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Sample records for plant-specific protein essential

  1. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

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    Muhammed Jamsheer K

    Full Text Available Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  2. SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

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    Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

    2011-02-04

    SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

  3. Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators

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    Shucai eWang

    2016-03-01

    Full Text Available OVATE FAMILY PROTEINS (OFPs are a class of proteins with a conserved OVATE domain. OVATE protein was first identified in tomato as a key regulator of fruit shape. OFPs are plant-specific proteins that are widely distributed in the plant kingdom including mosses and lycophytes. Transcriptional activity analysis of Arabidopsis OFPs (AtOFPs in protoplasts suggests that they act as transcription repressors. Functional characterization of OFPs from different plant species including Arabidopsis, rice, tomato, pepper and banana suggests that OFPs regulate multiple aspects of plant growth and development, which is likely achieved by interacting with different types of transcription factors including the KNOX and BELL classes, and/or directly regulating the expression of target genes such as Gibberellin 20 oxidase (GA20ox. Here, we examine how OVATE was originally identified, summarize recent progress in elucidation of the roles of OFPs in regulating plant growth and development, and describe possible mechanisms underpinning this regulation. Finally, we review potential new research directions that could shed additional light on the functional biology of OFPs in plants.

  4. ESSENTIAL DYNAMICS OF PROTEINS

    NARCIS (Netherlands)

    AMADEI, A; LINSSEN, ABM; BERENDSEN, HJC

    1993-01-01

    Analysis of extended molecular dynamics (MD) simulations of lysozyme in vacuo and in aqueous solution reveals that it is possible to separate the configurational space into two subspaces: (1) an ''essential'' subspace containing only a few degrees of freedom in which anharmonic motion occurs that

  5. DCD – a novel plant specific domain in proteins involved in development and programmed cell death

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    Doerks Tobias

    2005-07-01

    Full Text Available Abstract Background Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins. Results The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in development and cell death. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone. Conclusion It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.

  6. Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

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    Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

    2016-01-01

    The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

  7. An efficient method for sampling the essential subspace of proteins

    NARCIS (Netherlands)

    Amadei, A; Linssen, A.B M; de Groot, B.L.; van Aalten, D.M.F.; Berendsen, H.J.C.

    A method is presented for a more efficient sampling of the configurational space of proteins as compared to conventional sampling techniques such as molecular dynamics. The method is based on the large conformational changes in proteins revealed by the ''essential dynamics'' analysis. A form of

  8. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes.

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    Jiawei Luo

    Full Text Available Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC, based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID, of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.Experimental results based on three different PPI(protein-protein interaction networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC.LIDC is more effective for the prediction of essential proteins than other recently developed methods.

  9. A comparison of techniques for calculating protein essential dynamics

    NARCIS (Netherlands)

    van Aalten, D.M.F.; de Groot, B.L.; Findlay, J.B.C.; Berendsen, H.J.C.; Amadei, A

    1997-01-01

    Recently the basic theory of essential dynamics, a method for extracting large concerted motions from protein molecular dynamics trajectories, was described. Here, we introduce and test new aspects. A method for diagonalizing large covariance matrices is presented. We show that it is possible to

  10. Blood harmane concentrations and dietary protein consumption in essential tremor.

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    Louis, E D; Zheng, W; Applegate, L; Shi, L; Factor-Litvak, P

    2005-08-09

    Beta-carboline alkaloids (e.g., harmane) are highly tremorogenic chemicals. Animal protein (meat) is the major dietary source of these alkaloids. The authors previously demonstrated that blood harmane concentrations were elevated in patients with essential tremor (ET) vs controls. Whether this difference is due to greater animal protein consumption by patients or their failure to metabolize harmane is unknown. The aim of this study was to determine whether patients with ET and controls differ with regard to 1) daily animal protein consumption and 2) the correlation between animal protein consumption and blood harmane concentration. Data on current diet were collected with a semiquantitative food frequency questionnaire and daily calories and consumption of animal protein and other food types was calculated. Blood harmane concentrations were log-transformed (logHA). The mean logHA was higher in 106 patients than 161 controls (0.61 +/- 0.67 vs 0.43 +/- 0.72 g(-10)/mL, p = 0.035). Patients and controls consumed similar amounts of animal protein (50.2 +/- 19.6 vs 49.4 +/- 19.1 g/day, p = 0.74) and other food types (animal fat, carbohydrates, vegetable fat) and had similar caloric intakes. In controls, logHA was correlated with daily consumption of animal protein (r = 0.24, p = 0.003); in patients, there was no such correlation (r = -0.003, p = 0.98). The similarity between patients and controls in daily animal protein consumption and the absence of the normal correlation between daily animal protein consumption and logHA in patients suggests that another factor (e.g., a metabolic defect) may be increasing blood harmane concentration in patients.

  11. DiffSLC: A graph centrality method to detect essential proteins of a protein-protein interaction network.

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    Mistry, Divya; Wise, Roger P; Dickerson, Julie A

    2017-01-01

    Identification of central genes and proteins in biomolecular networks provides credible candidates for pathway analysis, functional analysis, and essentiality prediction. The DiffSLC centrality measure predicts central and essential genes and proteins using a protein-protein interaction network. Network centrality measures prioritize nodes and edges based on their importance to the network topology. These measures helped identify critical genes and proteins in biomolecular networks. The proposed centrality measure, DiffSLC, combines the number of interactions of a protein and the gene coexpression values of genes from which those proteins were translated, as a weighting factor to bias the identification of essential proteins in a protein interaction network. Potentially essential proteins with low node degree are promoted through eigenvector centrality. Thus, the gene coexpression values are used in conjunction with the eigenvector of the network's adjacency matrix and edge clustering coefficient to improve essentiality prediction. The outcome of this prediction is shown using three variations: (1) inclusion or exclusion of gene co-expression data, (2) impact of different coexpression measures, and (3) impact of different gene expression data sets. For a total of seven networks, DiffSLC is compared to other centrality measures using Saccharomyces cerevisiae protein interaction networks and gene expression data. Comparisons are also performed for the top ranked proteins against the known essential genes from the Saccharomyces Gene Deletion Project, which show that DiffSLC detects more essential proteins and has a higher area under the ROC curve than other compared methods. This makes DiffSLC a stronger alternative to other centrality methods for detecting essential genes using a protein-protein interaction network that obeys centrality-lethality principle. DiffSLC is implemented using the igraph package in R, and networkx package in Python. The python package can be

  12. Whey protein-based films incorporated with oregano essential oil

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    Sandra Prestes Lessa Fernandes Oliveira

    Full Text Available Abstract This study aimed to prepare whey protein-based films incorporated with oregano essential oil at different concentrations, and evaluate their properties and antimicrobial activity. Films were more flexible with increasing the concentration of oregano oil and water vapor permeability was higher in the films with oregano oil. Increasing the concentration of essential oil decreased the water solubility. The solubility of control film and film with 1.5% oregano oil was 20.2 and 14.0%, respectively. The addition of 1% of oregano oil improved the resistance of the films. The tensile strength for the control film was 66.0 MPa, while for the film with 1% of oregano oil was 108.7 MPa. Films containing 1.5% oregano oil showed higher antimicrobial activity. The zone of inhibition ranged from 0 to 1.7 cm. The results showed that the whey protein-based films incorporated with oregano essential oil has potential application as active packaging.

  13. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

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    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  14. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

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    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  15. Essential protein discovery based on a combination of modularity and conservatism.

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    Zhao, Bihai; Wang, Jianxin; Li, Xueyong; Wu, Fang-Xiang

    2016-11-01

    Essential proteins are indispensable for the survival of a living organism and play important roles in the emerging field of synthetic biology. Many computational methods have been proposed to identify essential proteins by using the topological features of interactome networks. However, most of these methods ignored intrinsic biological meaning of proteins. Researches show that essentiality is tied not only to the protein or gene itself, but also to the molecular modules to which that protein belongs. The results of this study reveal the modularity of essential proteins. On the other hand, essential proteins are more evolutionarily conserved than nonessential proteins and frequently bind each other. That is to say, conservatism is another important feature of essential proteins. Multiple networks are constructed by integrating protein-protein interaction (PPI) networks, time course gene expression data and protein domain information. Based on these networks, a new essential protein identification method is proposed based on a combination of modularity and conservatism of proteins. Experimental results show that the proposed method outperforms other essential protein identification methods in terms of a number essential protein out of top ranked candidates. Copyright © 2016. Published by Elsevier Inc.

  16. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Energy Technology Data Exchange (ETDEWEB)

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  17. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    International Nuclear Information System (INIS)

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-01-01

    Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  18. In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein

    DEFF Research Database (Denmark)

    Folador, Edson Luiz; de Carvalho, Paulo Vinícius Sanches Daltro; Silva, Wanderson Marques

    2016-01-01

    BACKGROUND: Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation...... of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were...... not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. CONCLUSIONS: The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing...

  19. Prediction of essential proteins based on subcellular localization and gene expression correlation.

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    Fan, Yetian; Tang, Xiwei; Hu, Xiaohua; Wu, Wei; Ping, Qing

    2017-12-01

    Essential proteins are indispensable to the survival and development process of living organisms. To understand the functional mechanisms of essential proteins, which can be applied to the analysis of disease and design of drugs, it is important to identify essential proteins from a set of proteins first. As traditional experimental methods designed to test out essential proteins are usually expensive and laborious, computational methods, which utilize biological and topological features of proteins, have attracted more attention in recent years. Protein-protein interaction networks, together with other biological data, have been explored to improve the performance of essential protein prediction. The proposed method SCP is evaluated on Saccharomyces cerevisiae datasets and compared with five other methods. The results show that our method SCP outperforms the other five methods in terms of accuracy of essential protein prediction. In this paper, we propose a novel algorithm named SCP, which combines the ranking by a modified PageRank algorithm based on subcellular compartments information, with the ranking by Pearson correlation coefficient (PCC) calculated from gene expression data. Experiments show that subcellular localization information is promising in boosting essential protein prediction.

  20. Identifying essential proteins based on sub-network partition and prioritization by integrating subcellular localization information.

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    Li, Min; Li, Wenkai; Wu, Fang-Xiang; Pan, Yi; Wang, Jianxin

    2018-06-14

    Essential proteins are important participants in various life activities and play a vital role in the survival and reproduction of living organisms. Identification of essential proteins from protein-protein interaction (PPI) networks has great significance to facilitate the study of human complex diseases, the design of drugs and the development of bioinformatics and computational science. Studies have shown that highly connected proteins in a PPI network tend to be essential. A series of computational methods have been proposed to identify essential proteins by analyzing topological structures of PPI networks. However, the high noise in the PPI data can degrade the accuracy of essential protein prediction. Moreover, proteins must be located in the appropriate subcellular localization to perform their functions, and only when the proteins are located in the same subcellular localization, it is possible that they can interact with each other. In this paper, we propose a new network-based essential protein discovery method based on sub-network partition and prioritization by integrating subcellular localization information, named SPP. The proposed method SPP was tested on two different yeast PPI networks obtained from DIP database and BioGRID database. The experimental results show that SPP can effectively reduce the effect of false positives in PPI networks and predict essential proteins more accurately compared with other existing computational methods DC, BC, CC, SC, EC, IC, NC. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Utilization of NPP Krsko plant specific simulator

    International Nuclear Information System (INIS)

    Fifnja, I.; Pribozic, F.; Krajnc, J.

    2002-01-01

    NPP Krsko started with licensed operator training using its own plant-specific full scope simulator in April 2000. Today, two years after simulator was completed, the benefits of simulator use are visible in various fields. The simulator was effectively used to conduct licensed operator continuing training and practical examinations. Two-year continuous training program was designed to help maintain and improve operator performance. The simulator was also used to provide just-in-time training prior to plant evolutions. Together with licensed operators the non-licensed operators are also included into simulator training to provide affective team training opportunity and to foster good communication and increase scenario realism. Now, the first group of initial licensed operator training using plant-specific simulator is also almost completed. It is the first time that NPP Krsko training department conducted complete initial training and this will represent the great experience for future training. Besides training, the simulator was also utilized for procedure development and validation, operating standards development, testing of plant modifications and other activities, like emergency preparedness procedures validation and training exercises.(author)

  2. Predicting Essential Genes and Proteins Based on Machine Learning and Network Topological Features: A Comprehensive Review

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    Zhang, Xue; Acencio, Marcio Luis; Lemke, Ney

    2016-01-01

    Essential proteins/genes are indispensable to the survival or reproduction of an organism, and the deletion of such essential proteins will result in lethality or infertility. The identification of essential genes is very important not only for understanding the minimal requirements for survival of an organism, but also for finding human disease genes and new drug targets. Experimental methods for identifying essential genes are costly, time-consuming, and laborious. With the accumulation of sequenced genomes data and high-throughput experimental data, many computational methods for identifying essential proteins are proposed, which are useful complements to experimental methods. In this review, we show the state-of-the-art methods for identifying essential genes and proteins based on machine learning and network topological features, point out the progress and limitations of current methods, and discuss the challenges and directions for further research. PMID:27014079

  3. DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

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    Margarita Salas

    2016-08-01

    Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

  4. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

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    Jun Ren

    2014-01-01

    Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

  5. Targeting Plant Ethylene Responses by Controlling Essential Protein-Protein Interactions in the Ethylene Pathway.

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    Bisson, Melanie M A; Groth, Georg

    2015-08-01

    The gaseous plant hormone ethylene regulates many processes of high agronomic relevance throughout the life span of plants. A central element in ethylene signaling is the endoplasmic reticulum (ER)-localized membrane protein ethylene insensitive2 (EIN2). Recent studies indicate that in response to ethylene, the extra-membranous C-terminal end of EIN2 is proteolytically processed and translocated from the ER to the nucleus. Here, we report that the conserved nuclear localization signal (NLS) mediating nuclear import of the EIN2 C-terminus provides an important domain for complex formation with ethylene receptor ethylene response1 (ETR1). EIN2 lacking the NLS domain shows strongly reduced affinity for the receptor. Interaction of EIN2 and ETR1 is also blocked by a synthetic peptide of the NLS motif. The corresponding peptide substantially reduces ethylene responses in planta. Our results uncover a novel mechanism and type of inhibitor interfering with ethylene signal transduction and ethylene responses in plants. Disruption of essential protein-protein interactions in the ethylene signaling pathway as shown in our study for the EIN2-ETR1 complex has the potential to guide the development of innovative ethylene antagonists for modern agriculture and horticulture. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  6. A powerful tool for plant specific simulations

    International Nuclear Information System (INIS)

    Boucau, J.; Ofstun, R.

    1988-01-01

    Following the Three Mile Island (TMI) accident, special emphasis has been placed by the Utilities and Safety Authorities on the training of plant operators and engineers to make them more familiar with the dynamic behavior of the plant in post-accident conditions. With a view to providing transient response under realistic operating conditions, concerns were raised over: the use of conservative safety analysis models for analyzing operational and accident transients; the ability to dynamically simulate the expected operator actions, and the need to simulate the plant during long term cooling recovery. To meet these requirements, an interactive, user friendly, cost efficient dynamic model capable of simulating two-phase conditions is needed. In response to these post-TMI concerns, Westinghouse has developed, the Transient Real-Time Engineering Analysis Tool (TREAT) which is a general purpose interactive thermal-hydraulic network code for thermal-hydraulic transient analysis. The applications of TREAT are numerous and range from engineering studies to plant operator training using the newly developed TREAT SIMULATOR. The purpose of this paper is to describe the TREAT program and potential applications. Special emphasis will be drawn at a plant specific training application performed for a French 4-loop plant

  7. Plant specific basic principle simulator as a first step to plant specific full scope simulator

    International Nuclear Information System (INIS)

    Krajnc, B.; Pribozic, F.; Novsak, M.

    1996-01-01

    Nuklearna Elektrarna Krsko (NEK) decided to enhance the quality and scope of initial training of NEK technical personnel, mainly in so called Phase 1 and 2 of training for licensed personnel. This training is a prerequisite for further training on the full scope simulator for future operators and is also given to larger number of engineers, working in different important areas where thorough knowledge of nuclear technology and plant systems is required. Due to that it was decided that plant specific Basis Principle Simulators (BPS) should be developed. The other important reason for such decision was an indication that NEK specific full scope simulator will have to be purchased. Based on that it was concluded that BPS should serve as a good opportunity to learn about the state of the art approaches in the modeling area, to see in which direction development of software in conjunction with state of the art hardware is going and in particular to the extent possible verify the existence of required plant documentation in support BPS and later plant specific full scope simulator. In this paper the scope of NEK BPS simulation, experience in initial data gathering, experience with know-how transfer based on direct involvement of NEK and Izobrazevalni Center za Jedrsko Tehnnologijo (ICJT) personnel in modeling of instrumentation and control will be presented. Lessons learned, particularly in light of coming project for NEK full scope simulator, will also be addressed. The future use of the BPS in the NEK training programs will be described. It can be concluded that due to very complex technology, phase approaches in training of key NEK technical personnel, the development of NEK plant specific BPS is justifiable, regardless of the fact that NEK will also obtain specific full scope simulator. It has to be pointed out that BPS can not be supplement for plant specific full scope simulator, due to number of reasons discussed in the paper. (author)

  8. An essential nonredundant role for mycobacterial DnaK in native protein folding.

    Directory of Open Access Journals (Sweden)

    Allison Fay

    2014-07-01

    Full Text Available Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.

  9. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    Directory of Open Access Journals (Sweden)

    Kyle J. Hackney

    2014-07-01

    Full Text Available Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids stimulate anabolic pathways (e.g., muscle protein synthesis both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1 and spaceflight is associated with reduced energy intake (~20%, which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake.

  10. DNA binding by the plant-specific NAC transcription factors in crystal and solution

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Lindemose, Søren; Grossmann, J. Günter

    2012-01-01

    angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY...

  11. Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

    Science.gov (United States)

    Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

    2017-06-01

    Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

  12. Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

    NARCIS (Netherlands)

    Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

    2012-01-01

    The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

  13. A genetic replacement system for selection-based engineering of essential proteins

    Science.gov (United States)

    2012-01-01

    Background Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. Results We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. Conclusions We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies. PMID:22898007

  14. The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

    DEFF Research Database (Denmark)

    Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

    2005-01-01

    MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B...... carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and Mre....... subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became...

  15. The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

    International Nuclear Information System (INIS)

    Isidro, Anabela; Henriques, Adriano O.; Tavares, Paulo

    2004-01-01

    A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses

  16. Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein

    Directory of Open Access Journals (Sweden)

    Stout Jeffrey R

    2010-06-01

    Full Text Available Abstract Regardless of age or gender, resistance training or provision of adequate amounts of dietary protein (PRO or essential amino acids (EAA can increase muscle protein synthesis (MPS in healthy adults. Combined PRO or EAA ingestion proximal to resistance training, however, can augment the post-exercise MPS response and has been shown to elicit a greater anabolic effect than exercise plus carbohydrate. Unfortunately, chronic/adaptive response data comparing the effects of different protein sources is limited. A growing body of evidence does, however, suggest that dairy PRO, and whey in particular may: 1 stimulate the greatest rise in MPS, 2 result in greater muscle cross-sectional area when combined with chronic resistance training, and 3 at least in younger individuals, enhance exercise recovery. Therefore, this review will focus on whey protein supplementation and its effects on skeletal muscle mass when combined with heavy resistance training.

  17. Metalloido-porins: Essentiality of Nodulin 26-like intrinsic proteins in metalloid transport.

    Science.gov (United States)

    Pommerrenig, Benjamin; Diehn, Till Arvid; Bienert, Gerd Patrick

    2015-09-01

    Metalloids are a group of physiologically important elements ranging from the essential to the highly toxic. Arsenic, antimony, germanium, and tellurium are highly toxic to plants themselves and to consumers of metalloid-contaminated plants. Boron, silicon, and selenium fulfill essential or beneficial functions in plants. However, when present at high concentrations, boron and selenium cause toxicity symptoms that are detrimental to plant fitness and yield. Consequently, all plants require efficient membrane transport systems to control the uptake and extrusion of metalloids into or out of the plant and their distribution within the plant body. Several Nodulin 26-like intrinsic proteins (NIPs) that belong to the aquaporin plant water channel protein family facilitate the diffusion of uncharged metalloid species. Genetic, physiological, and molecular evidence is that NIPs from primitive to higher plants not only transport all environmentally important metalloids, but that these proteins have a major role in the uptake, translocation, and extrusion of metalloids in plants. As most of the metalloid-permeable NIP aquaporins are impermeable or are poorly permeable to water, these NIP channel proteins should be considered as physiologically essential metalloido-porins. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  18. Acyl-CoA binding protein is an essential protein in mammalian cell lines

    DEFF Research Database (Denmark)

    Knudsen, Jens; Færgeman, Nils J.

