of the European Commission Information for Society and Media.

  9. Cellular dynamics and embryonic morphogenesis (United States)

    Zallen, Jennifer


    The elongated body axis is a characteristic feature of many multicellular animals. Axis elongation occurs largely through cell rearrangements that are coordinated across a large cell population and driven by an asymmetric distribution of cytoskeletal and junctional proteins [1]. To visualize cellular dynamics during this process, we performed time-lapse confocal imaging of cell behavior in the Drosophila embryo. These studies revealed that rearranging cells display a steady increase in topological disorder that is accompanied by the formation of transient structures where 5-11 cells meet [2,3]. These multicellular rosettes form and resolve in a directional fashion to produce a local change in the aspect ratio of the cellular assembly, contributing to an overall change in tissue structure. We propose that higher-order rosette structures link local cell interactions to global tissue reorganization during morphogenesis. [1] J. Zallen and E. Wieschaus, Developmental Cell 6, 343 (2004). [2] J. Zallen and R. Zallen, J. Phys.: Condens. Matter 16, S5073 (2004). [3] J. Blankenship et al., Developmental Cell 11, 459 (2006).

  10. Cellular Therapy for Heart Failure (United States)

    Psaltis, Peter J.; Schwarz, Nisha; Toledo-Flores, Deborah; Nicholls, Stephen J.


    The pathogenesis of cardiomyopathy and heart failure (HF) is underpinned by complex changes at subcellular, cellular and extracellular levels in the ventricular myocardium. For all of the gains that conventional treatments for HF have brought to mortality and morbidity, they do not adequately address the loss of cardiomyocyte numbers in the remodeling ventricle. Originally conceived to address this problem, cellular transplantation for HF has already gone through several stages of evolution over the past two decades. Various cell types and delivery routes have been implemented to positive effect in preclinical models of ischemic and nonischemic cardiomyopathy, with pleiotropic benefits observed in terms of myocardial remodeling, systolic and diastolic performance, perfusion, fibrosis, inflammation, metabolism and electrophysiology. To a large extent, these salubrious effects are now attributed to the indirect, paracrine capacity of transplanted stem cells to facilitate endogenous cardiac repair processes. Promising results have also followed in early phase human studies, although these have been relatively modest and somewhat inconsistent. This review details the preclinical and clinical evidence currently available regarding the use of pluripotent stem cells and adult-derived progenitor cells for cardiomyopathy and HF. It outlines the important lessons that have been learned to this point in time, and balances the promise of this exciting field against the key challenges and questions that still need to be addressed at all levels of research, to ensure that cell therapy realizes its full potential by adding to the armamentarium of HF management. PMID:27280304

  11. Discrepancy between mRNA and protein abundance: insight from information retrieval process in computers. (United States)

    Wang, Degeng


    Discrepancy between the abundance of cognate protein and RNA molecules is frequently observed. A theoretical understanding of this discrepancy remains elusive, and it is frequently described as surprises and/or technical difficulties in the literature. Protein and RNA represent different steps of the multi-stepped cellular genetic information flow process, in which they are dynamically produced and degraded. This paper explores a comparison with a similar process in computers-multi-step information flow from storage level to the execution level. Functional similarities can be found in almost every facet of the retrieval process. Firstly, common architecture is shared, as the ribonome (RNA space) and the proteome (protein space) are functionally similar to the computer primary memory and the computer cache memory, respectively. Secondly, the retrieval process functions, in both systems, to support the operation of dynamic networks-biochemical regulatory networks in cells and, in computers, the virtual networks (of CPU instructions) that the CPU travels through while executing computer programs. Moreover, many regulatory techniques are implemented in computers at each step of the information retrieval process, with a goal of optimizing system performance. Cellular counterparts can be easily identified for these regulatory techniques. In other words, this comparative study attempted to utilize theoretical insight from computer system design principles as catalysis to sketch an integrative view of the gene expression process, that is, how it functions to ensure efficient operation of the overall cellular regulatory network. In context of this bird's-eye view, discrepancy between protein and RNA abundance became a logical observation one would expect. It was suggested that this discrepancy, when interpreted in the context of system operation, serves as a potential source of information to decipher regulatory logics underneath biochemical network operation.

  12. Cellular Response to Reagent-Free Electron-Irradiated Gelatin Hydrogels. (United States)

    Wisotzki, Emilia I; Friedrich, Ralf P; Weidt, Astrid; Alexiou, Christoph; Mayr, Stefan G; Zink, Mareike


    As a biomaterial, it is well established that gelatin exhibits low cytotoxicity and can promote cellular growth. However, to circumvent the potential toxicity of chemical crosslinkers, reagent-free crosslinking methods such as electron irradiation are highly desirable. While high energy irradiation has been shown to exhibit precise control over the degree of crosslinking, these hydrogels have not been thoroughly investigated for biocompatibility and degradability. Here, NIH 3T3 murine fibroblasts are seeded onto irradiated gelatin hydrogels to examine the hydrogel's influence on cellular viability and morphology. The average projected area of cells seeded onto the hydrogels increases with irradiation dose, which correlates with an increase in the hydrogel's shear modulus up to 10 kPa. Cells on these hydrogels are highly viable and exhibits normal cell cycles, particularly when compared to those grown on glutaraldehyde crosslinked gelatin hydrogels. However, proliferation is reduced on both types of crosslinked samples. To mimic the response of the hydrogels in physiological conditions, degradability is monitored in simulated body fluid to reveal strongly dose-dependent degradation times. Overall, given the low cytotoxicity, influence on cellular morphology and variability in degradation times of the electron irradiated gelatin hydrogels, there is significant potential for application in areas ranging from regenerative medicine to mechanobiology. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Stochastic fluctuations and distributed control of gene expression impact cellular memory.

    Directory of Open Access Journals (Sweden)

    Guillaume Corre

    Full Text Available Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.

  14. Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II) in Erlang Mountainous Chickens. (United States)

    Yin, H D; Tian, K; Li, D Y; Gilbert, E R; Xiao, L H; Chen, S Y; Wang, Y; Liu, Y P; Zhao, X L; Zhu, Q


    Cellular retinol-binding protein II (CRBP II) belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05) in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05) in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.

  15. Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II in Erlang Mountainous Chickens

    Directory of Open Access Journals (Sweden)

    H. D. Yin


    Full Text Available Cellular retinol-binding protein II (CRBP II belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05 in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05 in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.

  16. Elevated levels of transferrin receptor 2 mRNA, not transferrin receptor 1 mRNA, are associated with increased survival in acute myeloid leukaemia. (United States)

    Nakamaki, Tsuyoshi; Kawabata, Hiroshi; Saito, Bungo; Matsunawa, Manabu; Suzuki, Junko; Adachi, Daisuke; Tomoyasu, Shigeru; Phillip Koeffler, H


    Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-alpha (membrane form) and TfR2-beta (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-alpha (r = 0.771, P TfR2-beta (r = 0.534, P TfR2-alpha (r = -0.486, P TfR2-beta (r = -0.435, P = 0.0003). Only TfR2 expression was significantly associated with either serum iron (r = -0.270, P = 0.045) or serum ferritin (r = -0.364, P = 0.008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-alpha, but not TfR1 or TfR2-beta mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-alpha and TfR2-beta survived significantly longer than a group without high expression of both of them (P TfR2 (especially the alpha form) is a novel prognostic factor for patients with AML.

  17. mRIN for direct assessment of genome-wide and gene-specific mRNA integrity from large-scale RNA-sequencing data. (United States)

    Feng, Huijuan; Zhang, Xuegong; Zhang, Chaolin


    The volume of RNA-Seq data sets in public repositories has been expanding exponentially, providing unprecedented opportunities to study gene expression regulation. Because degraded RNA samples, such as those collected from post-mortem tissues, can result in distinct expression profiles with potential biases, a particularly important step in mining these data is quality control. Here we develop a method named mRIN to directly assess mRNA integrity from RNA-Seq data at the sample and individual gene level. We systematically analyse large-scale RNA-Seq data sets of the human brain transcriptome generated by different consortia. Our analysis demonstrates that 3' bias resulting from partial RNA fragmentation in post-mortem tissues has a marked impact on global expression profiles, and that mRIN effectively identifies samples with different levels of mRNA degradation. Unexpectedly, this process has a reproducible and gene-specific component, and transcripts with different stabilities are associated with distinct functions and structural features reminiscent of mRNA decay in living cells.

  18. The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

    Directory of Open Access Journals (Sweden)

    John M Morrison


    Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

  19. Proteasomal degradation of TRIM5alpha during retrovirus restriction.

    Directory of Open Access Journals (Sweden)

    Christopher James Rold


    Full Text Available The host protein TRIM5alpha inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5alpha. Here, we show that TRIM5alpha is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5alpha-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5alpha protein but not the nonrestrictive human TRIM5alpha protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5alpha was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5alpha and TRIMCyp proteins. We also detected degradation of endogenous TRIM5alpha in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5alpha degradation by a proteasome-dependent mechanism.

  20. The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability. (United States)

    Morais, Denis R; Reis, Sabrina T; Viana, Nayara; Piantino, Camila Berfort; Massoco, Cristina; Moura, Caio; Dip, Nelson; Silva, Iran A; Srougi, Miguel; Leite, Katia Rm


    MicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy. The bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma. miR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use

  1. Degradation of copepod fecal pellets

    DEFF Research Database (Denmark)

    Poulsen, Louise K.; Iversen, Morten


    amount of fecal pellets. The total degradation rate of pellets by the natural plankton community of Oresund followed the phytoplankton biomass, with maximum degradation rate during the spring bloom (2.5 +/- 0.49 d(-1)) and minimum (0.52 +/- 0.14 d(-1)) during late winter. Total pellet removal rate ranged...

  2. Live Imaging of mRNA Synthesis in Drosophila. (United States)

    Garcia, Hernan G; Gregor, Thomas


    mRNA synthesis is one of the earliest readouts of the activity of a transcribed gene, which is of particular interest in the context of metazoan cell fate specification. These processes are intrinsically dynamic and stochastic, which makes in vivo single-cell measurements inevitable. Here, we present the application of a technology that has been widely used in single celled organisms to measure transcriptional activity in developing embryos of the fruit fly Drosophila melanogaster. The method allows for quantification of instantaneous polymerase occupancy of active gene loci and thereby enables the development and testing of models of gene regulation in development.