    2002-01-01

    In the present work, small interference RNA was used to knock-down acyl-CoA binding protein (ACBP) in HeLa, HepG2 and Chang cells. Transfection with ACBP-specific siRNA stopped growth, detached cells from the growth surface and blocked thymidine and acetate incorporation. The results show...

  19. Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Svenja Günther

    2007-12-01

    Full Text Available Lipoic acid (LA is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1. Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%. Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2. Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.

  20. Tob38, a novel essential component in the biogenesis of β-barrel proteins of mitochondria

    Science.gov (United States)

    Waizenegger, Thomas; Habib, Shukry J; Lech, Maciej; Mokranjac, Dejana; Paschen, Stefan A; Hell, Kai; Neupert, Walter; Rapaport, Doron

    2004-01-01

    Insertion of β-barrel proteins into the outer membrane of mitochondria is mediated by the TOB complex. Known constituents of this complex are Tob55 and Mas37. We identified a novel component, Tob38. It is essential for viability of yeast and the function of the TOB complex. Tob38 is exposed on the surface of the mitochondrial outer membrane. It interacts with Mas37 and Tob55 and is associated with Tob55 even in the absence of Mas37. The Tob38–Tob55 core complex binds precursors of β-barrel proteins and facilitates their insertion into the outer membrane. Depletion of Tob38 results in strongly reduced levels of Tob55 and Mas37 and the residual proteins no longer form a complex. Tob38-depleted mitochondria are deficient in the import of β-barrel precursor proteins, but not of other outer membrane proteins or proteins of other mitochondrial subcompartments. We conclude that Tob38 has a crucial function in the biogenesis of β-barrel proteins of mitochondria. PMID:15205677

  1. IQGAP1 is a novel CXCR2-interacting protein and essential component of the "chemosynapse".

    Directory of Open Access Journals (Sweden)

    Nicole F Neel

    Full Text Available Chemotaxis is essential for a number of physiological processes including leukocyte recruitment. Chemokines initiate intracellular signaling pathways necessary for chemotaxis through binding seven transmembrane G protein-couple receptors. Little is known about the proteins that interact with the intracellular domains of chemokine receptors to initiate cellular signaling upon ligand binding. CXCR2 is a major chemokine receptor expressed on several cell types, including endothelial cells and neutrophils. We hypothesize that multiple proteins interact with the intracellular domains of CXCR2 upon ligand stimulation and these interactions comprise a "chemosynapse", and play important roles in transducing CXCR2 mediated signaling processes.In an effort to define the complex of proteins that assemble upon CXCR2 activation to relay signals from activated chemokine receptors, a proteomics approach was employed to identify proteins that co-associate with CXCR2 with or without ligand stimulation. The components of the CXCR2 "chemosynapse" are involved in processes ranging from intracellular trafficking to cytoskeletal modification. IQ motif containing GTPase activating protein 1 (IQGAP1 was among the novel proteins identified to interact directly with CXCR2. Herein, we demonstrate that CXCR2 co-localizes with IQGAP1 at the leading edge of polarized human neutrophils and CXCR2 expressing differentiated HL-60 cells. Moreover, amino acids 1-160 of IQGAP1 directly interact with the carboxyl-terminal domain of CXCR2 and stimulation with CXCL8 enhances IQGAP1 association with Cdc42.Our studies indicate that IQGAP1 is a novel essential component of the CXCR2 "chemosynapse".

  2. Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

    Directory of Open Access Journals (Sweden)

    Karina E. J. Tripodi

    2011-01-01

    Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

  3. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

    DEFF Research Database (Denmark)

    Lasonder, Edwin; Janse, Chris J; van Gemert, Geert-Jan

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature...... whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed...... three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may...

  4. Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

    International Nuclear Information System (INIS)

    Karim, Shahid; Ramakrishnan, Vijay G.; Tucker, James S.; Essenberg, Richard C.; Sauer, John R.

    2004-01-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in 'knocking down' nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands

  5. Use of plant specific PSA to evaluate incidents at nuclear power plants

    International Nuclear Information System (INIS)

    1991-06-01

    One of the possible applications of the plant specific probabilistic safety assessment (PSA) is its use in the analysis of operational events at the plant. The methodological development in that area was initiated recently in the framework of the IAEA's Incident Reporting System where determination of the safety significance of the event is essential for optimizing feedback of operating experience. This report provides details of the methodology and procedures to be used in event analysis. The report also contains three case studies which have been performed and summarizes lessons learned from those case studies. The results (event probabilities) obtained using plant specific PSA and the results of the analysis of the same events in the framework of the Accident Sequence Precursor (ASP) programmes (generic models) were compared and commented on. 6 refs, figs and tabs

  6. Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

    KAUST Repository

    Beck, K.

    2013-05-06

    The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

  7. Trypanosoma brucei Tb927.2.6100 Is an Essential Protein Associated with Kinetoplast DNA

    KAUST Repository

    Beck, K.; Acestor, N.; Schulfer, A.; Anupama, A.; Carnes, J.; Panigrahi, A. K.; Stuart, K.

    2013-01-01

    The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.

  8. Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

    Directory of Open Access Journals (Sweden)

    Noriko Umegaki-Arao

    Full Text Available Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2 expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

  9. Structure of an essential bacterial protein YeaZ (TM0874) from Thermotoga maritima at 2.5 Å resolution

    International Nuclear Information System (INIS)

    Xu, Qingping; McMullan, Daniel; Jaroszewski, Lukasz; Krishna, S. Sri; Elsliger, Marc-André; Yeh, Andrew P.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Duan, Lian; Feuerhelm, Julie; Grant, Joanna; Han, Gye Won; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Bedem, Henry van den; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    The crystal structure of an essential bacterial protein, YeaZ, from T. maritima identifies an interface that potentially mediates protein–protein interaction. YeaZ is involved in a protein network that is essential for bacteria. The crystal structure of YeaZ from Thermotoga maritima was determined to 2.5 Å resolution. Although this protein belongs to a family of ancient actin-like ATPases, it appears that it has lost the ability to bind ATP since it lacks some key structural features that are important for interaction with ATP. A conserved surface was identified, supporting its role in the formation of protein complexes

  10. Selective renal vasoconstriction, exaggerated natriuresis and excretion rates of exosomic proteins in essential hypertension

    DEFF Research Database (Denmark)

    Damkjaer, M.; Jensen, Pia Hønnerup; Schwämmle, Veit

    2014-01-01

    AimIn essential hypertension (EH), the regulation of renal sodium excretion is aberrant. We hypothesized that in mild EH, (i) abnormal dynamics of plasma renin concentration (PRC) and atrial natriuretic peptide (ANP) are responsible for the exaggerated natriuresis, and (ii) exosomic protein...... patterns reflect the renal tubular abnormality involved in the dysregulation of sodium excretion. MethodsAfter 2-week drug washout and 4-day diet, systemic and renal hemodynamics, cardio-renal hormones, glomerular filtration and renal excretion were studied in male patients during saline loading (SL...

  11. An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein

    International Nuclear Information System (INIS)

    Janaki Ramaiah, M.; Parnaik, Veena K.

    2006-01-01

    Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif

  12. An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites.

    Science.gov (United States)

    Absalon, Sabrina; Robbins, Jonathan A; Dvorin, Jeffrey D

    2016-04-28

    Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites.

  13. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    OpenAIRE

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential a...

  14. Molecular characterization of the Borrelia burgdorferi in vivo-essential protein PncA.

    Science.gov (United States)

    Jewett, Mollie W; Jain, Sunny; Linowski, Angelika K; Sarkar, Amit; Rosa, Patricia A

    2011-10-01

    The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD(+) homeostasis and contributes to numerous important biological processes in diverse organisms. In Borrelia burgdorferi, the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the in vivo importance of PncA, the current annotation for the pncA ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of B. burgdorferi PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that pncA translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this in vivo-essential protein.

  15. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita; Straschil, Ursula; Bateman, Alex; Bö hme, Ulrike; Cherevach, Inna; Gong, Peng; Pain, Arnab; Billker, Oliver

    2010-01-01

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  16. The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

    KAUST Repository

    Tewari, Rita

    2010-10-21

    Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

  17. Stability of lime essential oil microparticles produced with protein-carbohydrate blends.

    Science.gov (United States)

    Campelo, Pedro Henrique; Sanches, Edgar Aparecido; Fernandes, Regiane Victória de Barros; Botrel, Diego Alvarenga; Borges, Soraia Vilela

    2018-03-01

    The objective of this work was to analyze the influence of maltodextrin equivalent dextrose on the lime essential oil reconstitution, storage, release and protection properties. Four treatments were evaluated: whey protein concentrate (WPC), and blends of maltodextrin with dextrose equivalents of 5 (WM5), 10 (WM10) and 20 (WM20). The reconstitution and storage properties of the microparticles (solubility, wettability and density), water kinetics adsorption, sorption isotherms, thermogravimetric properties, controlled release and degradation kinetics of encapsulated lime essential oil were studied to measure the quality of the encapsulated materials. The results of the study indicated that the DE degree influences the characteristics of reconstitution, storage, controlled release and degradation characteristics of encapsulated bioactive compounds. The increase in dextrose equivalent improves microparticle solubility, wettability and density, mainly due to the size of the maltodextrin molecules. The adsorption kinetics and sorption isotherm curves confirmed the increase in the hygroscopicity of maltodextrins with higher degrees of polymerization. The size of the maltodextrin chains influenced the release and protection of the encapsulated lime essential oil. Finally, the maltodextrin polymerization degree can be considered a parameter that will influence the physicochemical properties of microencapsulated food. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

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    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  19. The α-tocopherol transfer protein is essential for vertebrate embryogenesis.

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    Galen W Miller

    Full Text Available The hepatic α-tocopherol transfer protein (TTP is required for optimal α-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460. TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP's significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos. We conclude that TTP is essential for early development of the vertebrate central nervous system.

  20. A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility.

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    Sabine Schramm

    2011-05-01

    Full Text Available The synaptonemal complex (SC is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.

  1. Dimerization of translationally controlled tumor protein is essential for its cytokine-like activity.

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    Miyoung Kim

    Full Text Available BACKGROUND: Translationally Controlled Tumor Protein (TCTP found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF. Human recombinant HRF (HrHRF has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn. METHODS AND FINDINGS: We found that only NH(2-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH(2-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP. Allergenic activity of Del-N11TCTP prompted us to see whether the NH(2-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice. CONCLUSIONS: Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.

  2. Functional dissection of Streptococcus pyogenes M5 protein: the hypervariable region is essential for virulence.

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    Johan Waldemarsson

    Full Text Available The surface-localized M protein of Streptococcus pyogenes is a major virulence factor that inhibits phagocytosis, as determined ex vivo. Because little is known about the role of M protein in vivo we analyzed the contribution of different M protein regions to virulence, using the fibrinogen (Fg-binding M5 protein and a mouse model of acute invasive infection. This model was suitable, because M5 is required for mouse virulence and binds mouse and human Fg equally well, as shown here. Mixed infection experiments with wild type bacteria demonstrated that mutants lacking the N-terminal hypervariable region (HVR or the Fg-binding B-repeat region were strongly attenuated, while a mutant lacking the conserved C-repeats was only slightly attenuated. Because the HVR of M5 is not required for phagocytosis resistance, our data imply that this HVR plays a major but unknown role during acute infection. The B-repeat region is required for phagocytosis resistance and specifically binds Fg, suggesting that it promotes virulence by binding Fg. However, B-repeat mutants were attenuated even in Fg-deficient mice, implying that the B-repeats may have a second function, in addition to Fg-binding. These data demonstrate that two distinct M5 regions, including the HVR, are essential to virulence during the early stages of an infection. In particular, our data provide the first in vivo evidence that the HVR of an M protein plays a major role in virulence, focusing interest on the molecular role of this region.

  3. Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

    Science.gov (United States)

    Xu, Kai; Nagy, Peter D

    2017-04-01

    Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are

  4. XIRP2, an Actin-Binding Protein Essential for Inner Ear Hair-Cell Stereocilia

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    Déborah I. Scheffer

    2015-03-01

    Full Text Available Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.

  5. Plant specific PTS analysis of Kori Unit 1

    Energy Technology Data Exchange (ETDEWEB)

    Sung-Yull, Hong; Changheui, Jang; Ill-Seok, Jeong [Korea Eletric Power Research Inst., Daejon (Korea, Republic of); Tae-Eun, Jin [Korea Power Engineering Company, Yonging (Korea, Republic of)

    1997-09-01

    Currently, a nuclear PLIM (Plant Lifetime Management) program is underway in Korea to extend the operation life of Kori-1 which was originally licensed for 30 years. For the life extension of nuclear power plants, the residual lives of major components should be evaluated for the extended operation period. According to the residual life evaluation of reactor pressure vessel, which was classified as one of the major components crucial to life extension, it was found by screening analysis that reference PTS temperature would exceed screening criteria before the target extended operation years. In order to deal with this problem, a plant-specific PTS analysis for Kori-1 RPV has been initiated. In this paper, the relationship between PTS analysis and Kori-1 PLIM program is briefly described. The plant-specific PTS analysis covers system transient analysis, downcomer mixing analysis, and probabilistic fracture mechanics analysis to check the integrity or RPV during various PTS transients. The step-by-step procedure of the analysis will be described in detail. Finally, various issues regarding RPV materials and its integrity will be briefly mentioned, and their implications on Kori-1 PTS analysis will be discussed. Despite of the screening analysis result concern, it is now expected that Kori-1 PTS issues can be handled through the plant-specific PTS analysis. (author). 14 refs, 4 figs, 2 tabs.

  6. The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

    Science.gov (United States)

    Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

    2005-06-01

    Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

  7. From hub proteins to hub modules: the relationship between essentiality and centrality in the yeast interactome at different scales of organization.

    Directory of Open Access Journals (Sweden)

    Jimin Song

    Full Text Available Numerous studies have suggested that hub proteins in the S. cerevisiae physical interaction network are more likely to be essential than other proteins. The proposed reasons underlying this observed relationship between topology and functioning have been subject to some controversy, with recent work suggesting that it arises due to the participation of hub proteins in essential complexes and processes. However, do these essential modules themselves have distinct network characteristics, and how do their essential proteins differ in their topological properties from their non-essential proteins? We aimed to advance our understanding of protein essentiality by analyzing proteins, complexes and processes within their broader functional context and by considering physical interactions both within and across complexes and biological processes. In agreement with the view that essentiality is a modular property, we found that the number of intracomplex or intraprocess interactions that a protein has is a better indicator of its essentiality than its overall number of interactions. Moreover, we found that within an essential complex, its essential proteins have on average more interactions, especially intracomplex interactions, than its non-essential proteins. Finally, we built a module-level interaction network and found that essential complexes and processes tend to have higher interaction degrees in this network than non-essential complexes and processes; that is, they exhibit a larger amount of functional cross-talk than their non-essential counterparts.

  8. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Science.gov (United States)

    Savage, Linda J; Imre, Kathleen M; Hall, David A; Last, Robert L

    2013-01-01

    The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

  9. Analysis of essential Arabidopsis nuclear genes encoding plastid-targeted proteins.

    Directory of Open Access Journals (Sweden)

    Linda J Savage

    Full Text Available The Chloroplast 2010 Project (http://www.plastid.msu.edu/ identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ~1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/. Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles

  10. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  11. The plant i-AAA protease controls the turnover of an essential mitochondrial protein import component.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Murcha, Monika W; Jańska, Hanna

    2018-01-29

    Mitochondria are multifunctional organelles that play a central role in energy metabolism. Owing to the life-essential functions of these organelles, mitochondrial content, quality and dynamics are tightly controlled. Across the species, highly conserved ATP-dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of the plant mitochondrial inner membrane-embedded AAA protease (denoted i -AAA) FTSH4, providing its first bona fide substrate. Here, we report that the abundance of the Tim17-2 protein, an essential component of the TIM17:23 translocase (Tim17-2 together with Tim50 and Tim23), is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23-dependent pathway. Taken together, with the observation that FTSH4 prevents accumulation of Tim17-2, our data point towards the role of this i -AAA protease in the regulation of mitochondrial biogenesis in plants. © 2018. Published by The Company of Biologists Ltd.

  12. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    Science.gov (United States)

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  13. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    2016-06-01

    Full Text Available Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise.

  14. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    International Nuclear Information System (INIS)

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

    2016-01-01

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

  15. The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

    Science.gov (United States)

    Takahashi, Chika; Miyatake, Koichi; Kusakabe, Morioh; Nishida, Eisuke

    2018-06-01

    Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

    2016-05-27

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

  17. Effects of enalapril on urinary protein excretion of essential and renal parenchymal hypertensive patients

    International Nuclear Information System (INIS)

    Mazzucca, N.; Falciani, C.; Morini, V.; Bigazzi, R.; Paparatto, P.; Setti, G.P.; Bianchi, S.; Baldari, G.; Valteriani, C.; Chiapponi, I.

    1988-01-01

    Angiotensin converting enzyme (ACE) inhibiting drugs are able to reduce urinary protein excretion in experimental hypertension and in hypertensive patients with diabetes. Fifteen essential (group I) and six renal parenchymal (group II) mild or moderate hypertensive patients were treated with the ACE inhibitor Enalapril in monotherapy or in combination with a diuretic. Twenty-four hour urinary protein excretion was measured by means of colorimetric and RIA methods. All patients of group I had a significant decrease of arterial pressure with Enalapril alone and this reduction was dosage dependent. Three out of six patients of group II required the addition of diuretic to achieve a good pressure control. Serum creatinine values were stable in group I, while one patient of group II, who already had high baseline creatinine levels, showed an impairment of renal function requiring discontinuation of therapy. Twenty-four hour urinary protein excretion did not change in group I, while after two months of therapy a significant decrease was observed in group II (P<0.05), which was even more evident after 4 months (P<0.03). In this group a good correlation between MAP and proteinuria was observed. Finally, compared to the colorimetric method, RIA method seems to be more sensitive to assess the variations under Enalapril treatment. In conclusion, Enalapril is an effective drug in patients with moderate or mild hypertension. Caution must be exercised in administering Enalapril to patients with severe renal failure. Also in hypertensive patients with mild renal failure ACE inhibition appears to induce an antiproteinuric effect during long term therapy. This fact could be related to an improved hemodynamic intraglomerular status due to the renal effects of the drug. Finally urinary albumin RIA method seems to be more sensitive than colorimetric evaluation to follow-up the variations of proteinuria under Enalapril treatment

  18. Nuclear utility education and training becoming too plant specific?

    International Nuclear Information System (INIS)

    Wicks, F.

    1986-01-01

    As the Supervisor of a university nuclear reactor and operations curriculum, the author has also been offering education and training programs for nuclear utility technical support and operations personnel. Similar results have been reported by other universities offering similar programs. These programs also provide very important benefits to university nuclear engineering departments in terms of much needed revenues during this time of declining student enrollment and also by the information flow from the nuclear utility participants to the university personnel, which can yield both improved courses and identify research opportunities. University programs serve an important complementary function to plant-specific programs and should be continued and supported

  19. The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish.

    Directory of Open Access Journals (Sweden)

    Ian G Woods

    2005-03-01

    Full Text Available Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

  20. M6 membrane protein plays an essential role in Drosophila oogenesis.

    Science.gov (United States)

    Zappia, María Paula; Brocco, Marcela Adriana; Billi, Silvia C; Frasch, Alberto C; Ceriani, María Fernanda

    2011-01-01

    We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.

  1. Whey protein isolate edible films with essential oils incorporated to improve the microbial quality of poultry.

    Science.gov (United States)

    Fernández-Pan, Idoya; Mendoza, Mauricio; Maté, Juan I

    2013-09-01

    Whey protein isolate edible films with oregano or clove essential oils (EOs) incorporated as natural antimicrobials have been developed, with the aim of enhancing the microbial quality of poultry. The effectiveness of the films was determined against both the whole and selected microbiota developed during different periods of cold storage on the surface of skinless chicken breast. Tests were conducted by using both turbidimetric and agar disc diffusion methods. The antimicrobial edible films developed showed high effectiveness against the main spoilers developed on the surface of skinless chicken breasts cold-stored for 8 days. The films based on oregano EO showed greater effectiveness than those based on clove EO. Still, clove EO could be part of an effective antimicrobial edible film. Enterobacteriaceae was the most susceptible to the effect of the films when lower concentrations of EO were incorporated. The largest inhibition surfaces obtained were provoked by films with the highest concentration of oregano EO incorporated against lactic acid bacteria. The antimicrobial edible films developed in this study inhibited the growth of the microbial populations that developed through storage of the chicken breast and caused its spoilage. The results of this research have direct application in the food industry to enhance the control of the development of spoilers such as Pseudomonas spp. or lactic acid bacteria. © 2013 Society of Chemical Industry.