  3. mRNA Transcript abundance during plant growth and the influence of Li(+) exposure. (United States)

    Duff, M C; Kuhne, W W; Halverson, N V; Chang, C-S; Kitamura, E; Hawthorn, L; Martinez, N E; Stafford, C; Milliken, C E; Caldwell, E F; Stieve-Caldwell, E


    Lithium (Li) toxicity in plants is, at a minimum, a function of Li(+) concentration, exposure time, species and growth conditions. Most plant studies with Li(+) focus on short-term acute exposures. This study examines short- and long-term effects of Li(+) exposure in Arabidopsis with Li(+) uptake studies and measured shoot mRNA transcript abundance levels in treated and control plants. Stress, pathogen-response and arabinogalactan protein genes were typically more up-regulated in older (chronic, low level) Li(+)-treatment plants and in the much younger plants from acute high-level exposures. The gene regulation behavior of high-level Li(+) resembled prior studies due to its influence on: inositol synthesis, 1-aminocyclopropane-1-carboxylate synthases and membrane ion transport. In contrast, chronically-exposed plants had gene regulation responses that were indicative of pathogen, cold, and heavy-metal stress, cell wall degradation, ethylene production, signal transduction, and calcium-release modulation. Acute Li(+) exposure phenocopies magnesium-deficiency symptoms and is associated with elevated expression of stress response genes that could lead to consumption of metabolic and transcriptional energy reserves and the dedication of more resources to cell development. In contrast, chronic Li(+) exposure increases expression signal transduction genes. The identification of new Li(+)-sensitive genes and a gene-based "response plan" for acute and chronic Li(+) exposure are delineated. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Q&A: Trash talk: disposal and remote degradation of neuronal garbage. (United States)

    Arnold, Meghan Lee; Melentijevic, Ilija; Smart, Anna Joelle; Driscoll, Monica


    Caenorhabditis elegans neurons have recently been found to throw out cellular debris for remote degradation and/or storage, adding an "extracellular garbage elimination" option to known intracellular protein and organelle degradation pathways. This Q&A describes initial insights into the biology of seemingly selective protein and organelle elimination by challenged neurons, highlighting mysteries of how garbage is distinguished and sorted in the sending neuron, how the garbage-filled "exophers" appear to elicit degradative responses as they transit neighboring tissue, and how non-digestible materials get thrown out of cells again via processes that may be highly relevant to human neurodegenerative disease mechanisms.

  5. Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential (United States)

    Boros, Gábor; Miko, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Georgina; Juhász, Attila; Juhász, István; van der Horst, Gijsbertus; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella


    UVB irradiation induces harmful photochemical reactions, including formation of cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine. PMID:24211294

  6. Molecular, cellular, and tissue engineering

    CERN Document Server

    Bronzino, Joseph D


    Known as the bible of biomedical engineering, The Biomedical Engineering Handbook, Fourth Edition, sets the standard against which all other references of this nature are measured. As such, it has served as a major resource for both skilled professionals and novices to biomedical engineering. Molecular, Cellular, and Tissue Engineering, the fourth volume of the handbook, presents material from respected scientists with diverse backgrounds in molecular biology, transport phenomena, physiological modeling, tissue engineering, stem cells, drug delivery systems, artificial organs, and personalized medicine. More than three dozen specific topics are examined, including DNA vaccines, biomimetic systems, cardiovascular dynamics, biomaterial scaffolds, cell mechanobiology, synthetic biomaterials, pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, nanobiomaterials for tissue engineering, biomedical imaging of engineered tissues, gene therapy, noninvasive targeted protein and peptide drug deliver...

  7. Thermomechanical characterisation of cellular rubber (United States)

    Seibert, H.; Scheffer, T.; Diebels, S.


    This contribution discusses an experimental possibility to characterise a cellular rubber in terms of the influence of multiaxiality, rate dependency under environmental temperature and its behaviour under hydrostatic pressure. In this context, a mixed open and closed cell rubber based on an ethylene propylene diene monomer is investigated exemplarily. The present article intends to give a general idea of the characterisation method and the considerable effects of this special type of material. The main focus lies on the experimental procedure and the used testing devices in combination with the analysis methods such as true three-dimensional digital image correlation. The structural compressibility is taken into account by an approach for a material model using the Theory of Porous Media with additional temperature dependence.

  8. Novel Materials for Cellular Nanosensors

    DEFF Research Database (Denmark)

    Sasso, Luigi

    without or with poor surface conductivity, providing a patternable conducting polymer deposition technique integrated with standard microfabrication techniques. Electropolymerization of pyrrole on planar interdigitated electrodes resulted in the creation of doped conducting polymer films. Different....... An in vivo investigation also gave evidence of how the peptide nanowires can be used as surface modification in implantable electrodes for neurological measurements. Conducting polymers were utilized in electrode modifications for electrochemical sensor surfaces. Both chemical and electrochemical deposition...... methods were used to optimize the polymer film with respect to sensitivity towards cellular analytes, each method chosen accordingly to specific electrode geometry and shape. Chemical polymerization of pyrrole was used to achieve conductive polymer film coatings for out-of-plane electrode structures...


    Barron, E. S. Guzman; Nelson, Leonard; Ardao, Maria Isabel


    Oxidizing agents of sulfhydryl groups such as iodosobenzoate, alkylating agents such as iodoacetamide, and mercaptide-forming agents such as cadmium chloride, mercuric chloride, p-chloromercuribenzoate, sodium arsenite, and p-carboxyphenylarsine oxide, added in small concentrations to a suspension of sea urchin sperm produced an increase in respiration. When the concentration was increased there was an inhibition. These effects are explained by postulating the presence in the cells of two kinds of sulfhydryl groups: soluble sulfhydryl groups, which regulate cellular respiration, and fixed sulfhydryl groups, present in the protein moiety of enzymes. Small concentrations of sulfhydryl reagents combine only with the first, thus producing an increase in respiration; when the concentration is increased, the fixed sulfhydryl groups are also attacked and inhibition of respiration is the consequence. Other inhibitors of cell respiration, such as cyanide and urethanes, which do not combine with —SH groups, did not stimulate respiration in small concentration. PMID:18891144

  10. Discrete geodesics and cellular automata

    CERN Document Server

    Arrighi, Pablo


    This paper proposes a dynamical notion of discrete geodesics, understood as straightest trajectories in discretized curved spacetime. The notion is generic, as it is formulated in terms of a general deviation function, but readily specializes to metric spaces such as discretized pseudo-riemannian manifolds. It is effective: an algorithm for computing these geodesics naturally follows, which allows numerical validation---as shown by computing the perihelion shift of a Mercury-like planet. It is consistent, in the continuum limit, with the standard notion of timelike geodesics in a pseudo-riemannian manifold. Whether the algorithm fits within the framework of cellular automata is discussed at length. KEYWORDS: Discrete connection, parallel transport, general relativity, Regge calculus.

  11. Cellular compartmentalization of secondary metabolism

    Directory of Open Access Journals (Sweden)

    H. Corby eKistler


    Full Text Available Fungal secondary metabolism is often considered apart from the essential housekeeping functions of the cell. However, there are clear links between fundamental cellular metabolism and the biochemical pathways leading to secondary metabolite synthesis. Besides utilizing key biochemical precursors shared with the most essential processes of the cell (e.g. amino acids, acetyl CoA, NADPH, enzymes for secondary metabolite synthesis are compartmentalized at conserved subcellular sites that position pathway enzymes to use these common biochemical precursors. Co-compartmentalization of secondary metabolism pathway enzymes also may function to channel precursors, promote pathway efficiency and sequester pathway intermediates and products from the rest of the cell. In this review we discuss the compartmentalization of three well-studied fungal secondary metabolite biosynthetic pathways for penicillin G, aflatoxin and deoxynivalenol, and summarize evidence used to infer subcellular localization. We also discuss how these metabolites potentially are trafficked within the cell and may be exported.

  12. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min


    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  13. Expression of type XXIII collagen mRNA and protein. (United States)

    Koch, Manuel; Veit, Guido; Stricker, Sigmar; Bhatt, Pinaki; Kutsch, Stefanie; Zhou, Peihong; Reinders, Elina; Hahn, Rita A; Song, Rich; Burgeson, Robert E; Gerecke, Donald R; Mundlos, Stefan; Gordon, Marion K


    Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.

  14. Drift Degradation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Dwayne C. Kicker


    A statistical description of the probable block sizes formed by fractures around the emplacement drifts has been developed for each of the lithologic units of the repository host horizon. A range of drift orientations with the drift azimuth varied in 15{sup o} increments has been considered in the static analysis. For the quasi-static seismic analysis, and the time-dependent and thermal effects analysis, two drift orientations have been considered: a drift azimuth of 105{sup o} and the current emplacement drift azimuth of 75{sup o}. The change in drift profile resulting from progressive deterioration of the emplacement drifts has been assessed both with and without backfill. Drift profiles have been determined for four different time increments, including static (i.e., upon excavation), 200 years, 2,000 years, and 10,000 years. The effect of seismic events on rock fall has been analyzed. Block size distributions and drift profiles have been determined for three seismic levels, including a 1,000-year event, a 5,000-year event, and a 10,000-year event. Data developed in this modeling and analysis activity have been entered into the TDMS (DTN: MO0109RDDAAMRR.003). The following conclusions have resulted from this drift degradation analysis: (1) The available fracture data are suitable for supporting a detailed key block analysis of the repository host horizon rock mass. The available data from the north-south Main Drift and the east-west Cross Drift provide a sufficient representative fracture sample of the repository emplacement drift horizon. However, the Tptpln fracture data are only available from a relatively small section of the Cross Drift, resulting in a smaller fracture sample size compared to the other lithologic units. This results in a lower degree of confidence that the key block data based on the Tptpln data set is actually representative of the overall Tptpln key block population. (2) The seismic effect on the rock fall size distribution for all events

  15. Posttranscriptional regulation of cellular gene expression by the c-myc oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, G.C.; Cole, M.D. (Princeton Univ., NJ (USA). Dept. of Biology)


    The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. The authors used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G/sub o/ fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. Their results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.

  16. Convergent synthesis of degradable dendrons based on L-malic acid

    DEFF Research Database (Denmark)

    Meyhoff, Ulrich; Riber, Ulla; Boas, Ulrik


    New degradable polyester dendrons based on the cellular tricarboxylic acid cycle component L-malic acid were synthesized up to the third generation by convergent synthesis. The dendron wedges could be introduced in a stepwise, highly regioselective fashion. HMBC-NMR revealed that the C1-carbonyl...... on malic acid was exclusively esterified, before the reaction of the second dendron wedge at C4 took place. Degradation studies on a first generation dendron analyzed by HPLC showed that hydrolytic degradation of the dendron most profoundly takes place at pH 4 and pH 9 with the highest degradation rate...... at alkaline pH. NMR shows that the dendron degrades to malic acid and fumaric acid derivatives. Preliminary studies performed in the cell culture show low toxicity of the dendrons in concentrations of up to 50 μg mL-1....

  17. Anaerobic benzene degradation by bacteria (United States)

    Vogt, Carsten; Kleinsteuber, Sabine; Richnow, Hans‐Hermann


    Summary Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation. PMID:21450012

  18. Ascorbic acid induced atrazine degradation. (United States)

    Hou, Xiaojing; Huang, Xiaopeng; Ai, Zhihui; Zhao, Jincai; Zhang, Lizhi


    In this study, we systematically investigated the degradation efficiency and the degradation mechanism of atrazine in the presence of ascorbic acid at different pH values. Although atrazine could be degraded by ascorbic acid in a wide pH range from 4 to 12, its degradation under either acidic (pH≤4) or alkaline (pH≥12) condition was more efficient than under neutral condition (pH=7). This pH dependent atrazine degradation was related to the reactive characteristic of atrazine and the reductive activity of ascorbic acid. The ascorbic acid induced atrazine degradation pathways at different pH were investigated by comparing the atrazine degradation intermediates with liquid chromatography-mass spectrometry, high performance liquid chromatography and ion chromatography. It was found that more products were detected in presence of ascorbic acid at alkaline condition. The appearance of chloride ions confirmed the dechlorination of atrazine by ascorbic acid in the absence of molecular oxygen, while its dechlorination efficiency reached highest at pH 12. These results can shed light on the application of AA for the organic pollutant remediation. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. The RNA-binding protein KSRP promotes decay of beta-catenin mRNA and is inactivated by PI3K-AKT signaling

    DEFF Research Database (Denmark)

    Gherzi, Roberto; Trabucchi, Michele; Ponassi, Marco


    Beta-catenin plays an essential role in several biological events including cell fate determination, cell proliferation, and transformation. Here we report that beta-catenin is encoded by a labile transcript whose half-life is prolonged by Wnt and phosphatidylinositol 3-kinase-AKT signaling. AKT...... phosphorylates the mRNA decay-promoting factor KSRP at a unique serine residue, induces its association with the multifunctional protein 14-3-3, and prevents KSRP interaction with the exoribonucleolytic complex exosome. This impairs KSRP's ability to promote rapid mRNA decay. Our results uncover an unanticipated...... level of control of beta-catenin expression pointing to KSRP as a required factor to ensure rapid degradation of beta-catenin in unstimulated cells. We propose KSRP phosphorylation as a link between phosphatidylinositol 3-kinase-AKT signaling and beta-catenin accumulation....


    Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A


    A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Modifications of RNA polymerase II CTD: Connections to the histone code and cellular function. (United States)

    Srivastava, Rakesh; Ahn, Seong Hoon


    At the onset of transcription, many protein machineries interpret the cellular signals that regulate gene expression. These complex signals are mostly transmitted to the indispensable primary proteins involved in transcription, RNA polymerase II (RNAPII) and histones. RNAPII and histones are so well coordinated in this cellular function that each cellular signal is precisely allocated to specific machinery depending on the stage of transcription. The carboxy-terminal domain (CTD) of RNAPII in eukaryotes undergoes extensive posttranslational modification, called the 'CTD code', that is indispensable for coupling transcription with many cellular processes, including mRNA processing. The posttranslational modification of histones, known as the 'histone code', is also critical for gene transcription through the reversible and dynamic remodeling of chromatin structure. Notably, the histone code is closely linked with the CTD code, and their combinatorial effects enable the delicate regulation of gene transcription. This review elucidates recent findings regarding the CTD modifications of RNAPII and their coordination with the histone code, providing integrative pathways for the fine-tuned regulation of gene expression and cellular function. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Biochemical characterization of the RNA helicase UPF1 involved in nonsense-mediated mRNA decay. (United States)

    Fiorini, Francesca; Bonneau, Fabien; Le Hir, Hervé


    Degradation of eukaryotic mRNAs harboring a premature translation termination codon is ensured by the process of nonsense-mediated mRNA decay (NMD). The main effector of this quality-control pathway is the conserved RNA helicase UPF1 that forms a surveillance complex with the proteins UPF2 and UPF3. In all the organisms tested, the ATPase activity of UPF1 is essential for NMD. Here, we describe the expression of active recombinant UPF proteins and the reconstitution of the surveillance complex in vitro. To understand how UPF1 is regulated during NMD, we developed different biochemical approaches. We describe methods to monitor UPF1 binding to RNA, ATP hydrolysis and RNA unwinding in the presence of its binding partner UPF2. This functional analysis is an important complement for structural studies of protein complexes containing RNA helicases. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Pseudomonas kuykendallii sp. nov.: A novel y-Proteobacteria isolated from a hexazinone degrading bioreactor (United States)

    Three strains of Gram-negative bacteria designated strains H2T, H6, and H7 were isolated from bioreactors that degraded the herbicide hexazinone. Similar morphological characteristics, cellular fatty acid profiles and 16S rRNA gene sequences show that the isolates are members of the same species. ...

  4. Performance Degradation of LSCF Cathodes

    Energy Technology Data Exchange (ETDEWEB)

    Alinger, Matthew


    This final report summarizes the progress made during the October 1, 2008 - September 30, 2013 period under Cooperative Agreement DE-NT0004109 for the U. S. Department of Energy/National Energy Technology Laboratory (USDOE/NETL) entitled “Performance Degradation of LSCF Cathodes”. The primary objective of this program is to develop a performance degradation mitigation path for high performing, cost-effective solid oxide fuel cells (SOFCs). Strategies to mitigate performance degradation are developed and implemented. In addition, thermal spray manufacturing of SOFCs is explored. Combined, this work establishes a basis for cost-effective SOFC cells.

  5. LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

    Directory of Open Access Journals (Sweden)

    Donald B Bloch

    Full Text Available The mRNA processing body (P-body is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB, the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

  6. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

    Directory of Open Access Journals (Sweden)

    Lee Jee


    Full Text Available Abstract Background The peroxisome proliferator-activated receptor (PPAR-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA, is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. Methods To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1 were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. Results We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK phosphorylation and activator protein 1 (AP1 activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. Conclusion ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism

  7. Effect of chronic ethanol feeding on digestive enzyme synthesis and mRNA content in rat pancreas. (United States)

    Perkins, P S; Rutherford, R E; Pandol, S J


    Chronic ethanol ingestion is a primary factor in the development of pancreatitis in humans. Alterations in both enzyme secretion and protein synthesis have been implicated as causative factors. We determined the effect of chronic ethanol feeding on the content and synthesis rates of digestive enzymes in dispersed acini from rats that were pair-fed isocaloric diets with or without ethanol for 3-6 months. Total protein content and synthesis were unchanged. The relative synthetic rates of individual digestive enzymes were analyzed using scanning laser densitometry of 1-D sodium dodecylsulfate (SDS) gels. The content of all measurable digestive enzymes except amylase increased in acini from ethanol-fed rats. Relative synthetic rates were examined in pancreatic acini labeled in vitro with [35S]methionine. Liquid scintillation counting of radiolabeled digestive enzymes extracted from gel slices revealed that amylase synthesis in ethanol-fed rats decreased 2.8-fold compared with controls whereas the synthetic rates of proelastase 1 and 2, chymotrypsinogen, and trypsinogen increased by 1.5-, 1.4-, 1.8-, and 1.6-fold, respectively. Total cellular RNA was extracted from control and ethanol-fed rats and subjected to Northern and dot blot analysis. Amylase mRNA decreased in ethanol-fed rats whereas chymotrypsinogen and trypsinogen mRNA content increased, indicating that the effect of ethanol on expression of these genes was regulated at a step prior to translation. Elastase mRNA content was not altered, suggesting that the increased synthesis of proelastase may be regulated posttranscriptionally.

  8. Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA splicing.

    Directory of Open Access Journals (Sweden)

    Guillaume Fournier


    Full Text Available Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.

  9. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Bloch, Donald B., E-mail:; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong


    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  10. mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration

    Directory of Open Access Journals (Sweden)

    Thomas C. Roberts


    Full Text Available Duchenne muscular dystrophy (DMD is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1 the determination of gene expression changes associated with dystrophic pathology, (2 identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3 investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis, and (4 prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO with the accession number GSE64420.

  11. On the cellular convexity of complexes. (United States)

    Kim, C E


    In this paper we discuss cellular convexity of complexes. A new definition of cellular convexity is given in terms of a geometric property. Then it is proven that a regular complex is celiularly convex if and only if there is a convex plane figure of which it is the cellular image. Hence, the definition of cellular convexity by Sklansky [7] is equivalent to the new definition for the case of regular complexes. The definition of Minsky and Papert [4] is shown to be equivalent to our definition. Therefore, aU definitions are virtually equivalent. It is shown that a regular complex is cellularly convex if and only if its minimum-perimeter polygon does not meet the boundary of the complex. A 0(n) time algorithm is presented to determine the cellular convexity of a complex when it resides in n × m cells and is represented by the run length code.

  12. Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts. (United States)

    Honorine, Romy; Mosrin-Huaman, Christine; Hervouet-Coste, Nadège; Libri, Domenico; Rahmouni, A Rachid


    The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery.

  13. Interaction of human decapping scavenger with 5' mRNA cap analogues: structural requirements for catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Zbigniew M [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Bojarska, Elzbieta [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kowalska, Joanna [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Lewdorowicz, Magdalena [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Jemielity, Jacek [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kalek, Marcin [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Stepinski, Janusz [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Davis, Richard E [Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 12801 East 17th Avenue, Aurora, CO 80045 (United States); Darzynkiewicz, Edward [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland)


    The cap structure is a specific feature of the 5' end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m{sup 7}GpppN and/or short oligonucleotides after the 3' to 5' exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m{sup 7}GMP, m{sup 7}GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (K{sub as}) and corresponding Gibbs free energies ({delta}G{sup 0}) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.

  14. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. (United States)

    Schwartz, Schraga; Mumbach, Maxwell R; Jovanovic, Marko; Wang, Tim; Maciag, Karolina; Bushkin, G Guy; Mertins, Philipp; Ter-Ovanesyan, Dmitry; Habib, Naomi; Cacchiarelli, Davide; Sanjana, Neville E; Freinkman, Elizaveta; Pacold, Michael E; Satija, Rahul; Mikkelsen, Tarjei S; Hacohen, Nir; Zhang, Feng; Carr, Steven A; Lander, Eric S; Regev, Aviv


    N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  15. RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

    Directory of Open Access Journals (Sweden)

    Mrinmoyee Majumder


    Full Text Available RNA-binding proteins (RBP regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1, plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4 RNA structure within both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1 and the non-coding RNA Telomerase RNA Component (TERC, and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.

  16. Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor β-1 in a fasting-adapted mammal (United States)

    Martinez, Bridget; Soñanez-Organis, José G.; Vázquez-Medina, José Pablo; Viscarra, Jose A.; MacKenzie, Duncan S.; Crocker, Daniel E.; Ortiz, Rudy M.


    SUMMARY Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5–7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic–pituitary–thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism. PMID:24307712

  17. Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor β-1 in a fasting-adapted mammal. (United States)

    Martinez, Bridget; Soñanez-Organis, José G; Vázquez-Medina, José Pablo; Viscarra, Jose A; MacKenzie, Duncan S; Crocker, Daniel E; Ortiz, Rudy M


    Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

  18. Cellular mechanism of metformin action. (United States)

    Grigorescu, F; Laurent, A; Chavanieu, A; Capony, J P


    Activation of the insulin receptor tyrosine kinase and tyrosine phosphorylation of intracellular substrates are important steps in insulin signalling. In order to elucidate the cellular mechanism of action of metformin (NN'dimethylbiguanide) we have focused towards the effects of metformin on the insulin receptor kinase, the phosphorylation cascade and the biological effect of insulin. Since annexins (lipocortins) have been recently recognized as substrates of several tyrosine kinases we have investigated the effect of metformin on phosphorylation of annexins after insulin stimulation or microinjection of pp60c-src kinase in Xenopus laevis oocytes. Insulin induced in oocytes progression through the cell cycle from late G2 to M phase (maturation). Microinjection of pp60c-src kinase or treatment with metformin potentiates both the rate and the level of insulin-induced oocyte maturation. In oocytes prelabeled with 32P orthophosphate metformin potentiates insulin induced phosphorylation of annexins. It is concluded that annexins are substrates of the phosphorylation cascade initiated by insulin which is synergistic to the action of pp60c-src kinase and that this early phosphorylation events correlate well with the enhanced biological effect of insulin during metformin treatment.