  2. Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

    Science.gov (United States)

    San Martin, Fabiana; Mechaly, Ariel E; Larrieux, Nicole; Wunder, Elsio A; Ko, Albert I; Picardeau, Mathieu; Trajtenberg, Felipe; Buschiazzo, Alejandro

    2017-03-01

    The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic species L. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

  3. Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

    International Nuclear Information System (INIS)

    Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

    2010-01-01

    Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

  4. Plant specific severe accident management - the implementation phase

    International Nuclear Information System (INIS)

    Prior, R.

    1999-01-01

    Many plants are in the process of developing on-site guidance for technical staff to respond to a severe accident situation severe accident management guidance (SAMG). Once the guidance is developed, the SAMG must be implemented at the plant site, and this involves addressing a number of additional aspects. In this paper, approaches to this implementation phase are reviewed, including review and verification of plant specific SAMG, organizational aspects and integration with the emergency plan, training of SAMG users, validation and self-assessment and SAMG maintenance. Examples draw on experience from assisting numerous plants to implement symptom based severe accident management guidelines based on the Westinghouse Owners Group approach, in Westinghouse, non-Westinghouse and VVER plant types. It is hoped that it will be of use to those plant operators about to perform these activities.(author)

  5. Flooding PSA with Plant Specific Operating Experiences of Korean PWRs

    International Nuclear Information System (INIS)

    Choi, Sun Yeong; Yang, Joon Yull

    2006-01-01

    The purpose of this paper is to update the flooding PSA with Korean plant specific operating experience data and the appropriate estimation method for the flooding frequency to improve the PSA quality. The existing flooding PSA used the NPE (Nuclear Power Experience) database up to 1985 for the flooding frequency. They are all USA plant operating experiences. So an upgraded flooding frequency with Korean specific plant operation experience is required. We also propose a method of only using the PWR (Pressurized Water Reactor) data for the flooding frequency estimation in the case of the flooding area in the primary building even though the existing flooding PSA used both PWR and BWR (Boiled Water Reactor) data for all kinds of plant areas. We evaluate the CDF (Core Damage Frequency) with the modified flooding frequency and compare the results with that of the existing flooding PSA method

  6. Properties and Antioxidant Capacity of Anchovy (Engraulis encrasicholus) By-Product Protein Films Containing Thyme Essential Oil.

    Science.gov (United States)

    Tural, Serpil; Turhan, Sadettin

    2017-03-01

    In this study, some properties and antioxidant capacity of anchovy ( Engraulis encrasicholus ) by-product protein films with added 0.5, 1.0 and 1.5% of thyme essential oil were investigated. The films with thyme essential oil had higher elongation at break, water vapour permeability and oxygen permeability, lower solubility and tensile strength than control film (p<0.05). The incorporation of thyme essential oil affected transparency values of the films, but only the addition of 1.5% of thyme essential oil significantly reduced the transparency (p<0.05). In the film matrix, molecular organisation and intermolecular interaction were changed by thyme essential oil addition. The films with thyme essential oil had a heterogeneous surface and a relatively smooth cross-section structure. Slightly higher phase transition and lower glass transition temperatures were observed in films with thyme essential oil. The antioxidant capacity of the films was improved by incorporating thyme essential oil depending on its volume fraction.

  7. A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

    Science.gov (United States)

    Peng, Zeyu; Green, Peter G; Arakane, Yasuyuki; Kanost, Michael R; Gorman, Maureen J

    2014-01-01

    Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

  8. Use of plant specific information in life management

    International Nuclear Information System (INIS)

    Simola, K.; Talja, H.

    2002-01-01

    In plant life management decisions are made on prevention and mitigation measures of ageing phenomena. In these processes, information from several sources has to be combined, and the decisions are based on data and analyses including lots of uncertainties. In order to make good decisions, the uncertainties and limitations related to both analyses and the raw data should be recognised. A schematic presentation of the information used in the decision making with an emphasis on data needs and analyses for the technical life assessment of a component is given. On the way from the raw data to the final decision on ageing management there are several steps where engineering judgement is used or more sophisticated analyses are made. In this paper we present a view upon the decision making process in managing the ageing of components, systems and structures. Further, we propose practices to improve the transparency of ageing analyses and means to improve the availability and usability of plant specific information for ageing management purposes. The availability of plant specific information and easy access to these records are vital for the efficient ageing management. Often, the data collection and record keeping has not been organised in an efficient way and the use of experience data is very laborious. As illustrated, a lot of plant specific information is needed in ageing assessment. It is also very important to ensure that the information is up-to-date, e.g. possible modifications are taken into account in lifetime predictions. As an example of improvement in the data collection practices we shortly summarise the pipeline analysis and monitoring system that VTT is developing together with TVO. The system is meant to contain all up-to-date information necessary to analyse and monitor piping systems of an operating plant. The core of the system contains five databases. Piping database contains information like geometry, material, loading, detected cracks etc. All

  9. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  10. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    International Nuclear Information System (INIS)

    Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian; Roitberg, Adrian E.

    2015-01-01

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

  11. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    Energy Technology Data Exchange (ETDEWEB)

    Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

    2015-06-28

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

  12. The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

    DEFF Research Database (Denmark)

    Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

    2009-01-01

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

  13. Preparation of plant-specific NDA reference material

    International Nuclear Information System (INIS)

    Abedin-Zadeh, R.; Beetle, T.; Kuhn, E.; Terrey, D.; Turel, S.; Busca, G.; Guardini, S.

    1983-01-01

    The importance of having suitable and well characterized non-destructive assay (NDA) reference materials for the verification activities of the safeguards control authorities is stressed. The Euratom Inspectorate and the IAEA have initiated an extensive programme for the procurement and preparation of Joint Euratom/IAEA safeguards NDA reference materials with the active participation of the Ispra Establishment of the Euratom Joint Research Centre. The different type and nature of materials, condition of measurements, and plant characteristics and provisions had to be taken into account for plant-specific NDA reference materials. The preparation of each reference material was planned case by case and specific criteria such as limitations in different facilities, measurement capabilities, conditions, product availability and population variability are being ascertained. A procurement scheme was prepared describing step-by-step procedures detailing responsibilities, measurement conditions, destructive analysis schemes, desired characteristics and methods of data evaluation. This paper describes the principles and procedures carried out for the preparation of a reference MOX pin, low enriched uranium reference rods, low enriched uranium reference drums, reference MTR assemblies, and THTR reference pebbles. The scheme for each characterization technique is presented. (author)

  14. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

    Science.gov (United States)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-08-24

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

  15. The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

    Directory of Open Access Journals (Sweden)

    Bond Charles S

    2011-03-01

    Full Text Available Abstract Background The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. Results To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. Conclusions Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.

  16. Cysteine residue is not essential for CPM protein thermal-stability assay.

    Science.gov (United States)

    Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

    2015-05-01

    A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

  17. Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

    Science.gov (United States)

    Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

    2013-12-01

    Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

  18. Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.

    Science.gov (United States)

    Gevorkyan-Airapetov, Lada; Zohary, Keren; Popov-Celeketic, Dusan; Mapa, Koyeli; Hell, Kai; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

    2009-02-20

    The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23. We established two independent methods, chemical cross-linking and surface plasmon resonance, to track their interaction. In addition, we identified mutations in Tim23 that abolish its interaction with Tim50 in vitro. These mutations also destabilized the interaction between the two proteins in vivo, leading to defective import of preproteins via the TIM23 complex and to cell death at higher temperatures. This is the first study to describe the reconstitution of the Tim50-Tim23 interaction in vitro and to identify specific residues of Tim23 that are vital for the interaction with Tim50.

  19. Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

    NARCIS (Netherlands)

    Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J; Iyer, K Swaminathan; Ho, Diwei; Newcombe, Estella A; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S; Fox, Archa H

    2015-01-01

    Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule

  20. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins.

    LENUS (Irish Health Repository)

    Morgan, Claire C

    2010-01-01

    ABSTRACT: BACKGROUND: Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. RESULTS: We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. CONCLUSION: Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene.

  1. Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

    International Nuclear Information System (INIS)

    Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana

    2005-01-01

    Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH 2 -terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process

  2. Comprehensive analysis of LANA interacting proteins essential for viral genome tethering and persistence.

    Directory of Open Access Journals (Sweden)

    Subhash C Verma

    Full Text Available Kaposi's sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi's sarcoma (KS, Primary Effusion Lymphoma (PEL and Multicentric Castleman's Disease (MCD. KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA. LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs. We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET to demonstrate their association.

  3. Renoprotective Effects of Low-protein Diet with the Use of Keto-analogues of Essential Amino Acids Using

    Directory of Open Access Journals (Sweden)

    O.I. Romadanova

    2013-10-01

    Full Text Available The article discusses the role of low-protein diet and keto-analogues of essential amino acids in patients with impaired renal function. On the basis of results of the study on regularities of monocyte chemoattractant protein level (MCP-1, depending on the origin of glomerular damage and stage of chronic kidney disease and the dynamics of its changes under the influence of different approaches in complex treatment, it is concluded that administration of low-protein diet with inclusion of Ketosteril is necessary already at the early stages of the diseases for renoprotective action. Use of Ketosteril should be prolonged and continuous in order to increase the predialysis period and improve the quality of life in patients with chronic kidney disease.

  4. Lipid and protein oxidation in the internal part of italian type salami containing basil essential oil (Ocimum basilicum L.

    Directory of Open Access Journals (Sweden)

    Alexandre José Cichoski

    2011-06-01

    Full Text Available Different concentrations of basil essential oil (Ocimum basilicum L. (0.19; 0.38; 0.75; 1.87; 3.75 and 6.00 mg.g-1 were evaluated in relation to their antioxidant activity using the DPPH● radical methodology. From the IC50 obtained data, the concentrations of 0.19; 0.38; 0.75; 1.87; 3.75; 6.00 and 12.00 mg.mL-1 were applied directly to the product and these were sensorially evaluated by the test of control difference. The concentrations related to the highest acceptability (0.19; 0.38 and 0.75 mg.g-1 were tested for antioxidant activity in the internal part of Italian type salami - during the processing and after 30 days of storage, in terms of lipid and protein oxidation. The oxidation of lipids was determined using the method of TBARS. The method of carbonyl compounds was employed for proteins oxidation. Five different formulations of salami were elaborated: blank (without the use of antioxidant; control (using sodium eritorbate as antioxidant; and adding 0.19; 0.38 and 0.75 mg.g-1 of basil essential oil. The product was kept between 25 ºC and 18 ºC and UR between 95% and 70%, for 28 days. Analyses were carried out on the processing day and after 2, 7, 14, 21 and 28 days, and also following 30 days of storage. The basil essential oil in vitro presented an antioxidant activity of IC50 12 mg.mL-1. In the internal part of the Italian type salami the commercial antioxidant (control and the formulation containing 0.75 mg.g-1 of basil essential oil presented antioxidant activity in relation to the lipids, but not to the proteins - during processing and storage.

  5. Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

    Science.gov (United States)

    Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

    2017-11-01

    The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

  6. Bumble bees regulate their intake of essential protein and lipid pollen macronutrients.

    Science.gov (United States)

    Vaudo, A D; Stabler, D; Patch, H M; Tooker, J F; Grozinger, C M; Wright, G A

    2016-12-15

    Bee population declines are linked to the reduction of nutritional resources due to land-use intensification, yet we know little about the specific nutritional needs of many bee species. Pollen provides bees with their primary source of protein and lipids, but nutritional quality varies widely among host-plant species. Therefore, bees might have adapted to assess resource quality and adjust their foraging behavior to balance nutrition from multiple food sources. We tested the ability of two bumble bee species, Bombus terrestris and Bombus impatiens, to regulate protein and lipid intake. We restricted B. terrestris adults to single synthetic diets varying in protein:lipid ratios (P:L). The bees over-ate protein on low-fat diets and over-ate lipid on high-fat diets to reach their targets of lipid and protein, respectively. The bees survived best on a 10:1 P:L diet; the risk of dying increased as a function of dietary lipid when bees ate diets with lipid contents greater than 5:1 P:L. Hypothesizing that the P:L intake target of adult worker bumble bees was between 25:1 and 5:1, we presented workers from both species with unbalanced but complementary paired diets to determine whether they self-select their diet to reach a specific intake target. Bees consumed similar amounts of proteins and lipids in each treatment and averaged a 14:1 P:L for B. terrestris and 12:1 P:L for B. impatiens These results demonstrate that adult worker bumble bees likely select foods that provide them with a specific ratio of P:L. These P:L intake targets could affect pollen foraging in the field and help explain patterns of host-plant species choice by bumble bees. © 2016. Published by The Company of Biologists Ltd.

  7. An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

    Science.gov (United States)

    Baumann, P; Jackson, S P

    1996-06-25

    Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

  8. The molluscan RING-finger protein L-TRIM is essential for neuronal outgrowth

    NARCIS (Netherlands)

    van Diepen, M. T.; Spencer, G.E.; Minnen, J.; Gouwenberg, Y.; Bouwman, J.G.; Smit, A. B.; van Kesteren, R.E.

    2005-01-01

    The tripartite motif proteins TRIM-2 and TRIM-3 have been put forward as putative organizers of neuronal outgrowth and structural plasticity. Here, we identified a molluscan orthologue of TRIM-2/3, named L-TRIM, which is up-regulated during in vitro neurite outgrowth of central neurons. In adult

  9. ACYLTRANSFERASE ACTIVITIES OF THE HIGH-MOLECULAR-MASS ESSENTIAL PENICILLIN-BINDING PROTEINS

    NARCIS (Netherlands)

    ADAM, M; DAMBLON, C; JAMIN, M; ZORZI, W; DUSART, [No Value; GALLENI, M; ELKHARROUBI, A; PIRAS, G; SPRATT, BG; KECK, W; COYETTE, J; GHUYSEN, JM; NGUYENDISTECHE, M; FRERE, JM

    1991-01-01

    The high-molecular-mass penicillin-binding proteins (HMM-PBPs), present in the cytoplasmic membranes of all eubacteria, are involved in important physiological events such as cell elongation, septation or shape determination. Up to now it has, however, been very difficult or impossible to study the

  10. Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Ji Qiongmei; Huang, C.-H.; Peng Jianbin; Hashmi, Sarwar; Ye Tianzhang; Chen Ying

    2007-01-01

    We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans

  11. Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

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    Mi Young Yang

    2013-05-01

    Full Text Available GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

  12. Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence

    Directory of Open Access Journals (Sweden)

    Zhanping Lu

    2018-02-01

    Full Text Available Yin yang 1 (YY1 is a ubiquitous transcription factor and mammalian polycomb group protein (PcG with important functions to regulate embryonic development, lineage differentiation, and cell proliferation. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in hematopoietic stem cells (HSCs decreases long-term repopulating activity and ectopic YY1 expression expands HSCs. Although the YY1 PcG domain is required for Igκ chain rearrangement in B cells, the YY1 mutant lacking the PcG domain retained the capacity to stimulate HSC self-renewal. YY1 deficiency deregulated the genetic network governing HSC cell proliferation and impaired stem cell factor/c-Kit signaling, disrupting mechanisms conferring HSC quiescence. These results reveal a mechanism for how a ubiquitously expressed transcriptional repressor mediates lineage-specific functions to control adult hematopoiesis.

  13. p63 protein is essential for the embryonic development of vibrissae and teeth

    International Nuclear Information System (INIS)

    Rufini, Alessandro; Weil, Miguel; McKeon, Frank; Barlattani, Alberto; Melino, Gerry; Candi, Eleonora

    2006-01-01

    Development of skin appendages strongly depends on epithelial-mesenchymal interactions. One of the genes involved in this process is p63, a member of the p53 family of transcription factors, essential for ectodermal development, as elucidated by the phenotype of p63 knock-out mice. Surprisingly, no information on p63 expression in tooth and hair is yet available. Here, we show p63 expression during teeth and vibrissae morphogenesis in mouse embryos and we also show a correlation with the expression patterns of the epithelial marker keratin 5 and the proliferation marker Ki67. Our results show that p63 colocalizes with both K5 and Ki67 in the epithelium of developing vibrissae, while in teeth p63 is expressed, together with K5, in the undifferentiated ectoderm (enamel organ), and in ameloblasts, a subpopulation of differentiated ectodermal cells. Moreover, p63 expression in tooth seems not to be fully colocalized with nuclear Ki67 expression

  14. Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Buechler, J.A.; Taylor, S.S.

    1988-01-01

    The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [ 14 C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified

  15. The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism

    OpenAIRE

    Van Knippenberg, Ingeborg Christine; Carlton-Smith, Charles; Elliott, Richard Michael

    2010-01-01

    This work is supported by UK MRC and BBRC Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to...

  16. Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression.

    Science.gov (United States)

    King, Amy M; Bartpho, Thanatchaporn; Sermswan, Rasana W; Bulach, Dieter M; Eshghi, Azad; Picardeau, Mathieu; Adler, Ben; Murray, Gerald L

    2013-08-01

    Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp., but knowledge of leptospiral pathogenesis remains limited. However, the development of mutagenesis systems has allowed the investigation of putative virulence factors and their involvement in leptospirosis. LipL41 is the third most abundant lipoprotein found in the outer membranes of pathogenic leptospires and has been considered a putative virulence factor. LipL41 is encoded on the large chromosome 28 bp upstream of a small open reading frame encoding a hypothetical protein of unknown function. This gene was named lep, for LipL41 expression partner. In this study, lipL41 was found to be cotranscribed with lep. Two transposon mutants were characterized: a lipL41 mutant and a lep mutant. In the lep mutant, LipL41 protein levels were reduced by approximately 90%. Lep was shown through cross-linking and coexpression experiments to bind to LipL41. Lep is proposed to be a molecular chaperone essential for the stable expression of LipL41. The roles of LipL41 and Lep in the pathogenesis of Leptospira interrogans were investigated; surprisingly, neither of these two unique proteins was essential for acute leptospirosis.

  17. The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

    Science.gov (United States)

    Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

    2007-05-01

    The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

  18. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  19. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

    Science.gov (United States)

    Wang, Dashan

    2018-06-01

    The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.

  20. The N-terminus of Bunyamwera orthobunyavirus NSs protein is essential for interferon antagonism.

    Science.gov (United States)

    van Knippenberg, Ingeborg; Carlton-Smith, Charlie; Elliott, Richard M

    2010-08-01

    Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses.

  1. Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

    Directory of Open Access Journals (Sweden)

    Shekhtman Alexander

    2009-04-01

    Full Text Available Abstract Background Human S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment. Results The aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface. Conclusion We have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

  2. The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

    1996-01-01

    Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

  3. Essential roles of protein-solvent many-body correlation in solvent-entropy effect on protein folding and denaturation: Comparison between hard-sphere solvent and water

    International Nuclear Information System (INIS)

    Oshima, Hiraku; Kinoshita, Masahiro

    2015-01-01

    In earlier works, we showed that the entropic effect originating from the translational displacement of water molecules plays the pivotal role in protein folding and denaturation. The two different solvent models, hard-sphere solvent and model water, were employed in theoretical methods wherein the entropic effect was treated as an essential factor. However, there were similarities and differences in the results obtained from the two solvent models. In the present work, to unveil the physical origins of the similarities and differences, we simultaneously consider structural transition, cold denaturation, and pressure denaturation for the same protein by employing the two solvent models and considering three different thermodynamic states for each solvent model. The solvent-entropy change upon protein folding/unfolding is decomposed into the protein-solvent pair (PA) and many-body (MB) correlation components using the integral equation theories. Each component is further decomposed into the excluded-volume (EV) and solvent-accessible surface (SAS) terms by applying the morphometric approach. The four physically insightful constituents, (PA, EV), (PA, SAS), (MB, EV), and (MB, SAS), are thus obtained. Moreover, (MB, SAS) is discussed by dividing it into two factors. This all-inclusive investigation leads to the following results: (1) the protein-water many-body correlation always plays critical roles in a variety of folding/unfolding processes; (2) the hard-sphere solvent model fails when it does not correctly reproduce the protein-water many-body correlation; (3) the hard-sphere solvent model becomes problematic when the dependence of the many-body correlation on the solvent number density and temperature is essential: it is not quite suited to studies on cold and pressure denaturating of a protein; (4) when the temperature and solvent number density are limited to the ambient values, the hard-sphere solvent model is usually successful; and (5) even at the ambient

  4. A Disulfide Bond in the Membrane Protein IgaA Is Essential for Repression of the RcsCDB System

    Directory of Open Access Journals (Sweden)

    M. Graciela Pucciarelli

    2017-12-01

    Full Text Available IgaA is an integral inner membrane protein that was discovered as repressor of the RcsCDB phosphorelay system in the intracellular pathogen Salmonella enterica serovar Typhimurium. The RcsCDB system, conserved in many members of the family Enterobacteriaceae, regulates expression of varied processes including motility, biofilm formation, virulence and response to envelope stress. IgaA is an essential protein to which, in response to envelope perturbation, the outer membrane lipoprotein RcsF has been proposed to bind in order to activate the RcsCDB phosphorelay. Envelope stress has also been reported to be sensed by a surface exposed domain of RcsF. These observations support a tight control of the RcsCDB system by RcsF and IgaA via mechanisms that, however, remain unknown. Interestingly, RcsF and IgaA have four conserved cysteine residues in loops exposed to the periplasmic space. Two non-consecutive disulfide bonds were shown to be required for RcsF function. Here, we report mutagenesis studies supporting the presence of one disulfide bond (C404-C425 in the major periplasmic loop of IgaA that is essential for repression of the RcsCDB phosphorelay. Our data therefore suggest that the redox state of the periplasm may be critical for the control of the RcsCDB system by its two upstream regulators, RcsF and IgaA.