  19. Perfluorinated alginate for cellular encapsulation. (United States)

    Gattás-Asfura, Kerim M; Fraker, Christopher A; Stabler, Cherie L


    Molecules of pentadecafluorooctanoyl chloride (PFC) were grafted onto alginate (Alg) using a linear poly(ethylene glycol) linker and amide bonds. The resulting Alg-PFC material was characterized by proton nuclear magnetic resonance and infrared spectroscopies. The degree of PFC functionalization significantly influenced the physical and chemical properties of Alg-PFC, particularly when the resulting polymer was ionically crosslinked into hydrogels. Alg-PFC hydrogel beads fabricated via Ba(2+) crosslinking were found to match the permeability properties of control alginate beads, except upon swelling over time in culture media. When used to encapsulate MIN6 cells, a beta cell line, Alg-PFC beads demonstrated enhanced cell proliferation over alginate control beads. These results indicate that Alg-PFC hydrogels retain some of the PFC's biological-relevant benefits, such as enhancement of mass transport and bioinertness, to enhance cellular viability within alginate three-dimensional hydrogel environments. We envision these functionalized hydrogels to be particularly useful in the encapsulation of cells with a high metabolic demand, such as pancreatic islets. Copyright © 2012 Wiley Periodicals, Inc.

  20. Cellular Senescence: A Translational Perspective

    Directory of Open Access Journals (Sweden)

    James L. Kirkland


    Full Text Available Cellular senescence entails essentially irreversible replicative arrest, apoptosis resistance, and frequently acquisition of a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP. Senescent cells accumulate in various tissues with aging and at sites of pathogenesis in many chronic diseases and conditions. The SASP can contribute to senescence-related inflammation, metabolic dysregulation, stem cell dysfunction, aging phenotypes, chronic diseases, geriatric syndromes, and loss of resilience. Delaying senescent cell accumulation or reducing senescent cell burden is associated with delay, prevention, or alleviation of multiple senescence-associated conditions. We used a hypothesis-driven approach to discover pro-survival Senescent Cell Anti-apoptotic Pathways (SCAPs and, based on these SCAPs, the first senolytic agents, drugs that cause senescent cells to become susceptible to their own pro-apoptotic microenvironment. Several senolytic agents, which appear to alleviate multiple senescence-related phenotypes in pre-clinical models, are beginning the process of being translated into clinical interventions that could be transformative.

  1. Cellular Senescence: A Translational Perspective. (United States)

    Kirkland, James L; Tchkonia, Tamara


    Cellular senescence entails essentially irreversible replicative arrest, apoptosis resistance, and frequently acquisition of a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). Senescent cells accumulate in various tissues with aging and at sites of pathogenesis in many chronic diseases and conditions. The SASP can contribute to senescence-related inflammation, metabolic dysregulation, stem cell dysfunction, aging phenotypes, chronic diseases, geriatric syndromes, and loss of resilience. Delaying senescent cell accumulation or reducing senescent cell burden is associated with delay, prevention, or alleviation of multiple senescence-associated conditions. We used a hypothesis-driven approach to discover pro-survival Senescent Cell Anti-apoptotic Pathways (SCAPs) and, based on these SCAPs, the first senolytic agents, drugs that cause senescent cells to become susceptible to their own pro-apoptotic microenvironment. Several senolytic agents, which appear to alleviate multiple senescence-related phenotypes in pre-clinical models, are beginning the process of being translated into clinical interventions that could be transformative. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Chitin Degradation In Marine Bacteria

    DEFF Research Database (Denmark)

    Paulsen, Sara; Machado, Henrique; Gram, Lone


    Introduction: Chitin is the most abundant polymer in the marine environment and the second most abundant in nature. Chitin does not accumulate on the ocean floor, because of microbial breakdown. Chitin degrading bacteria could have potential in the utilization of chitin as a renewable carbon...... and nitrogen source in the fermentation industry.Methods: Here, whole genome sequenced marine bacteria were screened for chitin degradation using phenotypic and in silico analyses.Results: The in silico analyses revealed the presence of three to nine chitinases in each strain, however the number of chitinases...... chitin regulatory system.Conclusions: This study has provided insight into the ecology of chitin degradation in marine bacteria. It also served as a basis for choosing a more efficient chitin degrading production strain e.g. for the use of chitin waste for large-scale fermentations....

  3. Plant biomass degradation by fungi. (United States)

    Mäkelä, Miia R; Donofrio, Nicole; de Vries, Ronald P


    Plant biomass degradation by fungi has implications for several fields of science. The enzyme systems employed by fungi for this are broadly used in various industrial sectors such as food & feed, pulp & paper, detergents, textile, wine, and more recently biofuels and biochemicals. In addition, the topic is highly relevant in the field of plant pathogenic fungi as they degrade plant biomass to either gain access to the plant or as carbon source, resulting in significant crop losses. Finally, fungi are the main degraders of plant biomass in nature and as such have an essential role in the global carbon cycle and ecology in general. In this review we provide a global view on the development of this research topic in saprobic ascomycetes and basidiomycetes and in plant pathogenic fungi and link this to the other papers of this special issue on plant biomass degradation by fungi. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Optimized Cellular Core for Rotorcraft Project (United States)

    National Aeronautics and Space Administration — Patz Materials and Technologies proposes to develop a unique structural cellular core material to improve mechanical performance, reduce platform weight and lower...

  5. Review of cellular mechanotransduction on micropost substrates. (United States)

    Geng, Yuxu; Wang, Zhanjiang


    As physical entities, living cells can sense and respond to various stimulations within and outside the body through cellular mechanotransduction. Any deviation in cellular mechanotransduction will not only undermine the orchestrated regulation of mechanical responses, but also lead to the breakdown of their physiological function. Therefore, a quantitative study of cellular mechanotransduction needs to be conducted both in experiments and in computational simulations to investigate the underlying mechanisms of cellular mechanotransduction. In this review, we present an overview of the current knowledge and significant progress in cellular mechanotransduction via micropost substrates. In the aspect of experimental studies, we summarize significant experimental progress and place an emphasis on the coupled relationship among cellular spreading, focal adhesion and contractility as well as the influence of substrate properties on force-involved cellular behaviors. In the other aspect of computational investigations, we outline a coupled framework including the biochemically motivated stress fiber model and thermodynamically motivated adhesion model and present their predicted biomechanical responses and then compare predicted simulation results with experimental observations to further explore the mechanisms of cellular mechanotransduction. At last, we discuss the future perspectives both in experimental technologies and in computational models, as well as facing challenges in the area of cellular mechanotransduction.

  6. Cellular Targets of Dietary Polyphenol Resveratrol

    National Research Council Canada - National Science Library

    Wu, Joseph M


    To test the hypothesis that resveratrol, a grape derived polyphenol, exerts its chemopreventive properties against prostate cancer by interacting with specific cellular targets, denoted resveratrol targeting proteins (RTPs...

  7. Degradation of CIGS solar cells


    Theelen, M.J.


    Large scale commercial introduction of CIGS photovoltaics (PV) requires modules with low costs, high efficiencies and long and predictable lifetimes. Unfortunately,knowledge about the lifetime of CIGS PV is limited, which is reflected in the results of field studies: degradation rates varying from 0% to about 4% per year have been observed. Since warrantees are given out that the modules will still yield 80% of their initial power after 20 years of field exposure, degradation rates are often ...

  8. Some Misconceptions About Plastic Degradation


    Raouf, Mohamed Imad N. Raouf


    In consistence with the importance of implementing best utilization of human resources towards maintaining suitable healthy environment for our next generations, concepts and fundamentals upon which most researches on degradation of plastics are based, as a solution of solid waste reduction, will be discussed. Proper understanding of plastic figures would better utilize human efforts toward useful tasks to control solid waste. Unfortunately, when plastics are made more degradable, they becom...

  9. Anaerobic Degradation of Phenolic Compounds


    Schink, Bernhard; Philipp, Bodo; Jochen A Müller


    Mononuclear aromatic compounds are degraded anaerobically through three main pathways, the benzoyl-CoA pathway, the resorcinol pathway, and the phloroglucinol pathway. Various modification reactions channel a broad variety of mononuclear aromatics including aromatic hydrocarbons into either one of these three pathways. Recently, a further pathway was discovered with hydroxyhydroquinone as central intermediate through which especially nitrate-reducing bacteria degrade phenolic compounds and so...

  10. Role of cellular elements in thrombus formation and dissolution. (United States)

    Wohner, N


    Although fibrin forms the core matrix of thrombi, their structure depends also on the cellular elements embedded in its meshwork. Platelets are essential in the initial stages of thrombus formation, because they adhere and aggregate at sites of blood vessel wall injury and then serve as a surface for coagulation reactions, the overall rate of which determines the final structure of fibrin. In addition, platelets affect fibrinolysis through their proteins and phospholipids, which modulate plasmin activity. Leukocytes form mixed aggregates with platelets and thus influence the structure of thrombi. After activation they secrete different proteases (elastase, cathepsin G, matrix metalloproteinases) that enhance the von Willebrand factor-dependent platelet adhesion. Leukocyte-derived enzymes, first of all elastase, effect fibrinolysis by direct digestion of fibrin or indirectly modulate it by partial degradation of zymogens and inhibitors of coagulation and fibrinolytic proteases.

  11. The cellular and biochemical rules of lipid antigen presentation. (United States)

    De Libero, Gennaro; Collmann, Anthony; Mori, Lucia


    The recognition of both protein and lipid antigens follows similar strategies that rely on different molecular mechanisms. APC present lipid antigens exploiting the same mechanisms implicated in lipid translocation, lipoprotein assembly and lipid degradation. An important issue is how the lipid structure contributes to antigenicity. Lipid hydrophobicity influences the modes of internalization by APC, the trafficking through different membrane compartments, the binding to CD1 molecules and the stability of antigenic complexes. Some glycolipids with large hydrophilic parts require processing of the sugar moieties exerted by lysosomal hydrolases. Finally, extraction of lipids from membranes, their solubilization and loading on CD1 molecules are facilitated by the same lysosomal lipid-binding proteins that are also instrumental in lipid catabolism. More recent investigations reveal how lipid-specific immunity is regulated during infections. In this review we describe the main cellular and biochemical rules of lipid antigen presentation and discuss their implications in anti-microbial and autoimmune responses.

  12. Working session 1: Tubing degradation

    Energy Technology Data Exchange (ETDEWEB)

    Kharshafdjian, G. [Atomic Energy of Canada, Mississauga, Ontario (Canada); Turluer, G. [IPSN, Fontenay-aux-Roses (France)


    A general introductory overview of the purpose of the group and the general subject area of SG tubing degradation was given by the facilitator. The purpose of the session was described as to {open_quotes}develop conclusions and proposals on regulatory and technical needs required to deal with the issues of SG tubing degradation.{close_quotes} Types, locations and characteristics of tubing degradation in steam generators were briefly reviewed. The well-known synergistic effects of materials, environment, and stress and strain/strain rate, subsequently referred to by the acronym {open_quotes}MESS{close_quotes} by some of the group members, were noted. The element of time (i.e., evolution of these variables with time) was emphasized. It was also suggested that the group might want to consider the related topics of inspection capabilities, operational variables, degradation remedies, and validity of test data, and some background information in these areas was provided. The presentation given by Peter Millet during the Plenary Session was reviewed; Specifically, the chemical aspects and the degradation from the secondary side of the steam generator were noted. The main issues discussed during the October 1995 EPRI meeting on secondary side corrosion were reported, and a listing of the potential SG tube degradations was provided and discussed.

  13. Expression and cellular distribution of ubiquitin in response to injury in the developing spinal cord of Monodelphis domestica

    DEFF Research Database (Denmark)

    Noor, Natassya M; Møllgård, Kjeld; Wheaton, Benjamin J


    Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to pos...

  14. In phyllodes tumors of the breast expression of SPARC (osteonectin/BM40) mRNA by in situ hybridization correlates with protein expression by immunohistochemistry and is associated with tumor progression. (United States)

    Kim, Nah Ihm; Kim, Ga-Eon; Lee, Ji Shin; Park, Min Ho


    Secreted protein acidic and rich in cysteine (SPARC) plays an essential role in tumor invasion and metastasis. The present work was undertaken to detect expression of SPARC mRNA in phyllodes tumors (PTs) and its association with SPARC protein expression. This study also evaluated expression of SPARC mRNA and its correlation between grade and clinical behavior of PTs. In addition, we assessed in PTs the association of expression of SPARC with that of matrix metalloproteinase (MMP)-2 and of MMP-9. SPARC mRNA expression was determined by RNAscope in situ hybridization (ISH) in 50 benign, 22 borderline, and 10 malignant PTs using a tissue microarray. Furthermore, we applied immunohistochemistry (IHC) to examine expression of SPARC, MMP-2, and MMP-9. SPARC mRNA appeared to be concentrated mainly in the stromal compartment of PTs. IHC staining patterns of SPARC protein showed concordance with SPARC mRNA ISH results. Stromal SPARC expression increased continuously as PTs progress from benign through borderline to malignant PTs, both at mRNA (using ISH) (P = 0.044) and protein level (using IHC) (P = 0.000). The recurrence percentage was higher in the stromal SPARC mRNA or protein-positive group than in the SPARC-negative group but this difference was not statistically significant. Stromal SPARC mRNA and protein expression was associated with PT grade and correlated with MMP-2 expression. These results indicate that SPARC-mediated degradation of the extracellular matrix, and its possible association with MMPs, might contribute to progression of PTs.

  15. MicroRNA-145 ModulatesN6-Methyladenosine Levels by Targeting the 3'-Untranslated mRNA Region of theN6-Methyladenosine Binding YTH Domain Family 2 Protein. (United States)

    Yang, Zhe; Li, Jiong; Feng, Guoxing; Gao, Shan; Wang, Yuan; Zhang, Shuqin; Liu, Yunxia; Ye, Lihong; Li, Yueguo; Zhang, Xiaodong


    N 6 -Methyladenosine (m 6 A) is a prevalent modification present in the mRNAs of higher eukaryotes. YTH domain family 2 (YTHDF2), an m 6 A "reader" protein, can recognize mRNA m 6 A sites to mediate mRNA degradation. However, the regulatory mechanism of YTHDF2 is poorly understood. To this end, we investigated the post-transcriptional regulation of YTHDF2. Bioinformatics analysis suggested that the microRNA miR-145 might target the 3'-untranslated region (3'-UTR) of YTHDF2 mRNA. The levels of miR-145 were negatively correlated with those of YTHDF2 mRNA in clinical hepatocellular carcinoma (HCC) tissues, and immunohistochemical staining revealed that YTHDF2 was closely associated with malignancy of HCC. Interestingly, miR-145 decreased the luciferase activities of 3'-UTR of YTHDF2 mRNA. Mutation of predicted miR-145 binding sites in the 3'-UTR of YTHDF2 mRNA abolished the miR-145-induced decrease in luciferase activity. Overexpression of miR-145 dose-dependently down-regulated YTHDF2 expression in HCC cells at the levels of both mRNA and protein. Conversely, inhibition of miR-145 resulted in the up-regulation of YTHDF2 in the cells. Dot blot analysis and immunofluorescence staining revealed that the overexpression of miR-145 strongly increased m 6 A levels relative to those in control HCC cells, and this increase could be blocked by YTHDF2 overexpression. Moreover, miR-145 inhibition strongly decreased m 6 A levels, which were rescued by treatment with a small interfering RNA-based YTHDF2 knockdown. Thus, we conclude that miR-145 modulates m 6 A levels by targeting the 3'-UTR of YTHDF2 mRNA in HCC cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. A Computational model for compressed sensing RNAi cellular screening

    Directory of Open Access Journals (Sweden)

    Tan Hua


    Full Text Available Abstract Background RNA interference (RNAi becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive. Results In this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi, which employs a unique combination of group of small interfering RNAs (siRNAs to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear, which is ill-posed in general. However, the recently developed compressed sensing (CS theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially. Conclusions This csRNAi system is very promising in saving both time and cost for large-scale RNAi

  17. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  18. The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase. (United States)

    Sanduja, Sandhya; Kaza, Vimala; Dixon, Dan A


    The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis.

  19. Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®). (United States)

    Kwon, Yong-Dae; Choi, Hyun-Jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter; Pae, Ahran


    The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants.

  20. Real-time PCR quantification of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) mRNA in diatoms and pelagophytes. (United States)

    Wawrik, B; Paul, J H; Tabita, F R


    Transcriptional activity is often used as a surrogate for gene expression in environmental microbial communities. We developed a real-time PCR assay in which the ABI-Prism (PE Applied Biosystems) detection system is used for quantification of large-subunit ribulose-1,5-bisphosphate caboxylase/oxygenase (rbcL) mRNA in diatoms and pelagophytes both in cultures and from natural phytoplankton communities. Plasmid DNA containing rbcL inserts, as well as in vitro transcribed mRNA of the plasmids, was used to generate standard curves with a dynamic range of more than 6 orders of magnitude with high accuracy and precision (R(2) = 0.998). Expression levels in a cultured diatom (Phaeodactylum tricornutum) were quantified through one light-dark cycle by using traditional 35S-labeled oligonucleotide hybridization and real-time PCR. The mRNA levels detected by the two techniques were similar and correlated well (R(2) = 0.95; slope = 1.2). The quantities obtained by hybridization were slightly, yet significantly, larger (t = 5.29; P = 0.0011) than the quantities obtained by real-time PCR. This was most likely because partially degraded transcripts were not detected by real-time PCR. rbcL mRNA detection by real-time PCR was 3 orders of magnitude more sensitive than rbcL mRNA detection by hybridization. Diatom and pelagophyte rbcL mRNAs were also quantified in a profile from an oligotrophic site in the Gulf of Mexico. We detected the smallest amount of diatom rbcL expression in the surface water and maximum expression at a depth that coincided with the depth of the subsurface chlorophyll maximum. These results indicate that real-time PCR may be utilized for quantification of microbial gene expression in the environment.

  1. Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates, and cooperates with polynucleotide phosphorylase in 3' end maturation. (United States)

    Germain, Arnaud; Kim, Sang Hu; Gutierrez, Ryan; Stern, David B


    Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3'→5' exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non-viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two-step maturation of mRNA 3' ends, with RNR1 polishing 3' termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non-polysomal fractions, and half-life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3'→5' exoribonucleases in chloroplast mRNA 3' end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  2. Sphingosine kinase-1, S1P transporter spinster homolog 2 and S1P2 mRNA expressions are increased in liver with advanced fibrosis in human. (United States)

    Sato, Masaya; Ikeda, Hitoshi; Uranbileg, Baasanjav; Kurano, Makoto; Saigusa, Daisuke; Aoki, Junken; Maki, Harufumi; Kudo, Hiroki; Hasegawa, Kiyoshi; Kokudo, Norihiro; Yatomi, Yutaka


    The role of sphingosine 1-phosphate (S1P) in liver fibrosis or inflammation was not fully examined in human. Controversy exists which S1P receptors, S1P1 and S1P3 vs S1P2, would be importantly involved in its mechanism. To clarify these matters, 80 patients who received liver resection for hepatocellular carcinoma and 9 patients for metastatic liver tumor were enrolled. S1P metabolism was analyzed in background, non-tumorous liver tissue. mRNA levels of sphingosine kinase 1 (SK1) but not SK2 were increased in livers with fibrosis stages 3-4 compared to those with 0-2 and to normal liver. However, S1P was not increased in advanced fibrotic liver, where mRNA levels of S1P transporter spinster homolog 2 (SPNS2) but not S1P-degrading enzymes were enhanced. Furthermore, mRNA levels of S1P2 but not S1P1 or S1P3 were increased in advanced fibrotic liver. These increased mRNA levels of SK1, SPNS2 and S1P2 in fibrotic liver were correlated with α-smooth muscle actin mRNA levels in liver, and with serum ALT levels. In conclusion, S1P may be actively generated, transported to outside the cells, and bind to its specific receptor in human liver to play a role in fibrosis or inflammation. Altered S1P metabolism in fibrotic liver may be their therapeutic target.

  3. Transcript copy number of genes for DNA repair and translesion synthesis in yeast: contribution of transcription rate and mRNA stability to the steady-state level of each mRNA along with growth in glucose-fermentative medium. (United States)

    Michán, Carmen; Monje-Casas, Fernando; Pueyo, Carmen


    We quantitated the copy number of mRNAs (NTG1, NTG2, OGG1, APN1, APN2, MSH2, MSH6, REV3, RAD30) encoding different DNA repair enzymes and translesion-synthesis polymerases in yeast. Quantitations reported examine how the steady-state number of each transcript is modulated in association with the growth in glucose-fermentative medium, and evaluate the respective contribution of the rate of mRNA degradation and transcription initiation to the specific mRNA level profile of each gene. Each transcript displayed a unique growth-related profile, therefore altering the relative abundance of mRNAs coding for proteins with similar functions, as cells proceed from exponential to stationary phase. Nonetheless, as general trend, they exhibited maximal levels when cells proliferate rapidly and minimal values when cells cease proliferation. We found that previous calculations on the stability of the investigated mRNAs might be biased, in particular regarding those that respond to heat shock stress. Overall, the mRNAs experienced drastic increments in their stabilities in response to gradual depletion of essential nutrients in the culture. However, differences among the mRNA stability profiles suggest a dynamic modulation rather than a passive process. As general rule, the investigated genes were much more frequently transcribed during the fermentative growth than later during the diauxic arrest and the stationary phase, this finding conciliating low steady-state levels with increased mRNA stabilities. Interestingly, while the rate at which each gene is transcribed appeared as the only determinant of the number of mRNA copies at the exponential growth, later, when cell growth is arrested, the rate of mRNA degradation becomes also a key factor for gene expression. In short, our results raise the question of how important the respective contribution of transcription and mRNA stability mechanisms is for the steady-state profile of a given transcript, and how this contribution may

  4. Pulsed feedback defers cellular differentiation.

    Directory of Open Access Journals (Sweden)

    Joe H Levine


    Full Text Available Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable "polyphasic" positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a "timer" that operates over timescales much longer than a cell cycle.