  5. The Activity of Menkes Disease Protein ATP7A Is Essential for Redox Balance in Mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Ashima; Yang, Haojun; Duffy, Megan; Robinson, Emily; Conrad-Antoville, Arianrhod; Lu, Ya-Wen; Capps, Tony; Braiterman, Lelita; Wolfgang, Michael; Murphy, Michael P.; Yi, Ling; Kaler, Stephen G.; Lutsenko, Svetlana; Ralle, Martina

    2016-05-16

    Copper-transporting ATPase ATP7A is essential for mammalian copper homeostasis. Loss of ATP7A activity is associated with fatal Menkes disease and various other pathologies. In cells, ATP7A inactivation disrupts copper transport from the cytosol into the secretory pathway. Using fibroblasts from Menkes disease patients and mouse 3T3-L1 cells with a CRISPR/Cas9-inactivated ATP7A, we demonstrate that ATP7A dysfunction is also damaging to mitochondrial redox balance. In these cells, copper accumulates in nuclei, cytosol, and mitochondria, causing distinct changes in their redox environment. Quantitative imaging of live cells using GRX1-roGFP2 and HyPer sensors reveals highest glutathione oxidation and elevation of H2O2 in mitochondria, whereas the redox environment of nuclei and the cytosol is much less affected. Decreasing the H2O2 levels in mitochondria with MitoQ does not prevent glutathione oxidation; i.e. elevated copper and not H2O2 is a primary cause of glutathione oxidation. Redox misbalance does not significantly affect mitochondrion morphology or the activity of respiratory complex IV but markedly increases cell sensitivity to even mild glutathione depletion, resulting in loss of cell viability. Thus, ATP7A activity protects mitochondria from excessive copper entry, which is deleterious to redox buffers. Mitochondrial redox misbalance could significantly contribute to pathologies associated with ATP7A inactivation in tissues with paradoxical accumulation of copper (i.e. renal epithelia).

  6. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  7. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    International Nuclear Information System (INIS)

    Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

    2010-01-01

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

  8. Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD

    Directory of Open Access Journals (Sweden)

    Ackermann Martin

    2011-05-01

    Full Text Available Abstract Background The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (pppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (pppGpp - abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (pppGpp, and to a termination of cell division. The combination of single-cell timelapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.

  9. Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection.

    Science.gov (United States)

    Furihata, Shunsuke; Tanaka, Kohjiro; Ryuda, Masasuke; Ochiai, Masanori; Matsumoto, Hitoshi; Csikos, Gyorge; Hayakawa, Yoichi

    2014-01-01

    Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Rising atmospheric CO2 is reducing the protein concentration of a floral pollen source essential for North American bees.

    Science.gov (United States)

    Ziska, Lewis H; Pettis, Jeffery S; Edwards, Joan; Hancock, Jillian E; Tomecek, Martha B; Clark, Andrew; Dukes, Jeffrey S; Loladze, Irakli; Polley, H Wayne

    2016-04-13

    At present, there is substantive evidence that the nutritional content of agriculturally important food crops will decrease in response to rising levels of atmospheric carbon dioxide, Ca However, whether Ca-induced declines in nutritional quality are also occurring for pollinator food sources is unknown. Flowering late in the season, goldenrod (Solidago spp.) pollen is a widely available autumnal food source commonly acknowledged by apiarists to be essential to native bee (e.g. Bombus spp.) and honeybee (Apis mellifera) health and winter survival. Using floral collections obtained from the Smithsonian Natural History Museum, we quantified Ca-induced temporal changes in pollen protein concentration of Canada goldenrod (Solidago canadensis), the most wide spread Solidago taxon, from hundreds of samples collected throughout the USA and southern Canada over the period 1842-2014 (i.e. a Ca from approx. 280 to 398 ppm). In addition, we conducted a 2 year in situtrial of S. Canadensis populations grown along a continuous Ca gradient from approximately 280 to 500 ppm. The historical data indicated a strong significant correlation between recent increases in Ca and reductions in pollen protein concentration (r(2)= 0.81). Experimental data confirmed this decrease in pollen protein concentration, and indicated that it would be ongoing as Ca continues to rise in the near term, i.e. to 500 ppm (r(2)= 0.88). While additional data are needed to quantify the subsequent effects of reduced protein concentration for Canada goldenrod on bee health and population stability, these results are the first to indicate that increasing Ca can reduce protein content of a floral pollen source widely used by North American bees. © 2016 The Author(s).

  11. The ERM protein Moesin is essential for neuronal morphogenesis and long-term memory in Drosophila.

    Science.gov (United States)

    Freymuth, Patrick S; Fitzsimons, Helen L

    2017-08-29

    Moesin is a cytoskeletal adaptor protein that plays an important role in modification of the actin cytoskeleton. Rearrangement of the actin cytoskeleton drives both neuronal morphogenesis and the structural changes in neurons that are required for long-term memory formation. Moesin has been identified as a candidate memory gene in Drosophila, however, whether it is required for memory formation has not been evaluated. Here, we investigate the role of Moesin in neuronal morphogenesis and in short- and long-term memory formation in the courtship suppression assay, a model of associative memory. We found that both knockdown and overexpression of Moesin led to defects in axon growth and guidance as well as dendritic arborization. Moreover, reduction of Moesin expression or expression of a constitutively active phosphomimetic in the adult Drosophila brain had no effect on short term memory, but prevented long-term memory formation, an effect that was independent of its role in development. These results indicate a critical role for Moesin in both neuronal morphogenesis and long-term memory formation.

  12. Huntingtin Protein is Essential for Mitochondrial Metabolism, Bioenergetics and Structure in Murine Embryonic Stem Cells

    Science.gov (United States)

    Ismailoglu, Ismail; Chen, Qiuying; Popowski, Melissa; Yang, Lili; Gross, Steven S.; Brivanlou, Ali H.

    2014-01-01

    Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt-/- mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3,700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt-/-, extended poly-Q (Htt-Q140/7), and wildtype mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells, did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt-/- mESCs, including: (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt-/- early embryonic lethality. PMID:24780625

  13. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

  14. The adaptor protein CIKS/Act1 is essential for IL-25-mediated allergic airway inflammation.

    Science.gov (United States)

    Claudio, Estefania; Sønder, Søren Ulrik; Saret, Sun; Carvalho, Gabrielle; Ramalingam, Thirumalai R; Wynn, Thomas A; Chariot, Alain; Garcia-Perganeda, Antonio; Leonardi, Antonio; Paun, Andrea; Chen, Amy; Ren, Nina Y; Wang, Hongshan; Siebenlist, Ulrich

    2009-02-01

    IL-17 is the signature cytokine of recently discovered Th type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production, and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (also known as Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses that these two cytokines elicit. We identify CD11c(+) macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo.

  15. Development of Nuclear Plant Specific Analysis Simulators with ATLAS

    International Nuclear Information System (INIS)

    Jakubowski, Z.; Draeger, P.; Horche, W.; Pointner, W.

    2006-01-01

    The simulation software ATLAS, based on the best-estimate code ATHLET, has been developed by the GRS for a range of applications in the field of nuclear plant safety analysis. Through application of versatile simulation tools and graphical interfaces the user should be able to analyse with ATLAS all essential accident scenarios. Detailed analysis simulators for several German and Russian NPPs are being constructed on the basis of ATLAS. An overview of the ATLAS is presented in the paper, describing its configuration, functions performed by main components and relationships among them. A significant part of any power plant simulator are the balance-of-plant (BOP) models, not only because all the plant transients and non-LOCA accidents can be initiated by operation of BOP systems, but also because the response of the plant to transients or accidents is strongly influenced by the automatic operation of BOP systems. Modelling aspects of BOP systems are shown in detail, also the interface between the process model and BOP systems. Special emphasis has been put on the BOP model builder based on the methodology developed in the GRS. The BOP modeler called GCSM-Generator is an object oriented tool which runs on the online expert system G2. It is equipped with utilities to edit the BOP models, to verification them and to generate a GCSM code, specific for the ATLAS. The communication system of ATLAS presents graphically the results of the simulation and allows interactively influencing the execution of the simulation process (malfunctions, manual control). Displays for communications with simulated processes and presentation of calculations results are also presented. In the framework of the verification of simulation models different tools are used e.g. the PC-codes MATHCAD for the calculation and documentation, ATLET-Input-Graphic for control of geometry data and the expert system G2 for development of BOP-Models. The validation procedure and selected analyses results

  16. Amino acids fortification of low-protein diet for broilers under tropical climate: ideal essential amino acids profile

    Directory of Open Access Journals (Sweden)

    Elmutaz Atta Awad

    2014-05-01

    Full Text Available A three-week trial was conducted to determine the effect of lowering dietary protein level (DPL with optimal amino acid (AA profile on growth performance, blood metabolites, and relative weights of abdominal fat and internal organs in broiler chickens raised under tropical hot and humid environment. Five isocaloric (3023 metabolisable energy/kg starter (1-21 days experimental diets were formulated in a gradual crude protein (CP decline from 22.2 (control to 16.2% by 1.5% interval. All diets were meeting or exceeding National Research Council recommendations except CP and metabolisable energy. The formulations were also adjusted to contain 1.1 digestible Lys to meet the ideal AA ratios concept. Body weights (BW, weight gains (WG, feed intake and feed conversion ratio of groups with 19.2, 20.7 and 22.2% DPL were not significantly different. However, BW and WG suppressed (P<0.05 with 16.2 and 17.7% DPL. Feeding the 16.2% CP diet significantly reduced serum total protein and uric acid, but increased serum triglyceride (P<0.05. Moreover, relative heart weights increased (P<0.05 but no changes occurred in liver and abdominal fat weights in chicks with 16.2% DPL. In summary, CP of broilers starter (1-21 days diet can be reduced till 19.2% with essential AA fortification and without any adverse effect on growth performance under the hot, humid tropics.

  17. Plasmodium falciparum Calcium-Dependent Protein Kinase 2 Is Critical for Male Gametocyte Exflagellation but Not Essential for Asexual Proliferation

    Directory of Open Access Journals (Sweden)

    Abhisheka Bansal

    2017-10-01

    Full Text Available Drug development efforts have focused mostly on the asexual blood stages of the malaria parasite Plasmodium falciparum. Except for primaquine, which has its own limitations, there are no available drugs that target the transmission of the parasite to mosquitoes. Therefore, there is a need to validate new parasite proteins that can be targeted for blocking transmission. P. falciparum calcium-dependent protein kinases (PfCDPKs play critical roles at various stages of the parasite life cycle and, importantly, are absent in the human host. These features mark them as attractive drug targets. In this study, using CRISPR/Cas9 we successfully knocked out PfCDPK2 from blood-stage parasites, which was previously thought to be an indispensable protein. The growth rate of the PfCDPK2 knockout (KO parasites was similar to that of wild-type parasites, confirming that PfCDPK2 function is not essential for the asexual proliferation of the parasite in vitro. The mature male and female gametocytes of PfCDPK2 KO parasites become round after induction. However, they fail to infect female Anopheles stephensi mosquitoes due to a defect(s in male gametocyte exflagellation and possibly in female gametes.

  18. A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

    Science.gov (United States)

    Nanjareddy, Kalpana; Blanco, Lourdes; Arthikala, Manoj-Kumar; Alvarado-Affantranger, Xóchitl; Quinto, Carmen; Sánchez, Federico; Lara, Miguel

    2016-11-01

    The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

    Directory of Open Access Journals (Sweden)

    Mota Maria M

    2011-03-01

    Full Text Available Abstract Background The Plasmodium Cysteine Repeat Modular Proteins (PCRMP are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions PCRMP3 and 4 play multiple roles during the Plasmodium life

  20. The effect of uncertainties in nuclear reactor plant-specific failure data on core damage frequency

    International Nuclear Information System (INIS)

    Martz, H.F.

    1995-05-01

    It is sometimes the case in PRA applications that reported plant-specific failure data are, in fact, only estimates which are uncertain. Even for detailed plant-specific data, the reported exposure time or number of demands is often only an estimate of the actual exposure time or number of demands. Likewise the reported number of failure events or incidents is sometimes also uncertain because incident or malfunction reports may be ambiguous. In this report we determine the corresponding uncertainty in core damage frequency which can b attributed to such uncertainties in plant-specific data using a simple but typical nuclear power reactor example

  1. Complementation of essential yeast GPI mannosyltransferase mutations suggests a novel specificity for certain Trypanosoma and Plasmodium PigB proteins.

    Directory of Open Access Journals (Sweden)

    Leslie K Cortes

    Full Text Available The glycosylphosphatidylinositol (GPI anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH2-CH2-CH2-PO4-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-myo-inositol-PO4-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2-linked fourth mannose (Man-IV that is side-branched to the third mannose (Man-III of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man4-GPIs being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the in vivo specificity of Gpi10/PigB proteins from several Plasmodium and Trypanosoma species by examining their ability to restore viability to Saccharomyces cerevisiae strains harboring lethal defects in Man-III (gpi10Δ or Man-IV (smp3Δ addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from T. cruzi, T. congolense and P. falciparum are each capable of separately complementing essential gpi10Δ and smp3Δ mutations, while PIGB genes from T. vivax and T. brucei only complement gpi10Δ. Additionally, we show the ability of T. cruzi PIGB to robustly complement a gpi10Δ/smp3Δ double mutant. Our data suggest that certain Plasmodium and Trypanosoma PigB mannosyltransferases can transfer more than one mannose to GPI precursors in vivo, and suggest a novel

  2. The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

    Science.gov (United States)

    Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V

    2015-07-01

    PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

  3. Stability of cardamom (Elettaria cardamomum) essential oil in microcapsules made of whey protein isolate, guar gum, and carrageenan.

    Science.gov (United States)

    Mehyar, Ghadeer F; Al-Ismail, Khalid M; Al-Isamil, Khalid M; Al-Ghizzawi, Hana'a M; Holley, Richard A

    2014-10-01

    The effects of microencapsulating cardamom essential oil (CEO) in whey protein isolate (WPI) alone and combined with guar gum (GG) and carrageen (CG) on microencapsulation efficiency, oil chemical stability, and microcapsule structure were investigated. Freeze-dried microcapsules were prepared from emulsions containing (w/w): 15% and 30% WPI; 0.1% GG, and 0.2% CG as wall materials with CEO (at 10% of polymer concentration) as core material, and physical properties and chemical stability were compared. Bulk density of microcapsules was highest in WPI without GG or CG and in 30% WPI + GG microcapsules, and was more affected by moisture content (r = -0.6) than by mean particle diameter (d43 ; r = -0.2) and span (r = 0.1). Microcapsules containing only WPI had the highest entrapped oil (7.5%) and microencapsulation efficiency (98.5%). The concentrations of 1,8-cineole and d-limonene were used as indicators for microcapsule chemical stability since they were the main components of CEO. Microcapsules retained higher (P ≤ 0.05) concentrations of both components than non-microencapsulated CEO during 16 wk storage at 20 ºC, but higher loss of both components was noted at 35 ºC. Microencapsulated d-limonene was reduced faster than 1,8-cineole regardless of temperature. The 30% WPI and 30% WPI + GG microcapsules retained CEO best throughout storage at both storage temperatures. Scanning electron micrographs revealed that WPI microcapsules had smooth surfaces, were relatively homogenous and regular in shape, whereas GG and CG addition increased visual surface porosity and reduced shape regularity. It was concluded that the best formulation for encapsulating CEO was 30% WPI. Encapsulating cardamom essential oil in whey protein isolate alone or combined with guar gum produced dried powders that effectively retained and chemically stabilized CEO, and therefore enhanced its handling and storability. © 2014 Institute of Food Technologists®

  4. Effects of vanillin, quillaja saponin, and essential oils on in vitro fermentation and protein-degrading microorganisms of the rumen.

    Science.gov (United States)

    Patra, Amlan K; Yu, Zhongtang

    2014-01-01

    This study investigated the effects of vanillin on methanogenesis and rumen fermentation, and the responses of ruminal protein-degrading bacteria to vanillin (at concentrations of 0, 0.76 and 1.52 g/L), essential oils (clove oil, 1 g/L; origanum oil, 0.50 g/L, and peppermint oil, 1 g/L), and quillaja saponin (at concentration of 0 and 6 g/L) in vitro. Methane production, degradabilities of feed substrate, and ammonia concentration decreased linearly with increasing doses of vanillin. Concentration of total volatile fatty acids also decreased, whereas proportion of butyrate tended to increase linearly with increasing doses of vanillin. Protozoa population decreased, but abundances of Ruminococcus flavefaciens, Prevotella bryantii, Butyrivibrio fibrisolvens, Prevotella ruminicola, Clostridium aminophilum, and Ruminobacter amylophilus increased with increasing doses of vanillin. Origanum and clove oils resulted in lower ammonia concentrations compared to control and peppermint oil. All the tested essential oils decreased abundances of protozoa, Selenomonas ruminantium, R. amylophilus, P. ruminicola and P. bryantii, with the largest decrease resulted from origanum oil followed by clove oil and peppermint oil. The abundances of Megasphaera elsdenii, C. aminophilum, and Clostridium sticklandii were deceased by origanum oil while that of B. fibrisolvens was lowered by both origanum and clove oils. Saponin decreased ammonia concentration and protozoal population, but increased the abundances of S. ruminantium, R. amylophilus, P. ruminicola, and P. bryantii, though the magnitude was small (less than one log unit). The results suggest that reduction of ammonia production by vanillin and saponin may not be caused by direct inhibition of major known proteolytic bacteria, and essential oils can have different inhibitory effects on different proteolytic bacteria, resulting in varying reduction in ammonia production.

  5. The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function.

    Science.gov (United States)

    Müller, Sabine; Smertenko, Andrei; Wagner, Vera; Heinrich, Maria; Hussey, Patrick J; Hauser, Marie-Theres

    2004-03-09

    Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.

  6. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development.

    Science.gov (United States)

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-12-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of >95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. Copyright © 2014. Published by Elsevier B.V.

  7. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Olivier Poupel

    Full Text Available The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their

  8. Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus.

    Science.gov (United States)

    Poupel, Olivier; Moyat, Mati; Groizeleau, Julie; Antunes, Luísa C S; Gribaldo, Simonetta; Msadek, Tarek; Dubrac, Sarah

    2016-01-01

    The WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cistronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcellular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene

  9. Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of); Park, Jong-Wan, E-mail: parkjw@snu.ac.kr [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  10. Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    International Nuclear Information System (INIS)

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun; Park, Jong-Wan

    2012-01-01

    Highlights: ► HIF-1α is expressed PRMT5-dependently in hypoxic cancer cells. ► The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. ► The de novo synthesis of HIF-1α depends on PRMT5. ► PRMT5 is involved in the HIF-1α translation initiated by 5′ UTR of HIF-1α mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1–8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1α in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1α protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1α transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1α translation initiated by the 5′ UTR of HIF-1α mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  11. Ability of commercially available dairy ration programs to predict duodenal flows of protein and essential amino acids in dairy cows.