  5. Redox regulation of insulin degradation by insulin-degrading enzyme.

    Directory of Open Access Journals (Sweden)

    Crystal M Cordes

    Full Text Available Insulin-degrading enzyme (IDE is a thiol sensitive peptidase that degrades insulin and amyloid β, and has been linked to type 2 diabetes mellitus and Alzheimer's disease. We examined the thiol sensitivity of IDE using S-nitrosoglutathione, reduced glutathione, and oxidized glutathione to distinguish the effects of nitric oxide from that of the redox state. The in vitro activity of IDE was studied using either partially purified cytosolic enzyme from male Sprague-Dawley rats, or purified rat recombinant enzyme. We confirm that nitric oxide inhibits the degrading activity of IDE, and that it affects proteasome activity through this interaction with IDE, but does not affect the proteasome directly. Oxidized glutathione inhibits IDE through glutathionylation, which was reversible by dithiothreitol but not by ascorbic acid. Reduced glutathione had no effect on IDE, but reacted with partially degraded insulin to disrupt its disulfide bonds and accelerate its breakdown to trichloroacetic acid soluble fragments. Our results demonstrate the sensitivity of insulin degradation by IDE to the redox environment and suggest another mechanism by which the cell's oxidation state may contribute to the development of, and the link between, type 2 diabetes and Alzheimer's disease.

  6. Stochastic processes, multiscale modeling, and numerical methods for computational cellular biology

    CERN Document Server


    This book focuses on the modeling and mathematical analysis of stochastic dynamical systems along with their simulations. The collected chapters will review fundamental and current topics and approaches to dynamical systems in cellular biology. This text aims to develop improved mathematical and computational methods with which to study biological processes. At the scale of a single cell, stochasticity becomes important due to low copy numbers of biological molecules, such as mRNA and proteins that take part in biochemical reactions driving cellular processes. When trying to describe such biological processes, the traditional deterministic models are often inadequate, precisely because of these low copy numbers. This book presents stochastic models, which are necessary to account for small particle numbers and extrinsic noise sources. The complexity of these models depend upon whether the biochemical reactions are diffusion-limited or reaction-limited. In the former case, one needs to adopt the framework of s...

  7. Cellular telephone use and cancer risk

    DEFF Research Database (Denmark)

    Schüz, Joachim; Jacobsen, Rune; Olsen, Jørgen H.


    BACKGROUND: The widespread use of cellular telephones has heightened concerns about possible adverse health effects. The objective of this study was to investigate cancer risk among Danish cellular telephone users who were followed for up to 21 years. METHODS: This study is an extended follow-up ...

  8. Cellular encoding for interactive evolutionary robotics

    NARCIS (Netherlands)

    F.C. Gruau; K. Quatramaran


    textabstractThis work reports experiments in interactive evolutionary robotics. The goal is to evolve an Artificial Neural Network (ANN) to control the locomotion of an 8-legged robot. The ANNs are encoded using a cellular developmental process called cellular encoding. In a previous work similar

  9. Anxiety disorders and accelerated cellular ageing

    NARCIS (Netherlands)

    Verhoeven, J.E.; Revesz, D.; van Oppen, P.C.; Epel, E.S.; Wolkowitz, O.M.; Penninx, B.W.


    Background: Anxiety disorders increase the risk of onset of several ageing-related somatic conditions, which might be the consequence of accelerated cellular ageing. Aims: To examine the association between anxiety status and leukocyte telomere length (LTL) as an indicator of cellular ageing.

  10. Digoxin up-regulates multidrug resistance transporter (MDR1) mRNA and simultaneously down-regulates steroid xenobiotic receptor mRNA. (United States)

    Takara, Kohji; Takagi, Kentaro; Tsujimoto, Masayuki; Ohnishi, Noriaki; Yokoyama, Teruyoshi


    A steroid xenobiotic receptor (SXR) is involved in the induction of MDR1/P-glycoprotein. MDR1 up-regulation by digoxin was previously demonstrated in human colon adenocarcinoma Caco-2 cells, but the participation of SXR remains unclear. Herein, the participation of SXR in MDR1 up-regulation was examined using reverse transcription-polymerase chain reaction in Caco-2 cells, and digoxin-tolerant cells (Caco/DX) as well as human colon carcinoma LS180 cells, which expressed SXR. MDR1 mRNA expression in Caco-2 or LS180 cells was increased by exposure to 1 microM digoxin for 24h, in a concentration-dependent manner, but SXR mRNA decreased concentration-dependently and was undetectable or significantly lower at 1 microM digoxin, indicating antithetical changes in MDR1 and SXR mRNA expression. Moreover, the MDR1 mRNA level was higher in Caco/DX cells than Caco-2 cells, whereas the SXR mRNA level was lower in Caco/DX cells. Consequently, digoxin was demonstrated to up-regulate MDR1 mRNA and simultaneously down-regulate SXR mRNA expression.

  11. CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation. (United States)

    Li, Xuan; Wu, Xiao-Qi; Deng, Rong; Li, Dan-Dan; Tang, Jun; Chen, Wen-Dan; Chen, Jing-Hong; Ji, Jiao; Jiao, Lin; Jiang, Shan; Yang, Fen; Feng, Gong-Kan; Senthilkumar, Ravichandran; Yue, Fei; Zhang, Hai-Liang; Wu, Rui-Yan; Yu, Yan; Xu, Xue-Lian; Mai, Jia; Li, Zhi-Ling; Peng, Xiao-Dan; Huang, Yun; Huang, Xiang; Ma, Ning-Fang; Tao, Qian; Zeng, Yi-Xin; Zhu, Xiao-Feng


    Autophagy is a degradative pathway that delivers cellular components to the lysosome for degradation. The role of autophagy in cell differentiation is poorly understood. Here we show that CaMKII can directly phosphorylate Beclin 1 at Ser90 to promote K63-linked ubiquitination of Beclin 1 and activation of autophagy. Meanwhile, CaMKII can also promote K63-linked ubiquitination of inhibitor of differentiation 1/2 (Id-1/2) by catalyzing phosphorylation of Id proteins and recruiting TRAF-6. Ubiquitinated Id-1/Id-2 can then bind to p62 and be transported to autolysosomes for degradation. Id degradation promotes the differentiation of neuroblastoma cells and reduces the proportion of stem-like cells. Our study proposes a mechanism by which autophagic degradation of Id proteins can regulate cell differentiation. This suggests that targeting of CaMKII and the regulation of autophagic degradation of Id may be an effective therapeutic strategy to induce cell differentiation in neuroblastoma.

  12. mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research. (United States)

    Li, Changqing; Guillén, Jaime; Rabah, Nadia; Blanjoie, Alexandre; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Coutard, Bruno


    required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Degraded RNA transcript stable regions (StaRs) as targets for enhanced forensic RNA body fluid identification. (United States)

    Lin, Meng-Han; Albani, Patricia P; Fleming, Rachel


    The detection of messenger RNA (mRNA) using reverse transcriptase PCR (RT-PCR) is becoming common practice for forensic body fluid identification. However, the degraded and scarce nature of RNA from forensic samples mean that mRNA transcripts are not consistently detected or remain undetected in practice. Conventional primer design for RT-PCR (and quantitative RT-PCR) includes targeting primers to span exon-exon boundaries or by having the primers on two separate exons, and satisfying common primer thermodynamic criteria. We have found that the conventional placement of primers is not always optimal for obtaining reproducible results from degraded samples. Using massively parallel sequencing data from degraded body fluids, we designed primers to amplify transcript regions of high read coverage, hence, higher stability, and compared these with primers designed using conventional methodology. Our findings are that primers designed for transcript regions of higher read coverage resulted in vastly improved detection of mRNA transcripts that were not previously detected or were not consistently detected in the same samples using conventional primers. We developed a new concept whereby primers targeted to transcript stable regions (StaRs) are able to consistently and specifically amplify a wide range of RNA biomarkers in various body fluids of varying degradation levels. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria. (United States)

    Agrawal, Anurag; Mabalirajan, Ulaganathan


    Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function are key aspects of pathogenesis in asthma and chronic obstructive pulmonary disease (COPD). This motif is not unique to obstructive airway disease, reported in related airway diseases such as bronchopulmonary dysplasia and parenchymal diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as MitoQ, has substantially improved mitochondrial health and restored respiratory function. Modulation of noncoding RNA or protein regulators of mitochondrial biogenesis, dynamics, or degradation has been found to be effective in models of fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy mitochondria to epithelial cells has been associated with remarkable therapeutic efficacy in models of acute lung injury and asthma. Together, these form a 3R model--repair, reprogramming, and replacement--for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function in lung health and disease, with a focus on asthma and COPD, and provides an overview of mitochondria-targeted strategies for rejuvenating cellular respiration and optimizing respiratory function in lung diseases. Copyright © 2016 the American Physiological Society.

  15. With the Help of MOM: Mitochondrial Contributions to Cellular Quality Control. (United States)

    Braun, Ralf J; Westermann, Benedikt


    Mitochondria are essential organelles because they have key roles in cellular energy metabolism and many other metabolic pathways. Several quality control systems have evolved to ensure that dysfunctional mitochondria are either repaired or eliminated. The activities of these pathways are crucial for cellular health because they maintain functional mitochondria. In addition, the cytosolic ubiquitin-proteasome system (UPS) and the mitochondria-associated degradation pathway (MAD) share some of their core components, are functionally tightly interconnected, and mutually modulate their activities. Thus, the mitochondrial outer membrane (MOM) actively supports quality control systems in extramitochondrial compartments. Furthermore, mitochondrial quality surveillance systems also act on cytosolic or endoplasmic reticulum (ER) substrates and modulate immune responses. Therefore, mitochondria contribute to cellular quality control and homeostasis on several levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28 mRNA by microRNA847 upregulates auxin signaling to modulate cell proliferation and lateral organ growth in Arabidopsis. (United States)

    Wang, Jing-Jing; Guo, Hui-Shan


    MicroRNAs function in a range of developmental processes. Here, we demonstrate that miR847 targets the mRNA of the auxin/indole acetic acid (Aux/IAA) repressor-encoding gene IAA28 for cleavage. The rapidly increased accumulation of miR847 in Arabidopsis thaliana coincided with reduced IAA28 mRNA levels upon auxin treatment. This induction of miR847 by auxin was abolished in auxin receptor tir1-1 and auxin-resistant axr1-3 mutants. Further analysis demonstrates that miR847 functions as a positive regulator of auxin-mediated lateral organ development by cleaving IAA28 mRNA. Importantly, the ectopic expression of miR847 increases the expression of cell cycle genes as well as the neoplastic activity of leaf cells, prolonging later-stage rosette leaf growth and producing leaves with serrated margins. Moreover, both miR847 and IAA28 mRNAs are specifically expressed in marginal meristems of rosette leaves and lateral root initiation sites. Our data indicate that auxin-dependent induction of miR847 positively regulates meristematic competence by clearing IAA28 mRNA to upregulate auxin signaling, thereby determining the duration of cell proliferation and lateral organ growth in Arabidopsis. IAA28 mRNA encodes an Aux/IAA repressor protein, which is degraded through the proteasome in response to auxin. Altered signal sensitization to IAA28 mRNA levels, together with targeted IAA28 degradation, ensures a robust signal derepression. © 2015 American Society of Plant Biologists. All rights reserved.

  17. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Papadopoulos, T.; Pfeifer, U. (Univ. of Wuerzburg (West Germany))


    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  18. Pronounced alterations of cellular metabolism and structure due to hyper- or hypo-osmosis. (United States)

    Mao, Lei; Hartl, Daniela; Nolden, Tobias; Koppelstätter, Andrea; Klose, Joachim; Himmelbauer, Heinz; Zabel, Claus


    Cell volume alteration represents an important factor contributing to the pathology of late-onset diseases. Previously, it was reported that protein biosynthesis and degradation are inversely (trans) regulated during cell volume regulation. Upon cell shrinkage, protein biosynthesis was up-regulated and protein degradation down-regulated. Cell swelling showed opposite regulation. Recent evidence suggests a decrease of protein biodegradation activity in many neurodegenerative diseases and even during aging; both also show prominent cell shrinkage. To clarify the effect of cell volume regulation on the overall protein turnover dynamics, we investigated mouse embryonic stem cells under hyper- and hypotonic osmotic conditions using a 2-D gel based proteomics approach. These conditions cause cell swelling and shrinkage, respectively. Our results demonstrate that the adaption to altered osmotic conditions and therefore cell volume alterations affects a broad spectrum of cellular pathways, including stress response, cytoskeleton remodeling and importantly, cellular metabolism and protein degradation. Interestingly, protein synthesis and degradation appears to be cis-regulated (same direction) on a global level. Our findings also support the hypothesis that protein alterations due to osmotic stress contribute to the pathology of neurodegenerative diseases due to a 60% expression overlap with proteins found altered in Alzheimer's, Huntington's, or Parkinson's disease. Eighteen percent of the proteins altered are even shared with all three disorders.

  19. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee


    Full Text Available 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.

  20. Operating principles of tristable circuits regulating cellular differentiation (United States)

    Jia, Dongya; Jolly, Mohit Kumar; Harrison, William; Boareto, Marcelo; Ben-Jacob, Eshel; Levine, Herbert


    Many cell-fate decisions during embryonic development are governed by a motif comprised of two transcription factors (TFs) A and B that mutually inhibit each other and may self-activate. This motif, called as a self-activating toggle switch (SATS), can typically have three stable states (phenotypes)—two corresponding to differentiated cell fates, each of which has a much higher level of one TF than the other—≤ft(A,~B\\right)=≤ft(1,~0\\right) or ≤ft(0,~1\\right) —and the third state corresponding to an ‘undecided’ stem-like state with similar levels of both A and B—≤ft(A,~B\\right)=≤ft(1/2,1/2\\right) . Furthermore, two or more SATSes can be coupled together in various topologies in different contexts, thereby affecting the coordination between multiple cellular decisions. However, two questions remain largely unanswered: (a) what governs the co-existence and relative stability of these three stable states? (b) What orchestrates the decision-making of coupled SATSes? Here, we first demonstrate that the co-existence and relative stability of the three stable states in an individual SATS can be governed by the relative strength of self-activation, external signals activating and/or inhibiting A and B, and mutual degradation between A and B. Simultaneously, we investigate the effects of these factors on the decision-making of two coupled SATSes. Our results offer novel understanding into the operating principles of individual and coupled tristable self-activating toggle switches (SATSes) regulating cellular differentiation and can yield insights into synthesizing three-way genetic circuits and understanding of cellular reprogramming.

  1. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages. (United States)

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D


    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

  2. Microbial surfactant mediated degradation of anthracene in aqueous phase by marine Bacillus licheniformis MTCC 5514

    Directory of Open Access Journals (Sweden)

    Sreethar Swaathy


    Full Text Available The present study emphasizes the biosurfactant mediated anthracene degradation by a marine alkaliphile Bacillus licheniformis (MTCC 5514. The isolate, MTCC 5514 degraded >95% of 300 ppm anthracene in an aqueous medium within 22 days and the degradation percentage reduced significantly when the concentration of anthracene increased to above 500 ppm. Naphthalene, naphthalene 2-methyl, phthalic acid and benzene acetic acid are the products of degradation identified based on thin layer chromatography, high performance liquid chromatography, gas chromatography and mass analyses. It has been observed that the degradation is initiated by the biosurfactant of the isolate for solubilization through micellation and then the alkali pH and intra/extra cellular degradative enzymes accomplish the degradation process. Encoding of genes responsible for biosurfactant production (licA3 as well as catabolic reactions (C23O made with suitable primers designed. The study concludes in situ production of biosurfactant mediates the degradation of anthracene by B. licheniformis.

  3. Identifying mRNA, MicroRNA and Protein Profiles of Melanoma Exosomes (United States)

    Chen, Yinlu; Taylor, Douglas D.; Rai, Shesh N.; Waigel, Sabine; Zacharias, Wolfgang; Hao, Hongying; McMasters, Kelly M.


    Background Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. Methodology/Principal Findings In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. Conclusions/Significance Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma. PMID:23056502

  4. Tools for translation: non-viral materials for therapeutic mRNA delivery (United States)

    Hajj, Khalid A.; Whitehead, Kathryn A.


    In recent years, messenger RNA (mRNA) has come into the spotlight as a versatile therapeutic with the potential to prevent and treat a staggering range of diseases. Billions of dollars have been invested in the commercial development of mRNA drugs, with ongoing clinical trials focused on vaccines (for example, influenza and Zika viruses) and cancer immunotherapy (for example, myeloma, leukaemia and glioblastoma). Although significant progress has been made in the design of in vitro-transcribed mRNA that retains potency while minimizing unwanted immune responses, the widespread use of mRNA drugs requires the development of safe and effective drug delivery vehicles. In this Review, we provide an overview of the field of mRNA therapeutics and describe recent advances in the development of synthetic materials that encapsulate and deliver mRNA payloads.

  5. Metalloproteinases in corneal diseases: degradation and processing. (United States)

    Sakimoto, Tohru; Sawa, Mitsuru


    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with the potential to degrade all types of extracellular matrix. The ADAM (a disintegrin and metalloproteinase) family of peptidases was recently identified as cleaving the extracellular domain of transmembrane proteins. This was termed ectodomain shedding. We investigated the MMP expression in patients with corneal diseases and the potential role of ADAMs in corneal pathophysiology. We detected upregulation of the active form of MMP-2 and MMP-9 in the tear fluid from patients with corneal melting or recurrent corneal erosion. Using human corneal epithelial cells, we observed ADAM17-dependent ectodomain shedding of soluble tumor necrosis factor receptor 1 and soluble interleukin-6 (IL-6) receptor (sIL-6R). The production of sIL-6R was also induced by messenger RNA splicing in the human corneal epithelial cells. IL-6/sIL-6R-induced signal transducer and activator of transcription 3 phosphorylation was observed in cultured human corneal fibroblasts, suggesting that IL-6 trans-signaling induced inflammatory cellular signaling in the human corneal fibroblasts. We demonstrated that MMPs are significantly upregulated in collagen-destructive disorders of the cornea. Additionally, we observed that ectodomain shedding by ADAMs in corneal epithelial cells mediated the production of soluble cytokine receptors. Trans-signaling of IL-6 can induce an inflammatory response in corneal stroma, indicating the significance of IL-6 trans-signaling in ocular surface inflammation. Thus, MMPs and ADAMs play an important role in the pathophysiology of corneal diseases.

  6. Global SUMO proteome responses guide gene regulation, mRNA biogenesis, and plant stress responses

    Directory of Open Access Journals (Sweden)

    Magdalena eMazur


    Full Text Available Small-ubiquitin-like MOdifier (SUMO is a key regulator of abiotic stress, disease resistance and development in plants. The identification of >350 plant SUMO targets has revealed many processes modulated by SUMO and potential consequences of SUMO on its targets. Importantly, highly related proteins are SUMO-modified in plants, yeast, and metazoans. Overlapping SUMO targets include heat-shock proteins, transcription regulators, histones, histone-modifying enzymes, proteins involved in DNA damage repair, but also proteins involved in mRNA biogenesis and nucleo-cytoplasmic transport. Proteomics studies indicate key roles for SUMO in gene repression by controlling histone (deacetylation activity at genomic loci. The responsible heavily sumoylated transcriptional repressor complexes are recruited by EAR (Ethylene-responsive element binding factor [ERF]-associated Amphiphilic Repression-motif containing transcription factors in plants. These transcription factors are not necessarily themselves a SUMO target. Conversely, SUMO acetylation prevents binding of downstream partners by preventing binding of SIMs (SUMO-interaction peptide motifs presents in these partners, while SUMO acetylation has emerged as mechanism to recruit specifically bromodomains; bromodomain are generally linked with gene activation. These findings strengthen the idea of a bidirectional sumo-/acetylation switch in gene regulation. Quantitative proteomics has highlighted that global sumoylation provides a dynamic response to protein damage involving SUMO chain-mediated protein degradation, but also SUMO E3 ligase-dependent transcription of HSP (Heat-shock protein genes. With these insights in SUMO function and novel technical advancements, we can now study SUMO dynamics in responses to (abiotic stress in plants.

  7. Radiation degradation of silk protein

    Energy Technology Data Exchange (ETDEWEB)

    Pewlong, W.; Sudatis, B. [Office of Atomic Energy for Peace, Bangkok (Thailand); Takeshita, Hidefumi; Yoshii, Fumio; Kume, Tamikazu [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment


    Silk fibroin fiber from the domesticated silkworm Bombyx mori was irradiated using an electron beam accelerator to investigate the application of the radiation degradation technique as a means to solubilize fibroin. The irradiation caused a significant degradation of the fiber. The tensile strength of fibroin fiber irradiated up to 2500 kGy decreased rapidly with increasing dose. The presence of oxygen in the irradiation atmosphere enhanced degradation of the tensile strength. The solubilization of irradiated fibroin fiber was evaluated using the following three kinds of solutions: a calcium chloride solution(CaCl{sub 2}/C{sub 2}H{sub 5}OH/H{sub 2}O=1:2:8 in mole ratio), a hydrochloric acid (0.5 N) and a distilled water. Dissolution of fibroin fiber into these solutions was significantly enhanced by irradiation. Especially, an appreciable amount of water soluble proteins was extracted by a distilled water. (author)

  8. Markers of cellular senescence. Telomere shortening as a marker of cellular senescence. (United States)

    Bernadotte, Alexandra; Mikhelson, Victor M; Spivak, Irina M


    The cellular senescence definition comes to the fact of cells irreversible proliferation disability. Besides the cell cycle arrest, senescent cells go through some morphological, biochemical, and functional changes which are the signs of cellular senescence. The senescent cells (including replicative senescence and stress-induced premature senescence) of all the tissues look alike. They are metabolically active and possess the set of characteristics in vitro and in vivo, which are known as biomarkers of aging and cellular senescence. Among biomarkers of cellular senescence telomere shortening is a rather elegant frequently used biomarker. Validity of telomere shortening as a marker for cellular senescence is based on theoretical and experimental data.