    Science.gov (United States)

    Pacheco, D; Patton, R A; Parys, C; Lapierre, H

    2012-02-01

    The objective of this analysis was to compare the rumen submodel predictions of 4 commonly used dairy ration programs to observed values of duodenal flows of crude protein (CP), protein fractions, and essential AA (EAA). The literature was searched and 40 studies, including 154 diets, were used to compare observed values with those predicted by AminoCow (AC), Agricultural Modeling and Training Systems (AMTS), Cornell-Penn-Miner (CPM), and National Research Council 2001 (NRC) models. The models were evaluated based on their ability to predict the mean, their root mean square prediction error (RMSPE), error bias, and adequacy of regression equations for each protein fraction. The models predicted the mean duodenal CP flow within 5%, with more than 90% of the variation due to random disturbance. The models also predicted within 5% the mean microbial CP flow except CPM, which overestimated it by 27%. Only NRC, however, predicted mean rumen-undegraded protein (RUP) flows within 5%, whereas AC and AMTS underpredicted it by 8 to 9% and CPM by 24%. Regarding duodenal flows of individual AA, across all diets, CPM predicted substantially greater (>10%) mean flows of Arg, His, Ile, Met, and Lys; AMTS predicted greater flow for Arg and Met, whereas AC and NRC estimations were, on average, within 10% of observed values. Overpredictions by the CPM model were mainly related to mean bias, whereas the NRC model had the highest proportion of bias in random disturbance for flows of EAA. Models tended to predict mean flows of EAA more accurately on corn silage and alfalfa diets than on grass-based diets, more accurately on corn grain-based diets than on non-corn-based diets, and finally more accurately in the mid range of diet types. The 4 models were accurate at predicting mean dry matter intake. The AC, AMTS, and NRC models were all sufficiently accurate to be used for balancing EAA in dairy rations under field conditions. Copyright © 2012 American Dairy Science Association

  12. The Hedgehog Signal Induced Modulation of Bone Morphogenetic Protein Signaling: An Essential Signaling Relay for Urinary Tract Morphogenesis

    Science.gov (United States)

    Nakagata, Naomi; Miyagawa, Shinichi; Suzuki, Kentaro; Kitazawa, Sohei; Yamada, Gen

    2012-01-01

    Background Congenital diseases of the urinary tract are frequently observed in infants. Such diseases present a number of developmental anomalies such as hydroureter and hydronephrosis. Although some genetically-modified mouse models of growth factor signaling genes reproduce urinary phenotypes, the pathogenic mechanisms remain obscure. Previous studies suggest that a portion of the cells in the external genitalia and bladder are derived from peri-cloacal mesenchymal cells that receive Hedgehog (Hh) signaling in the early developmental stages. We hypothesized that defects in such progenitor cells, which give rise to urinary tract tissues, may be a cause of such diseases. Methodology/Principal Findings To elucidate the pathogenic mechanisms of upper urinary tract malformations, we analyzed a series of Sonic hedgehog (Shh) deficient mice. Shh−/− displayed hydroureter and hydronephrosis phenotypes and reduced expression of several developmental markers. In addition, we suggested that Shh modulation at an early embryonic stage is responsible for such phenotypes by analyzing the Shh conditional mutants. Tissue contribution assays of Hh-responsive cells revealed that peri-cloacal mesenchymal cells, which received Hh signal secreted from cloacal epithelium, could contribute to the ureteral mesenchyme. Gain- and loss-of-functional mutants for Hh signaling revealed a correlation between Hh signaling and Bone morphogenetic protein (Bmp) signaling. Finally, a conditional ablation of Bmp receptor type IA (BmprIA) gene was examined in Hh-responsive cell lineages. This system thus made it possible to analyze the primary functions of the growth factor signaling relay. The defective Hh-to-Bmp signaling relay resulted in severe urinary tract phenotypes with a decrease in the number of Hh-responsive cells. Conclusions/Significance This study identified the essential embryonic stages for the pathogenesis of urinary tract phenotypes. These results suggested that Hh

  13. Fibroblast activation protein (FAP is essential for the migration of bone marrow mesenchymal stem cells through RhoA activation.

    Directory of Open Access Journals (Sweden)

    Kuei-Min Chung

    Full Text Available BACKGROUND: The ability of human bone marrow mesenchymal stem cells (BM-MSCs to migrate and localize specifically to injured tissues is central in developing therapeutic strategies for tissue repair and regeneration. Fibroblast activation protein (FAP is a cell surface serine protease expressed at sites of tissue remodeling during embryonic development. It is also expressed in BM-MSCs, but not in normal tissues or cells. The function of FAP in BM-MSCs is not known. PRINCIPAL FINDINGS: We found that depletion of FAP proteins significantly inhibited the migration of BM-MSCs in a transwell chemotaxis assay. Such impaired migration ability of BM-MSCs could be rescued by re-expressing FAP in these cells. We then demonstrated that depletion of FAP activated intracellular RhoA GTPase. Consistently, inhibition of RhoA activity using a RhoA inhibitor rescued its migration ability. Inhibition of FAP activity with an FAP-specific inhibitor did not affect the activation of RhoA or the migration of BM-MSCs. Furthermore, the inflammatory cytokines interleukin-1beta (IL-1β and transforming growth factor-beta (TGF-β upregulated FAP expression, which coincided with better BM-MSC migration. CONCLUSIONS: Our results indicate FAP plays an important role in the migration of BM-MSCs through modulation of RhoA GTPase activity. The peptidase activity of FAP is not essential for such migration. Cytokines IL-1β and TGF-β upregulate the expression level of FAP and thus enhance BM-MSC migration.

  14. Protein Kinase A Regulatory Subunit Isoforms Regulate Growth and Differentiation in Mucor circinelloides: Essential Role of PKAR4

    Science.gov (United States)

    Ocampo, J.; McCormack, B.; Navarro, E.; Moreno, S.; Garre, V.

    2012-01-01

    The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway. PMID:22635921

  15. Viral Genome-Linked Protein (VPg Is Essential for Translation Initiation of Rabbit Hemorrhagic Disease Virus (RHDV.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available Rabbit hemorrhagic disease virus (RHDV, the causative agent of rabbit hemorrhagic disease, is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, limiting the study of the pathogenesis of RHDV. In addition, the mechanisms underlying RHDV translation and replication are largely unknown compared with other caliciviridae viruses. The RHDV replicon recently constructed in our laboratory provides an appropriate model to study the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon, we demonstrated that the viral genome-linked protein (VPg is essential for RHDV translation in RK-13 cells for the first time. In addition, we showed that VPg interacts with eukaryotic initiation factor 4E (eIF4E in vivo and in vitro and that eIF4E silencing inhibits RHDV translation, suggesting the interaction between VPg and eIF4E is involved in RHDV translation. Our results support the hypothesis that VPg serves as a novel cap substitute during the initiation of RHDV translation.

  16. Antimicrobial effectiveness of oregano and sage essential oils incorporated into whey protein films or cellulose-based filter paper.

    Science.gov (United States)

    Royo, Maite; Fernández-Pan, Idoya; Maté, Juan I

    2010-07-01

    In this study the antimicrobial effectiveness of oregano and sage essential oils (EOs) incorporated into two different matrices, whey protein isolate (WPI) and cellulose-based filter paper, was analysed. Antimicrobial properties of WPI-based films containing oregano and sage EOs were tested against Listeria innocua, Staphylococcus aureus and Salmonella enteritidis. Oregano EO showed antimicrobial activity against all three micro-organisms. The highest inhibition zones were against L. innocua. However, sage EO did not show antimicrobial activity against any of the micro-organisms. Antimicrobial activity was confirmed for both EOs using cellulose-based filter paper as supporting matrix, although it was significantly more intense for oregano EO. Inhibition surfaces were significantly greater when compared with those of the WPI films. This finding is likely due to the higher porosity and diffusivity of the active compounds in the filter paper. The interactions between the EOs and the films have a critical effect on the diffusivity of the active compounds and therefore on the final antimicrobial activity. As a result, to obtain active edible films, it is necessary to find the equilibrium point between the nature and concentration of the active compounds in the EO and the formulation of the film.

  17. Use of plant-specific PRA in an EOP scope audit

    International Nuclear Information System (INIS)

    O'Brien, J.J.

    1991-01-01

    Traditionally, decisions on which accident scenarios to proceduralize as emergency operating procedures (EOPs) have been based on existing design basis analyses, engineering judgment, and probabilistic risk assessments (PRAs) on generic plants. This approach has important strengths and limits. The major limitation of generic PRAs is their inability to account for plant-specific features. Use of plant-specific PRA to determine the impact of proceduralizing, or not proceduralizing, responses to scenarios considers plant-specific features. This helps to eliminate unnecessary EOPs, thus allowing resources to be concentrated on scenarios that are more important for a particular plant. In preparation for a US Nuclear Regulatory Commission audit, a plant-specific PRA was used to assess and quantify the plant's previous decision not to implement six reference emergency response guidelines (ERGs) as procedures. The original justification for nonimplementation of the ERGs was based on engineering judgment. The PRA provided a quantitative justification for implementation/nonimplementation of each guidelines. This analysis accounted for plant-specific design features not common to all reference plants

  18. Effect of the essential amino acids upon inclusion in vitro of 14C-phenylalanine and 14C-leucine in the protein of mammary gland

    International Nuclear Information System (INIS)

    Alexandrov, S.; Ivanov, N.; Sirakov, L.

    1983-01-01

    It is admitted that the essential amino acids could be divided into two groups, depending on the need of them for synthesis of milk protein: group i - amino acids, which are absorbed in quantities precisely corresponding to their content in milk protein (methionine, phenyl-alanine, histidine, thyrosine and triptophane), and group ii - amino acids, which are absorbed in quantities greater than their content in milk protein and which, because of this, could fullfil other metabolic functions in the mammary gland (threonine, valine, isoleucine, lysine and arginine). According to this concept, tissue slices of lactating mammary gland of guinea-pigs were incubated in the presence of grour i or group ii essential amino acids. Slices were incubated for 60 min at 37+-0.5 deg C, In a Crebs-Ringer phosphate buffer plus 0.2 glucose and 3.7 KBq/ml incubation medium DL-(I- 14 C)-phenylalanine or L-(U- 14 C)-leucine and their incorporation in the tissue proteins of mammary gland was measured in vitro. Group ii essential amino acids provoked significantly more intensive (P<0.001) inclusion in protein synthesis of these labelled amino acids in the tissue of mammary gland, as compared with group i essential amino acids

  19. Identification of amino-acid residues in the V protein of peste des petits ruminants essential for interference and suppression of STAT-mediated interferon signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Xusheng, E-mail: maxushengtt@163.com [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Yang, Xing [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Nian, Xiaofeng [Institute of Pathogen Biology and Immunology, Hebei North University, Zhangjiakou 07500 (China); Zhang, Zhidong; Dou, Yongxi [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Zhang, Xuehu [Gansu Agricultural University, Lanzhou (China); Luo, Xuenong; Su, Junhong; Zhu, Qiyun [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China); Cai, Xuepeng, E-mail: caixp@vip.163.com [State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730030 (China)

    2015-09-15

    Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.

  20. The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

    OpenAIRE

    Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

    2008-01-01

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recen...

  1. Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

    Science.gov (United States)

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-12-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the

  2. Intraflagellar Transporter Protein (IFT27), an IFT25 binding partner, Is Essential For Male Fertility and Spermiogenesis In Mice

    Science.gov (United States)

    Zhang, Yong; Liu, Hong; Li, Wei; Zhang, Zhengang; Shang, Xuejun; Zhang, David; Li, Yuhong; Zhang, Shiyang; Liu, Junpin; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-01-01

    Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27:Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survive to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymis contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2” axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional

  3. Whey protein and essential amino acids promote the reduction of adipose tissue and increased muscle protein synthesis during caloric restriction-induced weight loss in elderly, obese individuals

    Directory of Open Access Journals (Sweden)

    Coker Robert H

    2012-12-01

    Full Text Available Abstract Background Excess adipose tissue and sarcopenia presents a multifaceted clinical challenge that promotes morbidity and mortality in the obese, elderly population. Unfortunately, the mortality risks of muscle loss may outweigh the potential benefits of weight loss in the elderly. We have previously demonstrated the effectiveness of whey protein and essential amino acids towards the preservation of lean tissue, even under the conditions of strict bedrest in the elderly. Methods In the context of caloric restriction-based weight loss, we hypothesized that a similar formulation given as a meal replacement (EAAMR would foster the retention of lean tissue through an increase in the skeletal muscle fractional synthesis rate (FSR. We also proposed that EAAMR would promote the preferential loss of adipose tissue through the increased energy cost of skeletal muscle FSR. We recruited and randomized 12 elderly individuals to an 8 week, caloric restriction diet utilizing equivalent caloric meal replacements (800 kcal/day: 1 EAAMR or a 2 competitive meal replacement (CMR in conjunction with 400 kcal of solid food that totaled 1200 kcal/day designed to induce 7% weight loss. Combined with weekly measurements of total body weight and body composition, we also measured the acute change in the skeletal muscle FSR to EAAMR and CMR. Results By design, both groups lost ~7% of total body weight. While EAAMR did not promote a significant preservation of lean tissue, the reduction in adipose tissue was greater in EAAMR compared to CMR. Interestingly, these results corresponded to an increase in the acute skeletal muscle protein FSR. Conclusion The provision of EAAMR during caloric restriction-induced weight loss promotes the preferential reduction of adipose tissue and the modest loss of lean tissue in the elderly population.

  4. Growth and clinical variables in nitrogen-restricted piglets fed an adjusted essential amino acid mix: Effects using partially intact protein-based diets

    Science.gov (United States)

    Current recommendations for protein levels in infant formula ensure that growth matches or exceeds growth of breast-fed infants, but may provide a surplus of amino acids (AA). Recent studies in infants using AA-based formulas support specific adjustment of the essential AA (EAA) composition allowing...

  5. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

    NARCIS (Netherlands)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-01-01

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the

  6. IFT25, an intraflagellar transporter protein dispensable for ciliogenesis in somatic cells, is essential for sperm flagella formation.

    Science.gov (United States)

    Liu, Hong; Li, Wei; Zhang, Yong; Zhang, Zhengang; Shang, Xuejun; Zhang, Ling; Zhang, Shiyang; Li, Yanwei; Somoza, Andres V; Delpi, Brandon; Gerton, George L; Foster, James A; Hess, Rex A; Pazour, Gregory J; Zhang, Zhibing

    2017-05-01

    Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the "9+2" microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.

  7. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

    2017-01-01

    Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

  8. Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase.

    Science.gov (United States)

    Kato, Junya; Lefebre, Matthew; Galán, Jorge E

    2015-09-01

    Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. CmRBP50 protein phosphorylation is essential for assembly of a stable phloem-mobile high-affinity ribonucleoprotein complex.

    Science.gov (United States)

    Li, Pingfang; Ham, Byung-Kook; Lucas, William J

    2011-07-01

    RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

  10. 40 CFR 60.1170 - What plant-specific training must I provide?

    Science.gov (United States)

    2010-07-01

    ...) December 6, 2001. (3) The date before an employee assumes responsibilities that affect operation of the... 40 Protection of Environment 6 2010-07-01 2010-07-01 false What plant-specific training must I provide? 60.1170 Section 60.1170 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR...

  11. 40 CFR 62.15110 - Who must complete the plant-specific training course?

    Science.gov (United States)

    2010-07-01

    ...-specific training course? All employees with responsibilities that affect how a municipal waste combustion... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Who must complete the plant-specific training course? 62.15110 Section 62.15110 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...

  12. 40 CFR 60.1655 - Who must complete the plant-specific training course?

    Science.gov (United States)

    2010-07-01

    ...-specific training course? All employees with responsibilities that affect how a municipal waste combustion... 40 Protection of Environment 6 2010-07-01 2010-07-01 false Who must complete the plant-specific training course? 60.1655 Section 60.1655 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...

  13. High Protein Intake Does Not Prevent Low Plasma Levels of Conditionally Essential Amino Acids in Very Preterm Infants Receiving Parenteral Nutrition.

    Science.gov (United States)

    Morgan, Colin; Burgess, Laura

    2017-03-01

    We have shown that increasing protein intake using a standardized, concentrated, added macronutrients parenteral (SCAMP) nutrition regimen improves head growth in very preterm infants (VPIs) compared with a control parenteral nutrition (PN) regimen. VPIs are at risk of conditionally essential amino acid (CEAA) deficiencies because of current neonatal PN amino acid (AA) formulations. We hypothesized that the SCAMP regimen would prevent low plasma levels of CEAAs. To compare the plasma AA profiles at approximately day 9 of life in VPIs receiving SCAMP vs a control PN regimen. VPIs (parenteral and enteral protein, energy, and individual AA intake and the first plasma AA profile. Plasma profiles of the 20 individual protogenic AA levels were measured using ion exchange chromatography. Plasma AA profiles were obtained at median (interquartile range [IQR]) age of 9 (8-10) days in both SCAMP (n = 59) and control (n = 67) groups after randomizing 150 VPIs. Median (IQR) plasma levels of individual essential AAs were higher than the reference population mean (RPM) in both groups, especially for threonine. SCAMP infants had higher plasma levels of essential AAs than did the controls. Median (IQR) plasma levels of glutamine, arginine, and cysteine (CEAAs) were lower than the RPM in both groups. Plasma AA levels in PN-dependent VPIs indicate there is an imbalance in essential and CEAA provision in neonatal PN AA formulations that is not improved by increasing protein intake.

  14. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

    Science.gov (United States)

    Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

    2011-12-13

    Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

  15. Protein synthesis is essential not only for consolidation but also for maintenance and post-retrieval reconsolidation of acrobatic motor skill in rats.

    Science.gov (United States)

    Peng, Ji-Yun; Li, Bao-Ming

    2009-05-28

    It has been reported that consolidation of motor skill, a type of non-declarative memories, requires protein synthesis, as hippocampus-dependent declarative memory does. However, little is known about the importance of protein synthesis in maintenance and especially post-retrieval reconsolidation of acrobatic motor skill. Here, we show that protein synthesis is essential not only for the consolidation but also for the maintenance and reconsolidation of a rotarod-running skill. Intra-ventricle infusion of the protein synthesis inhibitor anisomycin 0 h but not 2 h post-training caused a severe deficit in the acquisition of the rotarod-running skill. Protein synthesis inhibition (PSI) also caused a deficit in the maintenance of the rotarod-running skill, as well-trained rats demonstrated a deficit in the rotarod-running performance upon treatment with anisomycin. Similarly, PSI impaired the post-retrieval reconsolidation of the rotarod-running skill: well-trained rats treated with anisomycin 0 h but not 0.5, 2 and 4 h after the task performance exhibited amnesia for the running skill later on. Interestingly, rats treated with anisomycin 6 and 12 h post-retrieval exhibited amnesia for the running skill. Thus, protein synthesis is essential not only for the consolidation but also for the maintenance and post-retrieval reconsolidation of rotarod-running acrobatic motor skill.

  16. Protein synthesis is essential not only for consolidation but also for maintenance and post-retrieval reconsolidation of acrobatic motor skill in rats

    Directory of Open Access Journals (Sweden)

    Peng Ji-Yun

    2009-05-01

    Full Text Available Abstract It has been reported that consolidation of motor skill, a type of non-declarative memories, requires protein synthesis, as hippocampus-dependent declarative memory does. However, little is known about the importance of protein synthesis in maintenance and especially post-retrieval reconsolidation of acrobatic motor skill. Here, we show that protein synthesis is essential not only for the consolidation but also for the maintenance and reconsolidation of a rotarod-running skill. Intra-ventricle infusion of the protein synthesis inhibitor anisomycin 0 h but not 2 h post-training caused a severe deficit in the acquisition of the rotarod-running skill. Protein synthesis inhibition (PSI also caused a deficit in the maintenance of the rotarod-running skill, as well-trained rats demonstrated a deficit in the rotarod-running performance upon treatment with anisomycin. Similarly, PSI impaired the post-retrieval reconsolidation of the rotarod-running skill: well-trained rats treated with anisomycin 0 h but not 0.5, 2 and 4 h after the task performance exhibited amnesia for the running skill later on. Interestingly, rats treated with anisomycin 6 and 12 h post-retrieval exhibited amnesia for the running skill. Thus, protein synthesis is essential not only for the consolidation but also for the maintenance and post-retrieval reconsolidation of rotarod-running acrobatic motor skill.

  17. Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

    DEFF Research Database (Denmark)

    Sleebs, Brad E; Lopaticki, Sash; Marapana, Danushka S

    2014-01-01

    , Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed...... surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp....... and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target....

  18. Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

    Science.gov (United States)

    Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

    2018-02-15

    Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

  19. SuHeXiang Wan essential oil alleviates amyloid beta induced memory impairment through inhibition of tau protein phosphorylation in mice.

    Science.gov (United States)

    Jeon, Songhee; Hur, Jinyoung; Jeong, Ha Jin; Koo, Byung-Soo; Pak, Sok Cheon

    2011-01-01

    SuHeXiang Wan (SHXW), a traditional Chinese medicine, has been used orally for the treatment of seizures, infantile convulsions and stroke. Previously, we reported the effects of a modified SHXW essential oil in terms of sedative effect, anticonvulsant activity and antioxidative activity. The purpose of this study was to evaluate the potential beneficial effects of SHXW essential oil in neurodegenerative diseases such as Alzheimer's disease (AD). SHXW essential oil was extracted from nine herbs. The mouse AD model was induced by a single injection of amyloid β protein (Aβ(1-42)) into the hippocampus. The animals were divided into four groups, the negative control group injected with Aβ(42-1), the Aβ group injected with Aβ(1-42), the SHXW group inhaled SHXW essential oil and received Aβ(1-42) injection, and the positive control group administered with docosahexaenoic acid (DHA, 10 mg/kg) and with subsequent Aβ(1-42) injection. Mice were analyzed by behavioral tests and immunological examination in the hippocampus. An additional in vitro investigation was performed to examine whether SHXW essential oil inhibits Aβ(1-42) induced neurotoxicity in a human neuroblastoma cell line, SH-SY5Y cells. Pre-inhalation of SHXW essential oil improved the Aβ(1-42) induced memory impairment and suppressed Aβ(1-42) induced JNK, p38 and Tau phosphorylation in the hippocampus. SHXW essential oil suppressed Aβ-induced apoptosis and ROS production via an up-regulation of HO-1 and Nrf2 expression in SH-SY5Y cells. The present study suggests that SHXW essential oil may have potential as a therapeutic inhalation drug for the prevention and treatment of AD.

  20. Essential amino acid enriched high-protein enteral nutrition modulates insulin-like growth factor-1 system function in a rat model of trauma-hemorrhagic shock.

    Directory of Open Access Journals (Sweden)

    Xianfeng Xia

    Full Text Available Nutrition support for critically ill patients supplemented with additional modular protein may promote skeletal muscle protein anabolism in addition to counteracting acute nitrogen loss. The present study was designed to investigate whether the essential amino acid (EAA enriched high-protein enteral nutrition (EN modulates the insulin-like growth factor-1 (IGF-1 system and activates the mammalian target of rapamycin (mTOR anabolic signaling pathway in a trauma-hemorrhagic shock (T-HS rat model.Male Sprague-Dawley rats (n = 90, 278.18 ± 0.94 g were randomly assigned to 5 groups: (1 normal control, (2 pair-fed, (3 T-HS, (4 T-HS and standard EN, and (5 T-HS and EAA enriched high-protein EN. Six animals from each group were harvested on days 2, 4, and 6 for serum, gastrocnemius, soleus, and extensor digitorum longus sample collection. T-HS significantly reduced muscle mass. Nutrition support maintained muscle mass, especially the EAA enriched high-protein EN. Meanwhile, a pronounced derangement in IGF-1-IGFBPs axis as well as impaired mTOR transduction was observed in the T-HS group. Compared with animals receiving standard EN, those receiving EAA enriched high-protein EN presented 18% higher serum free IGF-1 levels following 3 days of nutrition support and 22% higher after 5 days. These changes were consistent with the concomitant elevation in serum insulin and reduction in corticosterone levels. In addition, phosphorylations of downstream anabolic signaling effectors - including protein kinase B, mTOR, and ribosomal protein S6 kinase1 - increased significantly in rats receiving EAA enriched high-protein EN.Our findings firstly demonstrate the beneficial effect of EAA enriched high-protein EN on the metabolic modulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction.

  1. Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera

    OpenAIRE

    Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

    2017-01-01

    Abstract Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO cri...

  2. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Science.gov (United States)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  3. The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

    International Nuclear Information System (INIS)

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin

    2014-01-01

    The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. - Highlights: • mNLS mutant HPV16 L2 protein traffics pseudogenome to the TGN but fails to egress. • Cyclophilin activity is required for trafficking of the L2/DNA complex to the TGN. • Majority of L1 protein is shed from the L2/DNA complex prior to reaching the TGN

  4. The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

    Energy Technology Data Exchange (ETDEWEB)

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin, E-mail: msapp1@lsuhsc.edu

    2014-06-15

    The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. - Highlights: • mNLS mutant HPV16 L2 protein traffics pseudogenome to the TGN but fails to egress. • Cyclophilin activity is required for trafficking of the L2/DNA complex to the TGN. • Majority of L1 protein is shed from the L2/DNA complex prior to reaching the TGN.

  5. The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation

    International Nuclear Information System (INIS)

    Jin, S.; Roitsch, T.; Ankenbauer, R.G.; Gordon, M.P.; Nester, E.W.

    1990-01-01

    The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. The authors overproduced truncated VirA proteins in Escherichia coli be deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [γ- 32 P]ATP but not with [γ- 32 P]GTP or [α- 32 P]ATP, which suggests an ATP γ-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens

  6. Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

    Science.gov (United States)

    Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

    2018-02-01

    New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend. © 2018 Wiley-VHCA AG, Zurich, Switzerland.

  7. Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.

    Science.gov (United States)

    Walters, Alison D; Chong, James P J

    2017-05-01

    The single minichromosome maintenance (MCM) protein found in most archaea has been widely studied as a simplified model for the MCM complex that forms the catalytic core of the eukaryotic replicative helicase. Organisms of the order Methanococcales are unusual in possessing multiple MCM homologues. The Methanococcus maripaludis S2 genome encodes four MCM homologues, McmA-McmD. DNA helicase assays reveal that the unwinding activity of the three MCM-like proteins is highly variable despite sequence similarities and suggests additional motifs that influence MCM function are yet to be identified. While the gene encoding McmA could not be deleted, strains harbouring individual deletions of genes encoding each of the other MCMs display phenotypes consistent with these proteins modulating DNA damage responses. M. maripaludis S2 is the first archaeon in which MCM proteins have been shown to influence the DNA damage response.

  8. Intravenous supplementation of acetate, glucose or essential amino acids to an energy and protein deficient diet in lactating dairy goats

    DEFF Research Database (Denmark)

    Safayi, S.; Nielsen, M. O.

    2013-01-01

    amino acid supply is suboptimal. Goats were fed a basal diet deficient in energy (90% of requirements) and protein (80% of requirements), and were randomly allocated to 4 treatments in a balanced 4 x 4 Latin square design. The treatments consisted of 4-d continuous intravenous infusions of isoosmotic...... and close to significantly by ACE, but not by GLU treatment. GLU reduced milk protein percentage compared to all other treatments. High milk protein yields on EM and ACE treatments were associated with higher arterial AVD for acetate and oxygen (not significant for ACE), and higher AVD also for beta......In the present experiment we aimed to study, if milk synthesis is more sensitive toward deficiency in supply of amino acids in early (EL) versus late lactation (LL), and if energy yielding substrates in the form of acetate (but not glucose) can contribute to sustain milk (protein) synthesis, when...

  9. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids.

    Science.gov (United States)

    Dickinson, Jared M; Fry, Christopher S; Drummond, Micah J; Gundermann, David M; Walker, Dillon K; Glynn, Erin L; Timmerman, Kyle L; Dhanani, Shaheen; Volpi, Elena; Rasmussen, Blake B

    2011-05-01

    The relationship between mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis during instances of amino acid surplus in humans is based solely on correlational data. Therefore, the goal of this study was to use a mechanistic approach specifically designed to determine whether increased mTORC1 activation is requisite for the stimulation of muscle protein synthesis following L-essential amino acid (EAA) ingestion in humans. Examination of muscle protein synthesis and signaling were performed on vastus lateralis muscle biopsies obtained from 8 young (25 ± 2 y) individuals who were studied prior to and following ingestion of 10 g of EAA during 2 separate trials in a randomized, counterbalanced design. The trials were identical except during 1 trial, participants were administered a single oral dose of a potent mTORC1 inhibitor (rapamycin) prior to EAA ingestion. In response to EAA ingestion, an ~60% increase in muscle protein synthesis was observed during the control trial, concomitant with increased phosphorylation of mTOR (Ser(2448)), ribosomal S6 kinase 1 (Thr(389)), and eukaryotic initiation factor 4E binding protein 1 (Thr(37/46)). In contrast, prior administration of rapamycin completely blocked the increase in muscle protein synthesis and blocked or attenuated activation of mTORC1-signaling proteins. The inhibition of muscle protein synthesis and signaling was not due to differences in either extracellular or intracellular amino acid availability, because these variables were similar between trials. These data support a fundamental role for mTORC1 activation as a key regulator of human muscle protein synthesis in response to increased EAA availability. This information will be useful in the development of evidence-based nutritional therapies targeting mTORC1 to counteract muscle wasting associated with numerous clinical conditions.

  10. A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

    Science.gov (United States)

    Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

    2008-10-01

    Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

  11. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels

    NARCIS (Netherlands)

    van Tiel, Claudia M.; Westerman, Jan; Paasman, Marten A.; Hoebens, Martha M.; Wirtz, Karel W. A.; Snoek, Gerry T.

    2002-01-01

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165)

  12. SpoVT: From Fine-Tuning Regulator in Bacillus subtilis to Essential Sporulation Protein in Bacillus cereus.

    Science.gov (United States)

    Eijlander, Robyn T; Holsappel, Siger; de Jong, Anne; Ghosh, Abhinaba; Christie, Graham; Kuipers, Oscar P

    2016-01-01

    Sporulation is a highly sophisticated developmental process adopted by most Bacilli as a survival strategy to withstand extreme conditions that normally do not support microbial growth. A complicated regulatory cascade, divided into various stages and taking place in two different compartments of the cell, involves a number of primary and secondary regulator proteins that drive gene expression directed toward the formation and maturation of an endospore. Such regulator proteins are highly conserved among various spore formers. Despite this conservation, both regulatory and phenotypic differences are observed between different species of spore forming bacteria. In this study, we demonstrate that deletion of the regulatory sporulation protein SpoVT results in a severe sporulation defect in Bacillus cereus , whereas this is not observed in Bacillus subtilis . Although spores are initially formed, the process is stalled at a later stage in development, followed by lysis of the forespore and the mother cell. A transcriptomic investigation of B. cereus Δ spoVT shows upregulation of genes involved in germination, potentially leading to premature lysis of prespores formed. Additionally, extreme variation in the expression of species-specific genes of unknown function was observed. Introduction of the B. subtilis SpoVT protein could partly restore the sporulation defect in the B. cereus spoVT mutant strain. The difference in phenotype is thus more than likely explained by differences in promoter targets rather than differences in mode of action of the conserved SpoVT regulator protein. This study stresses that evolutionary variances in regulon members of sporulation regulators can have profound effects on the spore developmental process and that mere protein homology is not a foolproof predictor of similar phenotypes.

  13. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

    Science.gov (United States)

    Robscheit-Robbins, F S; Miller, L L; Whipple, G H

    1947-02-28

    Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

  14. Interactions among predators and plant specificity protect herbivores from top predators.

    Science.gov (United States)

    Bosc, Christopher; Pauw, Anton; Roets, Francois; Hui, Cang

    2018-05-04

    The worldwide loss of top predators from natural and agricultural systems has heightened the need to understand how important they are in controlling herbivore abundance. The effect of top predators on herbivore species is likely to depend on 1) the importance of the consumption of intermediate predators by top predators (intra-guild predation; IGP), but also on 2) plant specificity by herbivores, because specialists may defend themselves better (enemy-free space; EFS). Insectivorous birds, as top predators, are generally known to effectively control herbivorous insects, despite also consuming intermediate predators such as spiders, but how this effect varies among herbivore species in relation to the cascading effects of IGP and EFS is not known. To explore this, we excluded birds from natural fynbos vegetation in South Africa using large netted cages and recorded changes in abundance relative to control plots for 199 plant-dwelling intermediate predator and 341 herbivore morpho-species that varied in their estimated plant specificity. We found a strong negative effect of birds on the total abundance of all intermediate predators, with especially clear effects on spiders (strong IGP). In contrast with previous studies, which document a negative effect of birds on herbivores, we found an overall neutral effect of birds on herbivore abundance, but the effect varied among species: some species were negatively affected by birds, suggesting that they were mainly consumed by birds, whereas others, likely released from spiders by IGP, were positively affected. Some species were also effectively neutrally affected by birds. These tended to be more specialized to plants compared to the other species, which may imply that some plant specialists benefited from protection provided by EFS from both birds and spiders. These results suggest that the response of herbivore species to top predators may depend on cascading effects of interactions among predators and on their degree

  15. Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

    NARCIS (Netherlands)

    van der Sligte, Naomi E.; Kampen, Kim R.; ter Elst, Arja; Scherpen, Frank J. G.; Meeuwsen-de Boer, Tiny G. J.; Guryev, Victor; van Leeuwen, Frank N.; Kornblau, Steven M.; de Bont, Eveline S. J. M.

    2015-01-01

    Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric

  16. The Fe/S Cluster Assembly Protein Isd11 Is Essential for tRNA Thiolation in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Paris, Zdeněk; Changmai, Piya; RUBIO, M. A. T.; Zíková, Alena; Stuart, K. D.; Alfonzo, J. D.; Lukeš, Julius

    2010-01-01

    Roč. 285, č. 29 (2010), s. 22394-22402 ISSN 0021-9258 R&D Projects: GA ČR GA204/09/1667 Institutional research plan: CEZ:AV0Z60220518 Keywords : IRON-SULFUR PROTEINS * SACCHAROMYCES-CEREVISIAE * CYSTEINE DESULFURASE * THIO- MODIFICATION * FRATAXIN Subject RIV: EB - Genetic s ; Molecular Biology Impact factor: 5.328, year: 2010

  17. ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

    Science.gov (United States)

    Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

    2013-01-25

    The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

  18. Metals and ions in protein structures - from essential to marginal questions in identification of ions in enzymes and receptors

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan

    2013-01-01

    Roč. 20, č. 1 (2013), s. 14-15 ISSN 1211-5894. [Discussions in Structural Molecular Biology /11./. 14.03.2013-16.03.2013, Nové Hrady] R&D Projects: GA ČR GA310/09/1407 Institutional support: RVO:61389013 Keywords : protein * X-ray diffraction * metal Subject RIV: EE - Microbiology, Virology

  19. Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression

    Science.gov (United States)

    Translational control of ATF4 through upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While ATF4 translation is typically induced by inhibitory phosphorylation of eIF2, ATF4 translation can be also induced by expression of a new translational inhibitor protein, eIF5-mimi...

  20. The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sergi Portolés

    2010-11-01

    Full Text Available Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1. CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2 are essential for clock temperature compensation in Arabidopsis.

  1. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    International Nuclear Information System (INIS)

    Xu, H. Howard; Real, Lilian; Bailey, Melissa Wu

    2006-01-01

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats

  2. Identification of pH-sensing Sites in the Light Harvesting Complex Stress-related 3 Protein Essential for Triggering Non-photochemical Quenching in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Ballottari, Matteo; Truong, Thuy B; De Re, Eleonora; Erickson, Erika; Stella, Giulio R; Fleming, Graham R; Bassi, Roberto; Niyogi, Krishna K

    2016-04-01

    Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green algaChlamydomonas reinhardtii Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesisin vivoandin vitrofor identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp(117), Glu(221), and Glu(224)were shown to be essential for LHCSR3-dependent NPQ induction inC. reinhardtii Analysis of recombinant proteins carrying the same mutations refoldedin vitrowith pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

    Science.gov (United States)

    Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

    2017-10-19

    The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

    DEFF Research Database (Denmark)

    John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

    2010-01-01

    The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

  5. Tetratricopeptide repeat protein Pyg7 is essential for photosystem I assembly by interacting with PsaC in Arabidopsis.

    Science.gov (United States)

    Yang, Huixia; Li, Pin; Zhang, Aihong; Wen, Xiaogang; Zhang, Lixin; Lu, Congming

    2017-09-01

    Although progress has been made in determining the structure and understanding the function of photosystem I (PSI), the PSI assembly process remains poorly understood. PsaC is an essential subunit of PSI and participates in the transfer of electrons to ferredoxin. However, how PsaC is assembled during accumulation of the PSI complex is unknown. In the present study, we showed that Pyg7 localized to the stromal thylakoid and associated with the PSI complex. We also showed that Pyg7 interacted with PsaC. Furthermore, we found that the PSI assembly process was blocked following formation of the PsaAB heterodimer in the pyg7 mutant. In addition, the analyses of PSI stability in Pyg7RNAi plants showed that Pyg7 is involved in maintaining the assembled PSI complex under excess-light conditions. Moreover, we demonstrated that decreased Pyg7 content resulted in decreased efficiency of PSI assembly in Pyg7RNAi plants. These findings suggest that the role of Pyg7 in PSI biogenesis has evolved as an essential assembly factor by interacting with PsaC in Arabidopsis, in addition to being a stability factor for PSI as seen in Synechocystis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  6. The Candida albicans Ddr48 protein is essential for filamentation, stress response, and confers partial antifungal drug resistance.

    Science.gov (United States)

    Dib, Leila; Hayek, Peter; Sadek, Helen; Beyrouthy, Berna; Khalaf, Roy A

    2008-06-01

    Candida albicans is a dimorphic pathogenic fungus that causes mucosal and systemic infections. C. albicans pathogenicity is attributed to its ability to exist in different morphologic states and to respond to stress by up regulating several key genes. DDR48 is a stress-associated gene involved in DNA repair and in response to antifungal drug exposure. One allele of DDR48 was knocked out by homologous recombination that inserted a marker cassette in its position. Furthermore, reintroducing DDR48 on a plasmid created a revertant strain. Strains were grown on filamentation inducing and noninducing media, subjected to an oxidative stress challenge, injected into mice to assess virulence, and assayed for antifungal susceptibility by the E-test method. DDR48 was found to be haploid insufficient and possibly essential, since only a heterozygote, but not a homozygous, null mutant was generated. The mutant was filamentation defective on all hyphal media tested including serum and corn meal agar. Discrepancies in drug resistance profiles also were present: compared with the parental strain, DDR48/ddr48 heterozygote strain was susceptible in a dose-dependent manner to itraconazole and fluconazole and susceptible to ketoconazole. The mutant also appeared to be hypersensitive to a potentially lethal hydrogen peroxide challenge. However, no reduction in virulence of the mutant was observed. The present findings provide evidence that DDR48 is essential for filamentation, stress response, and possibly viability of C. albicans, making it a prime target for antifungal drug design.

  7. EGF-CFC proteins are essential coreceptors for the TGF-β signals Vg1 and GDF1

    Science.gov (United States)

    Cheng, Simon K.; Olale, Felix; Bennett, James T.; Brivanlou, Ali H.; Schier, Alexander F.

    2003-01-01

    The TGF-β signals Nodal, Activin, GDF1, and Vg1 have been implicated in mesoderm induction and left-right patterning. Nodal and Activin both activate Activin receptors, but only Nodal requires EGF-CFC coreceptors for signaling. We report that Vg1 and GDF1 signaling in zebrafish also depends on EGF-CFC proteins, but not on Nodal signals. Correspondingly, we find that in Xenopus Vg1 and GDF1 bind to and signal through Activin receptors only in the presence of EGF-CFC proteins. These results establish that multiple TGF-β signals converge on Activin receptor/EGF-CFC complexes and suggest a more widespread requirement for coreceptors in TGF-β signaling than anticipated previously. PMID:12514096

  8. ngs (Notochord Granular Surface) Gene Encodes a Novel Type of Intermediate Filament Family Protein Essential for Notochord Maintenance in Zebrafish*

    Science.gov (United States)

    Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

    2013-01-01

    The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins. PMID:23132861

  9. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    OpenAIRE

    Kowalewski-Nimmerfall, Elisabeth; Sch?hs, Philipp; Maresch, Daniel; Rendic, Dubravko; Kr?mer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be subst...

  10. Essential corrections of interference reactions in the determination of protein contents of Nigeria foodstuffs using fast neutron activation analysis

    International Nuclear Information System (INIS)

    Adesanmi, C.A.; Essiett, A.A.; Balogun, F.A.

    2003-01-01

    Using the 14 N(n, zn) 13 N reaction, the protein contents of some Nigeria staple foods were estimated. The contribution of some elements in these foods such as Br, P, K and the recoil proton to the 511 KeV photopeak commonly used in this technique was investigated. While Br was found absent from the high carbohydrate foods such as the cassava tuber based 'gari', maize and rice, this element was shown to produce significant interference in broad beans and soya beans. The contribution of Br to the 511 Kev line in protein measurement in the latter set of food materials ranged between 12% and 62%. Proton recoil were found to be a significant source of interference in virtually all the food stuffs investigated. The result of protein contents using 14 MeV compared favourably, within experimental errors, with that of the wet kjeldahl method, except in the case of soya beans which indicates an underestimation of the contribution from the recoil proton

  11. Studies on limiting essential amino acids in grieves as source of dietary protein for rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Pfeffer, E; Mandel, S; Rodehutscord, M

    1994-01-01

    Diets were computed to contain equal concentrations of digestible crude protein either of wheat gluten (diet 1) or of grieves (diets 2-8). Per kg dry diet, 41 g crystalline amino acids were supplemented. All diets contained at least 1.2 g Lys per MJ digestible energy (DE). In diet 2, ratios of Met + Cys, Trp, Leu, Ile and Phe to Lys were about equal to those in diet 1. In each of diets 3-7, one of the respective amino acids, in diet 8 all five were replaced by Glu in the supplemented mixture of amino acids. Each diet was fed to triplicate groups of 20 trout during a trial lasting 66 days. Trout fed the diet containing wheat gluten consumed more dry matter and showed higher growth rates as well as higher protein contents in their gained body mass than trout fed diets based on grieves. Supplementing Met plus Trp significantly improved dry matter intake, growth rate and protein content of gain, though not to the level of trout fed the wheat gluten diet, whereas Leu, Ile and Phe showed no such effect. When grieves were not supplemented with both Met and Trp, gain in body mass contained significantly more lipids. DE required per kg gain by trout fed wheat gluten, grieves + Met + Trp or grieves without supplementation of Met and Trp was 20.1, 21.2 and 29.9 MJ, respectively.