  9. Prolyl carboxypeptidase mRNA expression in the mouse brain. (United States)

    Jeong, Jin Kwon; Diano, Sabrina


    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain. © 2013 Published by Elsevier B.V.

  10. Cellular ATP release in the lung and airway

    Directory of Open Access Journals (Sweden)

    Satoru Ito


    Full Text Available Adenosine triphosphate (ATP is a universal energy source synthesized by mitochondrial oxidative phosphorylation and cytosolic glycolysis and transported by the vesicular nucleotide transporter for storage in secretory vesicles. Extracellular ATP regulates physiological functions and homeostasis of the respiratory system and is associated with pathogenesis of respiratory diseases. Thus, modulation of ATP and purinergic signaling may be a novel therapeutic approach to pulmonary disease. ATP is released from alveolar epithelial cells, airway epithelial cells, airway smooth muscle cells, fibroblasts and endothelial cells in response to various chemical and mechanical stimuli. In addition to conductive pathways such as connexins and pannexins, vesicular exocytosis is involved in the mechanisms of ATP release from the cells. Imaging approaches enable us to visualize ATP release from not only cultured cells but also lung tissue ex vivo. Extracellular vesicles, exosomes and membrane-derived microvesicles, containing cytoplasmic proteins, mRNA and microRNA, represent important mediators of cell-to-cell communication and the intercellular microenvironment. However, it is not known whether extracellular vesicles contain ATP as an intercellular messenger. Future studies are necessary to elucidate the mechanisms of cellular ATP release and purinergic signaling in the respiratory system.

  11. Trypsin-mediated enzymatic degradation of type II collagen in the human vitreous (United States)

    van Deemter, Mariëlle; Kuijer, Roel; Harm Pas, Hendri; Jacoba van der Worp, Roelofje; Hooymans, Johanna Martina Maria


    Purpose Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation. Methods Immunohistochemistry and western blotting were used for detecting and locating trypsin isoforms in the vitreous and retina of human donor eyes. The capability of the retina to produce these trypsins was analyzed with polymerase chain reaction. Whether the different trypsins degraded type II collagen was tested in vitro. The sizes of the in vitro induced type II collagen degradation products were compared to those present in the vitreous of human eyes of different ages. Results Trypsin-1 and trypsin-2 were detected in the vitreous. In the retina, messenger ribonucleic acid (mRNA) coding for trypsin-2, -3, and -4 was present. Using immunohistochemistry, trypsin-2 was detected in microglial cells located in the vitreous and the retina. All trypsin isoforms degraded type II collagen and produced degradation products of similar sizes as those present in the vitreous. Conclusions Trypsin-1 and trypsin-2 appear to have a function in the degradation of vitreous type II collagen. They could therefore have a role in the development of vitreous liquefaction. PMID:23882137

  12. Are microRNAs true sensors of ageing and cellular senescence? (United States)

    Williams, Justin; Smith, Flint; Kumar, Subodh; Vijayan, Murali; Reddy, P Hemachandra


    All living beings are programmed to death due to aging and age-related processes. Aging is a normal process of every living species. While all cells are inevitably progressing towards death, many disease processes accelerate the aging process, leading to senescence. Pathologies such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, cardiovascular disease, cancer, and skin diseases have been associated with deregulated aging. Healthy aging can delay onset of all age-related diseases. Genetics and epigenetics are reported to play large roles in accelerating and/or delaying the onset of age-related diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent molecular biology discoveries have revealed that microRNAs (miRNAs) are potential sensors of aging and cellular senescence. Due to miRNAs capability to bind to the 3' untranslated region (UTR) of mRNA of specific genes, miRNAs can prevent the translation of specific genes. The purpose of our article is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging and senescence. Our article discusses the current understanding of cellular senescence, its interplay with miRNAs regulation, and how they both contribute to disease processes. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Growth-based determination and biochemical confirmation of genetic requirements for protein degradation in Saccharomyces cerevisiae. (United States)

    Watts, Sheldon G; Crowder, Justin J; Coffey, Samuel Z; Rubenstein, Eric M


    Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for

  14. Increase in Catalase mRNA in Wounded Sweet Potato Tuberous Root Tissue


    Shigeru, Sakajo; Kenzo, Nakamura; Tadashi, Asahi; Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University


    Catalase protein, as well as its activity, increases in wounded sweet potato tuberous root tissue [Esaka et al. (1983) Plant Cell Physiol. 24: 615]. Whether catalase mRNA increases in wounded tissue was examined with a hybridization probe of a cDNA for sweet potato catalase mRNA. The content of catalase mRNA in the tissue increased after a lag phase of 10 h to reach a maximum at 30 h after wounding, whereas total RNA content increased without a lag phase. The increase in the mRNA content afte...

  15. Coupling mRNA processing with transcription in time and space. (United States)

    Bentley, David L


    Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3' end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes.

  16. TS mRNA levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer. (United States)

    Zhang, Qun; Shen, Jie; Wang, Hao; Hu, Jing; Yu, Lixia; Xie, Li; Wei, Jia; Liu, Baorui; Guan, Wenxian; Qian, Xiaoping


    The purpose of the study is to analyze the relationship between tumor thymidylate synthase (TS) mRNA expression levels and raltitrexed/pemetrexed/5-FU sensitivity. We collected freshly removed colorectal tumor specimens from 50 patients. Chemosensitivities to anticancer drugs were evaluated by histoculture drug response assay. We adopted quantitative reverse transcription polymerase chain reaction for TS mRNA detection and immunohistochemical staining for assessing TS expression in tumor tissues. There is a significant relationship between TS mRNA expression levels and in vitro chemosensitivity of freshly removed colorectal tumor specimens to pemetrexed (P TS mRNA expression levels can predict pemetrexed and raltitrexed sensitivity in colorectal cancer.


    Directory of Open Access Journals (Sweden)



    Full Text Available Cellular interactions involve many types of cell surface molecules and operate via homophilic and/or heterophilic protein-protein and protein-carbohydrate binding. Our investigations in different model-systems (marine invertebrates and mammals have provided direct evidence that a novel class of primordial proteoglycans, named by us gliconectins, can mediate cell adhesion via a new alternative molecular mechanism of polyvalent carbohydrate-carbohydrate binding. Biochemical characterization of isolated and purified glyconectins revealed the presence of specific carbohydrate structures, acidic glycans, different from classical glycosaminoglycans. Such acidic glycans of high molecular weight containing fucose, glucuronic or galacturonic acids, and sulfate groups, originally found in sponges and sea urchin embryos, may represent a new class of carbohydrate carcino-embryonal antigens in mice and humans. Such interactions between biological macromolecules are usually investigated by kinetic binding studies, calorimetric methods, X-ray diffraction, nuclear magnetic resonance, and other spectroscopic analyses. However, these methods do not supply a direct estimation of the intermolecular binding forces that are fundamental for the function of the ligand-receptor association. Recently, we have introduced atomic force microscopy to quantify the binding strength between cell adhesion proteoglycans. Measurement of binding forces intrinsic to cell adhesion proteoglycans is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. As a model, we selected the glyconectin 1, a cell adhesion proteoglycan isolated from the marine sponge Microciona prolifera. This glyconectin mediates in vivo cell recognition and aggregation via homophilic, species-specific, polyvalent, and calcium ion-dependent carbohydrate-carbohydrate interactions. Under physiological conditions, an adhesive force of up to 400 piconewtons

  18. The Science of Battery Degradation

    Energy Technology Data Exchange (ETDEWEB)

    Sullivan, John P. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Physics; El Gabaly Marquez, Farid [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Physics; McCarty, Kevin [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Physics; Sugar, Joshua Daniel [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Physics; Talin, Alec A. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Materials Physics; Fenton, Kyle R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Power Sources Design and Development; Nagasubramanian, Ganesan [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Power Sources Design and Development; Harris, Charles Thomas [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Nanosystems Synthesis/Analysis; Jungjohann, Katherine Leigh [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Nanosystems Synthesis/Analysis; Hayden, Carl C. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Combustion Chemistry Dept.; Kliewer, Christopher Jesse [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Combustion Chemistry Dept.; Hudak, Nicholas S. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Power Sources Research and Development; Leung, Kevin [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Nanostructure Physics; McDaniel, Anthony H. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Hydrogen and Combustion Technology; Tenney, Craig M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Chemical and Biological Systems; Zavadil, Kevin R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Advanced Materials Lab.


    This report documents work that was performed under the Laboratory Directed Research and Development project, Science of Battery Degradation. The focus of this work was on the creation of new experimental and theoretical approaches to understand atomistic mechanisms of degradation in battery electrodes that result in loss of electrical energy storage capacity. Several unique approaches were developed during the course of the project, including the invention of a technique based on ultramicrotoming to cross-section commercial scale battery electrodes, the demonstration of scanning transmission x-ray microscopy (STXM) to probe lithium transport mechanisms within Li-ion battery electrodes, the creation of in-situ liquid cells to observe electrochemical reactions in real-time using both transmission electron microscopy (TEM) and STXM, the creation of an in-situ optical cell utilizing Raman spectroscopy and the application of the cell for analyzing redox flow batteries, the invention of an approach for performing ab initio simulation of electrochemical reactions under potential control and its application for the study of electrolyte degradation, and the development of an electrochemical entropy technique combined with x-ray based structural measurements for understanding origins of battery degradation. These approaches led to a number of scientific discoveries. Using STXM we learned that lithium iron phosphate battery cathodes display unexpected behavior during lithiation wherein lithium transport is controlled by nucleation of a lithiated phase, leading to high heterogeneity in lithium content at each particle and a surprising invariance of local current density with the overall electrode charging current. We discovered using in-situ transmission electron microscopy that there is a size limit to lithiation of silicon anode particles above which particle fracture controls electrode degradation. From electrochemical entropy measurements, we discovered that entropy

  19. Regulation of Cellular Identity in Cancer. (United States)

    Roy, Nilotpal; Hebrok, Matthias


    Neoplastic transformation requires changes in cellular identity. Emerging evidence increasingly points to cellular reprogramming, a process during which fully differentiated and functional cells lose aspects of their identity while gaining progenitor characteristics, as a critical early step during cancer initiation. This cell identity crisis persists even at the malignant stage in certain cancers, suggesting that reactivation of progenitor functions supports tumorigenicity. Here, we review recent findings that establish the essential role of cellular reprogramming during neoplastic transformation and the major players involved in it with a special emphasis on pancreatic cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Cryptographic primitives based on cellular transformations

    Directory of Open Access Journals (Sweden)

    B.V. Izotov


    Full Text Available Design of cryptographic primitives based on the concept of cellular automata (CA is likely to be a promising trend in cryptography. In this paper, the improved method performing data transformations by using invertible cyclic CAs (CCA is considered. Besides, the cellular operations (CO as a novel CAs application in the block ciphers are introduced. Proposed CCAs and COs, integrated under the name of cellular transformations (CT, suit well to be used in cryptographic algorithms oriented to fast software and cheap hardware implementation.