  12. Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer

    OpenAIRE

    O'Sullivan, Aine G.; Mulvaney, Eamon P.; Hyland, Paula B.; Kinsella, B. Therese

    2015-01-01

    The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TP? and TP? isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TP?/TP?-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenet...

  13. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

    DEFF Research Database (Denmark)

    Becker-Heck, Anita; Zohn, Irene E; Okabe, Noriko

    2011-01-01

    Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right...... organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40...

  14. Plant specification of a generic human-error data through a two-stage Bayesian approach

    International Nuclear Information System (INIS)

    Heising, C.D.; Patterson, E.I.

    1984-01-01

    Expert judgement concerning human performance in nuclear power plants is quantitatively coupled with actuarial data on such performance in order to derive plant-specific human-error rate probability distributions. The coupling procedure consists of a two-stage application of Bayes' theorem to information which is grouped by type. The first information type contains expert judgement concerning human performance at nuclear power plants in general. Data collected on human performance at a group of similar plants forms the second information type. The third information type consists of data on human performance in a specific plant which has the same characteristics as the group members. The first and second information types are coupled in the first application of Bayes' theorem to derive a probability distribution for population performance. This distribution is then combined with the third information type in a second application of Bayes' theorem to determine a plant-specific human-error rate probability distribution. The two stage Bayesian procedure thus provides a means to quantitatively couple sparse data with expert judgement in order to obtain a human performance probability distribution based upon available information. Example calculations for a group of like reactors are also given. (author)

  15. Cytosolic iron-sulphur protein assembly is functionally conserved and essential in procyclic and bloodstream Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Basu, Somsuvro; Netz, D. J.; Haindrich, A. C.; Herlerth, N.; Lagny, T. J.; Pierik, A. J.; Lill, R.; Lukeš, Julius

    2014-01-01

    Roč. 93, č. 5 (2014), s. 897-910 ISSN 0950-382X R&D Projects: GA ČR(CZ) GAP305/11/2179; GA MŠk LH12104; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : inducible expression system * Cfd1- Nbp35 complex * DNA metabolism * Fe/S proteins * transfer-RNA * cluster * mitochondrial * maturation * biogenesis * yeast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.419, year: 2014

  16. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence.

    Science.gov (United States)

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R; Kehn-Hall, Kylene; Omichinski, James G

    2015-05-12

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH.

  17. GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

    Science.gov (United States)

    Tafesse, Fikadu G; Guimaraes, Carla P; Maruyama, Takeshi; Carette, Jan E; Lory, Stephen; Brummelkamp, Thijn R; Ploegh, Hidde L

    2014-08-29

    A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. WHITE PANICLE3, a Novel Nucleus-Encoded Mitochondrial Protein, Is Essential for Proper Development and Maintenance of Chloroplasts and Mitochondria in Rice

    Directory of Open Access Journals (Sweden)

    Hongchang Li

    2018-06-01

    Full Text Available Mitochondria and chloroplasts are interacting organelles that play important roles in plant development. In addition to a small number proteins encoded by their own genomes, the majority of mitochondrial and chloroplast proteins are encoded in the cell nucleus and imported into the organelle. As a consequence, coordination between mitochondria, chloroplasts, and the nucleus is of crucial importance to plant cells. Variegated mutants are chloroplast-defective mutants and are considered to be ideal models for studying the intercommunication between these organelles. Here, we report the isolation of WHITE PANICLE3 (WP3, a nuclear gene involved in variegation, from a naturally occurring white panicle rice mutant. Disrupted expression of WP3 in the mutant leads to severe developmental defects in both chloroplasts and mitochondria, and consequently causes the appearance of white-striped leaves and white panicles in the mutant plants. Further investigation showed that WP3 encodes a protein most likely targeted to mitochondria and is specifically expressed in rice panicles. Interestingly, we demonstrate that the recessive white-panicle phenotype in the wp3 mutant is inherited in a typical Mendelian manner, while the white-striped leaf phenotype in wp3 is maternally inherited. Our data collectively suggest that the nucleus-encoded mitochondrial protein, WP3, plays an essential role in the regulation of chloroplast development in rice panicles by maintaining functional mitochondria. Therefore, the wp3 mutant is an excellent model in which to explore the communication between the nucleus, mitochondria, and chloroplasts in plant cells.

  19. The lysosomal enzyme receptor protein (LERP is not essential, but is implicated in lysosomal function in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Medina Hasanagic

    2015-10-01

    Full Text Available The lysosomal enzyme receptor protein (LERP of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, LerpF6, which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30–40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy.

  20. Geranylgeranyl diphosphate synthase in fission yeast is a heteromer of farnesyl diphosphate synthase (FPS), Fps1, and an FPS-like protein, Spo9, essential for sporulation.

    Science.gov (United States)

    Ye, Yanfang; Fujii, Makoto; Hirata, Aiko; Kawamukai, Makoto; Shimoda, Chikashi; Nakamura, Taro

    2007-09-01

    Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1(+) and spo9(+), whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1(+), but not spo9(+), suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1(+) nor spo9(+) expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9Delta cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9Delta cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

  1. Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

    Science.gov (United States)

    Aguiar, Bruno G; Padmanabhan, Prasad K; Dumas, Carole; Papadopoulou, Barbara

    2018-06-12

    VCP/p97/Cdc48 is one of the best-characterized type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homolog (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, L. infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress and a temperature sensitive VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP mRNA undergoes 3'UTR-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasize the crucial role LiVCP plays under heat stress and during the parasite intracellular development. This article is protected by copyright. All rights reserved.

  2. Nucleic acid labeling with [3H]orotic acid and nucleotide profile in rats in protein deprivation, enteral and parenteral essential amino acid administration, and 5-fluorouracil treatment

    International Nuclear Information System (INIS)

    Jakobsson, B.; el Hag, I.A.; Andersson, M.; Christensson, P.I.; Stenram, U.

    1990-01-01

    Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of [3H]orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes

  3. Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates.

    Science.gov (United States)

    Ning, Jue; Otto, Thomas D; Pfander, Claudia; Schwach, Frank; Brochet, Mathieu; Bushell, Ellen; Goulding, David; Sanders, Mandy; Lefebvre, Paul A; Pei, Jimin; Grishin, Nick V; Vanderlaan, Gary; Billker, Oliver; Snell, William J

    2013-05-15

    Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

  4. Identification of amino acids essential for the human parainfluenza type 2 virus V protein to lower the intracellular levels of the STAT2

    International Nuclear Information System (INIS)

    Kozuka, Yuji; Yamashita, Yasufumi; Kawano, Mitsuo; Tsurudome, Masato; Ito, Morihiro; Nishio, Machiko; Komada, Hiroshi; Ito, Yasuhiko

    2003-01-01

    The V protein of SV41 targets STAT1, while a specific loss of STAT2 is induced by the hPIV2 V protein. We established HeLa cells constitutively expressing various chimeric proteins between the hPIV2 and SV41 V proteins, and which STAT (STAT1 or 2) was expressed in these cells was analyzed. Both the P-V common domain and the V specific domain of hPIV2 V protein are necessary for STAT2 lowering. The internal domain (aa145-173) containing a large number of nonidentical amino acids between hPIV2 and SV41 does not direct STAT tropism, and the regions necessary for STAT2 lowering are discontinuous. The N-terminal domain (aa1-104) and the internal domain (aa126-196) of the hPIV2 V protein do not determine STAT tropism. HeLa cells expressing A105E or H108P show distinct expression of STAT2, but do show low expression or a loss of STAT1, indicating that the amino acid residues 105 and 108 of the hPIV2 V protein are essential for STAT2 lowering. Interestingly, there is an important amino acid(s) in the region (aa121-125) for STAT2 lowering, and the presence of either amino acid residue 123 or 125 of the hPIV2 V protein is necessary for lowering of STAT2. In addition, HeLa cells expressing S216D or 1217R expressed STAT2, but no STAT1, indicating that the amino acid residues 216 and 217 of the hPIV2 V protein are indispensable for STAT2 lowering. HeLa/hPIV2V cells and HeLa/S104/P are resistant to IFN-β, while they are sensitive to IFN-γ. On the other hand, HeLa/SV41V, HeLa/S216D, and HeLa1217R cells are resistant to both IFNs. Intriguingly, HeLa/A105E and HeLa/H108P cells were found to be sensitive to IFN-γ

  5. Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor: a study on possible competition for the semi-essential amino acid cysteine

    OpenAIRE

    Pedersen, Sindre Andre

    2007-01-01

    In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are ...

  6. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao

    2015-03-01

    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  7. Shewanella oneidensis MR-1 chemotaxis proteins and electron-transport chain components essential for congregation near insoluble electron acceptors.

    Science.gov (United States)

    Harris, H Wayne; El-Naggar, Mohamed Y; Nealson, Kenneth H

    2012-12-01

    Shewanella oneidensis MR-1 cells utilize a behaviour response called electrokinesis to increase their speed in the vicinity of IEAs (insoluble electron acceptors), including manganese oxides, iron oxides and poised electrodes [Harris, El-Naggar, Bretschger, Ward, Romine, Obraztsova and Nealson (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 326-331]. However, it is not currently understood how bacteria remain in the vicinity of the IEA and accumulate both on the surface and in the surrounding medium. In the present paper, we provide results indicating that cells that have contacted the IEAs swim faster than those that have not recently made contact. In addition, fast-swimming cells exhibit an enhancement of swimming reversals leading to rapid non-random accumulation of cells on, and adjacent to, mineral particles. We call the observed accumulation near IEAs 'congregation'. Congregation is eliminated by the loss of a critical gene involved with EET (extracellular electron transport) (cymA, SO_4591) and is altered or eliminated in several deletion mutants of homologues of genes that are involved with chemotaxis or energy taxis in Escherichia coli. These genes include chemotactic signal transduction protein (cheA-3, SO_3207), methyl-accepting chemotaxis proteins with the Cache domain (mcp_cache, SO_2240) or the PAS (Per/Arnt/Sim) domain (mcp_pas, SO_1385). In the present paper, we report studies of S. oneidensis MR-1 that lend some insight into how microbes in this group can 'sense' the presence of a solid substrate such as a mineral surface, and maintain themselves in the vicinity of the mineral (i.e. via congregation), which may ultimately lead to attachment and biofilm formation.

  8. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

  9. Evaluation of Risk Metrics for KHNP Reference Plants Using the Latest Plant Specific Data

    International Nuclear Information System (INIS)

    Jeon, Ho Jun; Hwang, Seok Won; Ghi, Moon Goo

    2010-01-01

    As Risk-Informed Applications (RIAs) are actively implemented in the nuclear industry, an issue associated with the technical adequacy of the Probabilistic Safety Assessment (PSA) arises in its data sources. The American Society of Mechanical Engineers (ASME) PRA standard suggests the use of component failure data that represent the as-built and as-operated plant conditions. Furthermore, the peer reviews for the KHNP reference plants stated that the component failure data should be updated to reflect the latest plant specific data available. For ensuring the technical adequacy in PSA data elements, we try to update component failure data to reflect the as-operated plant conditions, and a trend analysis of the failure data is implemented. In addition, by applying the updated failure data to the PSA models of the KHNP reference plants, the risk metrics of Core Damage Frequency (CDF) and Large Early Release Frequency (LERF) are evaluated

  10. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang

    2017-01-01

    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  11. Comparison and lessons learned from plant specific PSA of German NPP

    International Nuclear Information System (INIS)

    Balfanz, Hans-Peter; Berg, H.P.

    2000-01-01

    PSA are launched in frame of Periodic Safety Reviews (PSR) in Germany. The aims are to identify overall safety level and relative weak points. Some backfitting measures have been realized for older plants to remove relative weak points and to bring these plants to the state of the art. In this field PSA is well accepted today and is seen as a valuable tool supplementing the deterministic analysis. Main application of PSA within PSR is planned to become mandatory as part of the revision of the German Atomic Energy Act. According to the German PSA Guideline plant specific PSA level 1+ were performed for all 19 In comparison with international practice German PSA are very detailed. Otherwise they do not handle all external events, non-power states and accident management measures as discussed before. The New PSA guideline will cover these aspects and therefore analysts have to take them into account in further PSA. Moreover gathering of plant specific data is needed. The development in this field is driven by the utilities (for instance in frame of their so-called ZEDB project). Public discussion about quantitative risk of industrial hazards is quite limited in Germany and PSA results have only few impacts to this respect. Independent from this PSA for NPP is understood as a diverse tool in supporting the deterministic licensing and supervision process. Risk based decision making as well as informed regulation are just only of the beginning. State of PSA of NPP in Germany, comparison of PSA result of different NPP, German PSA guideline and state of discussion of further development and recommendation of further development of PSA of NPP are discussed in this paper in more detail. (S.Y.)

  12. Comparison and lessons learned from plant specific PSA of German NPP

    Energy Technology Data Exchange (ETDEWEB)

    Balfanz, Hans-Peter [TUEV Nord, Hamburg (Germany); Berg, H.P.

    2000-07-01

    PSA are launched in frame of Periodic Safety Reviews (PSR) in Germany. The aims are to identify overall safety level and relative weak points. Some backfitting measures have been realized for older plants to remove relative weak points and to bring these plants to the state of the art. In this field PSA is well accepted today and is seen as a valuable tool supplementing the deterministic analysis. Main application of PSA within PSR is planned to become mandatory as part of the revision of the German Atomic Energy Act. According to the German PSA Guideline plant specific PSA level 1+ were performed for all 19 In comparison with international practice German PSA are very detailed. Otherwise they do not handle all external events, non-power states and accident management measures as discussed before. The New PSA guideline will cover these aspects and therefore analysts have to take them into account in further PSA. Moreover gathering of plant specific data is needed. The development in this field is driven by the utilities (for instance in frame of their so-called ZEDB project). Public discussion about quantitative risk of industrial hazards is quite limited in Germany and PSA results have only few impacts to this respect. Independent from this PSA for NPP is understood as a diverse tool in supporting the deterministic licensing and supervision process. Risk based decision making as well as informed regulation are just only of the beginning. State of PSA of NPP in Germany, comparison of PSA result of different NPP, German PSA guideline and state of discussion of further development and recommendation of further development of PSA of NPP are discussed in this paper in more detail. (S.Y.)

  13. Na+/K+-ATPase α1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role in the barrier integrity.

    Directory of Open Access Journals (Sweden)

    Yue Yang

    2011-01-01

    Full Text Available The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+/K(+-ATPase α1 (ATP1A1 with protein kinase C eta (PKCη and occludin is one of the mechanisms of loud sound-induced vascular permeability increase.Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma.The results presented here provide a novel method for

  14. The moderate essential amino acid restriction entailed by low-protein vegan diets may promote vascular health by stimulating FGF21 secretion.

    Science.gov (United States)

    McCarty, Mark F

    2016-02-12

    The serum total and LDL cholesterol levels of long-term vegans tend to be very low. The characteristically low ratio of saturated to unsaturated fat in vegan diets, and the absence of cholesterol in such diets, clearly contribute to this effect. But there is reason to suspect that the quantity and composition of dietary protein also play a role in this regard. Vegan diets of moderate protein intake tend to be relatively low in certain essential amino acids, and as a result may increase hepatic activity of the kinase GCN2, which functions as a gauge of amino acid status. GCN2 activation boosts the liver's production of fibroblast growth factor 21 (FGF21), a factor which favorably affects serum lipids and metabolic syndrome. The ability of FGF21 to decrease LDL cholesterol has now been traced to at least two mechanisms: a suppression of hepatocyte expression of sterol response element-binding protein-2 (SREBP-2), which in turn leads to a reduction in cholesterol synthesis; and up-regulated expression of hepatocyte LDL receptors, reflecting inhibition of a mechanism that promotes proteasomal degradation of these receptors. In mice, the vascular benefits of FGF21 are also mediated by favorable effects on adipocyte function - most notably, increased adipocyte secretion of adiponectin, which directly exerts anti-inflammatory effects on the vasculature which complement the concurrent reduction in LDL particles in preventing or reversing atherosclerosis. If, as has been proposed, plant proteins preferentially stimulate glucagon secretion owing to their amino acid composition, this would represent an additional mechanism whereby plant protein promotes FGF21 activity, as glucagon acts on the liver to boost transcription of the FGF21 gene.

  15. The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

    Science.gov (United States)

    Arsenault, Patrick R; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S; Lee, Frank S

    2016-09-15

    Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. The adaptor protein CIKS/Act1 is essential for IL-25-mediated allergic airway inflammation1

    Science.gov (United States)

    Claudio, Estefania; Sønder, Søren Ulrik; Saret, Sun; Carvalho, Gabrielle; Ramalingam, Thirumalai R; Wynn, Thomas A; Chariot, Alain; Garcia-Perganeda, Antonio; Leonardi, Antonio; Paun, Andrea; Chen, Amy; Ren, Nina Y.; Wang, Hongshan; Siebenlist, Ulrich

    2008-01-01

    IL-17 is the signature cytokine of recently discovered T helper type 17 (Th17) cells, which are prominent in defense against extracellular bacteria and fungi as well as in autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis in animal models. IL-25 is a member of the IL-17 family of cytokines, but has been associated with Th2 responses instead and may negatively cross-regulate Th17/IL-17 responses. IL-25 can initiate an allergic asthma-like inflammation in the airways, which includes recruitment of eosinophils, mucus hypersecretion, Th2 cytokine production and airways hyperreactivity. We demonstrate that these effects of IL-25 are entirely dependent on the adaptor protein CIKS (a.k.a. Act1). Surprisingly, this adaptor is necessary to transmit IL-17 signals as well, despite the very distinct biologic responses these two cytokines elicit. We identify CD11c+ macrophage-like lung cells as physiologic relevant targets of IL-25 in vivo. PMID:19155511

  17. A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development1[OPEN

    Science.gov (United States)

    Blanco, Lourdes; Quinto, Carmen

    2016-01-01

    The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR’s role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean. PMID:27698253

  18. Arborvitae (Thuja plicata essential oil significantly inhibited critical inflammation- and tissue remodeling-related proteins and genes in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Xuesheng Han

    2017-06-01

    Full Text Available Arborvitae (Thuja plicata essential oil (AEO is becoming increasingly popular in skincare, although its biological activity in human skin cells has not been investigated. Therefore, we sought to study AEO's effect on 17 important protein biomarkers that are closely related to inflammation and tissue remodeling by using a pre-inflamed human dermal fibroblast culture model. AEO significantly inhibited the expression of vascular cell adhesion molecule 1 (VCAM-1, intracellular cell adhesion molecule 1 (ICAM-1, interferon gamma-induced protein 10 (IP-10, interferon-inducible T-cell chemoattractant (I-TAC, monokine induced by interferon gamma (MIG, and macrophage colony-stimulating factor (M-CSF. It also showed significant antiproliferative activity and robustly inhibited collagen-I, collagen-III, plasminogen activator inhibitor-1 (PAI-1, and tissue inhibitor of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2. The inhibitory effect of AEO on increased production of these protein biomarkers suggests it has anti-inflammatory property. We then studied the effect of AEO on the genome-wide expression of 21,224 genes in the same cell culture. AEO significantly and diversely modulated global gene expression. Ingenuity pathway analysis (IPA showed that AEO robustly affected numerous critical genes and signaling pathways closely involved in inflammatory and tissue remodeling processes. The findings of this study provide the first evidence of the biological activity and beneficial action of AEO in human skin cells.

  19. A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

    Science.gov (United States)

    Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

    2016-11-16

    Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  20. Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

    International Nuclear Information System (INIS)

    Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.; Garofoli, Daniella; Ewen, Catherine; Davidson, Courtney E.; Ghaffari, Mazyar; Kane, Kevin P.; Lacy, Paige; Logan, Michael R.; Befus, A. Dean; Bleackley, R. Chris; Moqbel, Redwan

    2008-01-01

    Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo

  1. The pH dependence of polymerization and bundling by the essential bacterial cytoskeletal protein FtsZ.

    Directory of Open Access Journals (Sweden)

    Raúl Pacheco-Gómez

    Full Text Available There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow. It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP hydrolysis to polymer formation, and electron microscopy (EM imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution--it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli--it stabilizes associations of protofilaments.

  2. Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

    Directory of Open Access Journals (Sweden)

    Scaife Jes R

    2008-02-01

    Full Text Available Abstract Background Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose (to stimulate insulin and essential amino acids (EAA would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of

  3. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

    Science.gov (United States)

    van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

    2002-06-21

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

  4. Essential Tremor

    Science.gov (United States)

    ... Treatment There is no definitive cure for essential tremor. Symptomatic drug therapy may include propranolol or other beta blockers and primidone, an anticonvulsant drug. Eliminating tremor "triggers" ...

  5. The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation.

    Science.gov (United States)

    Miguel-Arribas, Andrés; Hao, Jian-An; Luque-Ortega, Juan R; Ramachandran, Gayetri; Val-Calvo, Jorge; Gago-Córdoba, César; González-Álvarez, Daniel; Abia, David; Alfonso, Carlos; Wu, Ling J; Meijer, Wilfried J J

    2017-01-01

    Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

  6. The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

    Directory of Open Access Journals (Sweden)

    Andrés Miguel-Arribas

    2017-11-01

    Full Text Available Bacterial conjugation is the process by which a conjugative element (CE is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer (oriT, where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis. We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1LS20 and aux2LS20, and which we show are essential for conjugation. Both Aux1LS20 and Aux2LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1LS20 and Aux2LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriTLS20, although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1LS20 and/or Aux2LS20 are located upstream of almost 400 relaxase genes of the RelLS20 family (MOBL of relaxases. Thus, Aux1LS20 and Aux2LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

  7. The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

    Science.gov (United States)

    Fiuza, Maria; Canova, Marc J; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A

    2008-12-26

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

  8. The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

    Science.gov (United States)

    Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

    2008-01-01

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. PMID:18974047

  9. Use of egg white protein powder based films fortified with sage and lemon balm essential oils in the storage of lor cheese

    Directory of Open Access Journals (Sweden)

    Gökhan Kavas

    2016-03-01

    Full Text Available Edible film was produced by adding 3 % sorbitol (w/v to egg white protein powder (EWPP. The first group of lor cheese samples was coated with a film fortified by sage essential oil (SEO and the second group of samples was coated with films enriched by adding lemon balm essential oil (BEO at various concentrations [0.5 %, 1 %, 2 % (v/v]. The films were labeled as EWPPSEO(0.5, EWPPSEO(1, EWPPSEO(2, EWPPBEO(0.5, EWPPBEO(1, EWPPBEO(2 to indicate the type and the concentration of the additive. The third batch of the lor cheese samples was coated exclusively with non-fortified EWPP and the fourth batch was uncoated. All of the cheese samples were artificially contaminated with Escherichia coli O157:H7 (E. coli O157:H7, Listeria monocytogenes (L. monocytogenes and Staphylococcus aureus (S. aureus. Viable cell counts of these species, yeasts and moulds were determined after the cheese production. All the samples were stored at +4 °C. Their physicochemical and microbiological properties were examined on the 1st, 7th, 15th and 30th day of the storage. Thereat significant (P0.05. Physicochemical and antibacterial properties were more significant in SEO at all concentrations compared to BEO. However, the antifungal effect of BEO was higher than that of SEO. The antifungal effect of BEO was the same at 1 % (v/v and 2 % (v/v concentrations. E. coli O157:H7 was the most resistant microorganism to the essential oils while L. monocytogenes was the most sensitive. EWPP showed a bacteriostatic effect on the microorganisms and bactericidal effects were determined on the 30th day of the storage against L. monocytogenes and yeast-moulds.

  10. Crystallization and preliminary X-ray crystallographic analysis of MbtI, a protein essential for siderophore biosynthesis in Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Harrison, Anthony J.; Ramsay, Rochelle J.; Baker, Edward N.; Lott, J. Shaun

    2004-01-01

    MbtI, the putative isochorismate synthase essential for siderophore biosynthesis in M. tuberculosis, has been crystallized. Diffraction data have been collected to 1.8 Å resolution. Mycobacterium tuberculosis, the causative agent of tuberculosis, depends on the secretion of salicylate-based siderophores called mycobactins for the acquisition of extracellular iron, which is essential for the growth and virulence of the bacterium. The protein MbtI is thought to be the isochorismate synthase enzyme responsible for the conversion of chorismate to isochorismate, the first step in the salicylate production required for mycobactin biosynthesis. MbtI has been overexpressed in Escherichia coli, purified and crystallized. The crystals diffract to a maximum resolution of 1.8 Å. They belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 51.8, b = 163.4, c = 194.9 Å, consistent with the presence of either two, three or four molecules in the asymmetric unit

  11. Analytical support of plant specific SAMG development validation of SAMG using MELCOR 1.8.5

    International Nuclear Information System (INIS)

    Duspiva, Jiri

    2006-01-01

    They are two NPPs in operation in Czech Republic. Both of NPPs operated in CR have already implemented EOPs, developed under collaboration with the WESE. The project on SAMG development has started and follows the previous one for EOPs also with the WESE as the leading organization. Plant specific SAMGs for the Temelin as well as Dukovany NPPs are based on the WOG generic SAMGs. The analytical support of plant specific SAMGs development is performed by the NRI Rez within the validation process. Basic conditions as well as their filling by NRI Rez are focused on analyst, analytical tools and their applications. More detail description is attended to the approach of the preparation of the MELCOR code application to the evaluation of hydrogen risk, validation of recent set of hydrogen passive autocatalytic recombiners and definition of proposals to amend system of hydrogen removal. Such kind of parametric calculations will request to perform very wide set of runs. It could not be possible with the whole plant model and decoupling of such calculation with storing of mass and energy sources into the containment is only one way. The example of this decoupling for the LOCA scenario is shown. It includes seven sources - heat losses from primary and secondary circuits, fluid blowndown through cold leg break, fission products blowndown through cold leg break, fluid blowndown through break in reactor pressure vessel bottom head, fission products through break in reactor pressure vessel bottom head, melt ejection from reactor pressure vessel to cavity and gas masses and heat losses from corium in cavity. The stand alone containment analysis was tested in two configurations - with or without taking of fission products into account. Testing showed very good agreement of all calculations until lower head failure and acceptable agreement after that. Also some problematic features appeared. The stand alone test with fission product was possible only after the changes in source code

  12. KNIME essentials

    CERN Document Server

    Bakos, Gábor

    2013-01-01

    KNIME Essentials is a practical guide aimed at getting the results you want, as quickly as possible.""Knime Essentials"" is written for data analysts looking to quickly get up to speed using the market leader in data processing tools, KNIME. No knowledge of KNIME is required, but we will assume that you have some background in data processing.

  13. Senp1 Is Essential for Desumoylating Sumo1-Modified Proteins but Dispensable for Sumo2 and Sumo3 Deconjugation in the Mouse Embryo

    Directory of Open Access Journals (Sweden)

    Prashant Sharma

    2013-05-01

    Full Text Available Posttranslational modification with small ubiquitin-like modifier (Sumo regulates numerous cellular and developmental processes. Sumoylation is dynamic with deconjugation by Sumo-specific proteases (Senps regulating steady-state levels. Different Senps are found in distinct subcellular domains, which may limit their deconjugation activity to colocalizing Sumo-modified proteins. In vitro, Senps can discriminate between the different Sumo paralogs: Sumo1 versus the highly related Sumo2 and Sumo3 (Sumo2/3, which can form poly-Sumo chains. However, a full understanding of Senp specificity in vivo is still lacking. Here, using biochemical and genetic approaches, we establish that Senp1 has an essential, nonredundant function to desumoylate Sumo1-modified proteins during mouse embryonic development. Senp1 specificity for Sumo1 conjugates represents an intrinsic function and not simply a product of colocalization. In contrast, Senp1 has only a limited role in Sumo2/3 desumoylation, although it may regulate Sumo1-mediated termination of poly-Sumo2/3 chains.

  14. Cyclodextrin-Complexed Ocimum basilicum Leaves Essential Oil Increases Fos Protein Expression in the Central Nervous System and Produce an Antihyperalgesic Effect in Animal Models for Fibromyalgia

    Directory of Open Access Journals (Sweden)

    Simone S. Nascimento

    2014-12-01

    Full Text Available O. basilicum leaves produce essential oils (LEO rich in monoterpenes. The short half-life and water insolubility are limitations for LEO medical uses. β-Cyclodextrin (β-CD has been employed to improve the pharmacological properties of LEO. We assessed the antihyperalgesic profile of LEO, isolated or complexed in β-CD (LEO/β-CD, on an animal model for fibromyalgia. Behavioral tests: mice were treated every day with either LEO/β-CD (25, 50 or 100 mg/kg, p.o., LEO (25 mg/kg, p.o., tramadol (TRM 4 mg/kg, i.p. or vehicle (saline, and 60 min after treatment behavioral parameters were assessed. Therefore, mice were evaluated for mechanical hyperalgesia (von Frey, motor coordination (Rota-rod and muscle strength (Grip Strength Metter in a mice fibromyalgia model. After 27 days, we evaluated the central nervous system (CNS pathways involved in the effect induced by experimental drugs through immunofluorescence protocol to Fos protein. The differential scanning analysis (DSC, thermogravimetry/derivate thermogravimetry (TG/DTG and infrared absorption spectroscopy (FTIR curves indicated that the products prepared were able to incorporate the LEO efficiently. Oral treatment with LEO or LEO-βCD, at all doses tested, produced a significant reduction of mechanical hyperalgesia and we were able to significantly increase Fos protein expression. Together, our results provide evidence that LEO, isolated or complexed with β-CD, produces analgesic effects on chronic non-inflammatory pain as fibromyalgia.

  15. HuD and the Survival Motor Neuron Protein Interact in Motoneurons and Are Essential for Motoneuron Development, Function, and mRNA Regulation.

    Science.gov (United States)

    Hao le, Thi; Duy, Phan Q; An, Min; Talbot, Jared; Iyer, Chitra C; Wolman, Marc; Beattie, Christine E

    2017-11-29

    protein, HuD. Novel mutants reveal that HuD is also necessary for motor axonal branch and dendrite formation. Data also revealed that both SMN and HuD affect levels of an mRNA involved in axonal growth. Moreover, expressing HuD in SMN-deficient motoneurons can rescue the motoneuron development and motor defects caused by low levels of SMN. These data support that SMN:HuD complexes are essential for normal motoneuron development and indicate that mRNA handling is a critical component of SMA. Copyright © 2017 the authors 0270-6474/17/3711559-13$15.00/0.

  16. The KYxxL motif in Rad17 protein is essential for the interaction with the 9–1–1 complex

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Ikeuchi, Masayoshi; Nakayama, Yuji [Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414 (Japan); Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan)

    2016-09-02

    ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. - Highlights: • We have identified a conserved KYxxL motif in Rad17 protein. • The KYxxL motif is crucial for the interaction with the 9–1–1 complex. • The KYxxL motif is dispensable or inhibitory for UV-induced Rad17 phosphorylation. • Nucleotide binding of Rad17 is required for its nuclear localization.

  17. Acinetobacter baumannii universal stress protein A plays a pivotal role in stress response and is essential for pneumonia and sepsis pathogenesis.

    Science.gov (United States)

    Elhosseiny, Noha M; Amin, Magdy A; Yassin, Aymen S; Attia, Ahmed S

    2015-01-01

    Acinetobacter baumannii is one of the most significant threats to global public health. This threat is compounded by the fact that A. baumannii is rapidly becoming resistant to all relevant antimicrobials. Identifying key microbial factors through which A. baumannii resists hostile host environment is paramount to the development of novel antimicrobials targeting infections caused by this emerging pathogen. An attractive target could be a molecule that plays a role in the pathogenesis and stress response of A. baumannii. Accordingly, the universal stress protein A (UspA) was chosen to be fully investigated in this study. A platform of A. baumannii constructs, expressing various levels of the uspA gene ranging from zero to thirteen folds of wild-type level, and a recombinant E. coli strain, were employed to investigate the role of UspA in vitro stress and in vivo pathogenesis. The UspA protein plays a significant role in protecting A. baumannii from H(2)O(2), low pH, and the respiratory toxin 2,4-DNP. A. baumannii UspA protein plays an essential role in two of the deadliest types of infection caused by A. baumannii; pneumonia and sepsis. This distinguishes A. baumannii UspA from its closely related homolog, the Staphylococcus aureus Usp2, as well as from the less similar Burkholderia glumae Usps. Heterologous and overexpression experiments suggest that UspA mediates its role via an indirect mechanism. Our study highlights the role of UspA as an important contributor to the A. baumannii stress and virulence machineries, and polishes it as a plausible target for new therapeutics. Copyright © 2014 Elsevier GmbH. All rights reserved.

  18. Iron storage proteins are essential for the survival and pathogenesis of Mycobacterium tuberculosis in THP-1 macrophages and the guinea pig model of infection.

    Science.gov (United States)

    Reddy, P Vineel; Puri, Rupangi Verma; Khera, Aparna; Tyagi, Anil K

    2012-02-01

    Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ΔbfrA ΔbfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.

  19. Astronomy essentials

    CERN Document Server

    Brass, Charles O

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Astronomy includes the historical perspective of astronomy, sky basics and the celestial coordinate systems, a model and the origin of the solar system, the sun, the planets, Kepler'

  20. Conceptual Framework for Physical Protection Against Sabotage Considering Plant-specific Radiological Consequences

    International Nuclear Information System (INIS)

    Lee, Joung Hoon; Yu, Dong Han

    2010-01-01

    According to the Generation IV (Gen IV) Technology Roadmap, Gen IV nuclear energy systems (NESs) should highlight proliferation resistance and physical protection (PR and PP) as one of the four goals along with sustainability, safety and reliability, and economics. Especially, physical protection (PP) is the typical important characteristic of an NES that impedes the theft of materials suitable for nuclear explosives or radiation dispersal devices (RDD) and the sabotage of facilities and transportation by subnation entities and other non-Host State adversaries. These two subjects have been studied separately. Proliferation is commonly considered as an international concern and the past work on the PR assessments can be found. On the other hands, PP is regarded as a State security concern, much of which is classified and facility-dependent. Recently, more concern has been focused on the PP design and regulation because of rapid environment changes including radiological consequences by internal sabotage and nuclear terrorism by RDDs. The current PP Regulation has been applied intensively to the existing nuclear facilities and could be a possible guidance for the future GEN-IV NESs. This paper first reviews the IAEA guide document, INFCIRC/225, which was accepted as the standard international guideline in the physical protection area. It has been updated several times up to now, and is undergoing another revision. The paper introduces current substantial changes in the document regarding PP including the national nuclear security and sabotage in the nuclear facilities. Then, it presents a conceptual framework for physical protection against sabotage considering plant-specific radiological consequence after malicious acts within certain vital areas. The framework combines the newly developed method of vital area identification, the current PSA level 2 works, and physical protection concepts. This would help to improve a design concept of new physical protection

  1. Cernavoda NPP Unit 1: Operating experience program and plant specific performance indicators (level 2)

    International Nuclear Information System (INIS)

    Teodor, Vasile; Popa, Viorel

    1998-01-01

    The basis for the Operating Experience Program was set in place since early stages of the commissioning phase (1993), when a system based on the Canadian approach was implemented for reporting, reviewing, assessing and establishing of the necessary corrective action for unplanned events. This system provided excellent opportunity to train staff in unplanned event assessment methodology, and prepare the station for the formal reporting process following criticality in accordance with the licensing requirements. The formal process, set in place after criticality is described in Station Instruction Procedure SI-01365-P13 'Unplanned Event Report' and was developed under the supervision of Safety and Compliance Department. In parallel, a program for information exchange and trending of performance indicators was developed by Technical Services Department. The WANO recommendations following August 1997 Peer Review provided the opportunity for a better understanding and reconsideration of the Operating Experience Program. As a result, all the activities related to this topic were assigned to a new structure, within Safety and Compliance Department. As such an Operating Experience Group was created and a new program is now being developed in an integrated and centralized manner. The content of the paper is the following: - Overview; - Operating Experience Program; - Event Analysis (Unplanned Events Assessment System - UEIR Process- and Systematic Analysis of Operational Events - ACR Process); - Information Exchange Program; - Monitoring of Operating Experience - Plant Specific Performance Indicators; - Purpose; - Level 2 Performance Indicators. Four appendices are added containing: - A. Station performance indicators/targets (Level 2); - B. SPI (Station Performance Indicators - Level 2) - Graphics; - C. UEIR, LRS (Safety and Licensing Review Sheet), UEFR (Unplanned Event Follow-up Report), ACR and OPEX forms. (authors)

  2. Conceptual Framework for Physical Protection Against Sabotage Considering Plant-specific Radiological Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joung Hoon; Yu, Dong Han [Korea Institute of Nuclear Nonproliferation and Control, Daejeon (Korea, Republic of)

    2010-10-15

    According to the Generation IV (Gen IV) Technology Roadmap, Gen IV nuclear energy systems (NESs) should highlight proliferation resistance and physical protection (PR and PP) as one of the four goals along with sustainability, safety and reliability, and economics. Especially, physical protection (PP) is the typical important characteristic of an NES that impedes the theft of materials suitable for nuclear explosives or radiation dispersal devices (RDD) and the sabotage of facilities and transportation by subnation entities and other non-Host State adversaries. These two subjects have been studied separately. Proliferation is commonly considered as an international concern and the past work on the PR assessments can be found. On the other hands, PP is regarded as a State security concern, much of which is classified and facility-dependent. Recently, more concern has been focused on the PP design and regulation because of rapid environment changes including radiological consequences by internal sabotage and nuclear terrorism by RDDs. The current PP Regulation has been applied intensively to the existing nuclear facilities and could be a possible guidance for the future GEN-IV NESs. This paper first reviews the IAEA guide document, INFCIRC/225, which was accepted as the standard international guideline in the physical protection area. It has been updated several times up to now, and is undergoing another revision. The paper introduces current substantial changes in the document regarding PP including the national nuclear security and sabotage in the nuclear facilities. Then, it presents a conceptual framework for physical protection against sabotage considering plant-specific radiological consequence after malicious acts within certain vital areas. The framework combines the newly developed method of vital area identification, the current PSA level 2 works, and physical protection concepts. This would help to improve a design concept of new physical protection

  3. CRISPR/Cas9 allows efficient and complete knock-in of a destabilization domain-tagged essential protein in a human cell line, allowing rapid knockdown of protein function.

    Science.gov (United States)

    Park, Arnold; Won, Sohui T; Pentecost, Mickey; Bartkowski, Wojciech; Lee, Benhur

    2014-01-01

    Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to

  4. 77 FR 29391 - An Approach for Probabilistic Risk Assessment in Risk-Informed Decisions on Plant-Specific...

    Science.gov (United States)

    2012-05-17

    ... NUCLEAR REGULATORY COMMISSION [NRC-2012-0110] An Approach for Probabilistic Risk Assessment in Risk-Informed Decisions on Plant-Specific Changes to the Licensing Basis AGENCY: Nuclear Regulatory Commission. ACTION: Draft regulatory guide; request for comment. SUMMARY: The U.S. Nuclear Regulatory...

  5. Amino acids fortification of low-protein diet for broilers under tropical climate. 2. Nonessential amino acids and increasing essential amino acids

    Directory of Open Access Journals (Sweden)

    Elmutaz Atta Awad

    2014-08-01

    Full Text Available A three-week trial was carried out to evaluate the effect of nonessential amino acids (NEAA supplementation to a low-crude protein (CP diet with adequate essential amino acids (EAA level on growth performance, blood metabolites, and relative weights of abdominal fat, breast yield, and internal organs in broiler chickens raised under tropical hot and humid environment. Five isocaloric (3000 metabolisable energy/kg corn-soybean diets were administered (1 to 21 days to 5 groups of broilers (60 birds/group as follows: i 22.2% CP (positive control; PC; ii 16.2% CP+all EAA to meet or exceed the National Research Council (1994 recommendations (negative control; NC; iii NC+further EAA to equal the levels in the PC diet; iv NC+NEAA to equal the levels in the PC; v NC+EAA and NEAA to equal the amino acids levels in the PC diet. The results showed that the fortification of EAA alone, only improved feed intake (FI, whereas, addition of NEAA or EAA+NEAA significantly enhanced body weight, daily weight gain, and FI and decreased the feed conversion ratio to the same levels as in PC. Serum uric acid was significantly reduced and serum triglyceride increased in NC group. Dietary treatments had no significant effect on relative weights of heart, liver, abdominal fat, breast meat yield, serum albumin, and serum total protein. In conclusion, these results suggest that NEAA fortification may improve the growth performance of broilers fed an excessive low-CP diet under tropical hot and humid condition.

  6. Essential oils improved weight gain, growth and feed efficiency of young dairy calves fed 18 or 20% crude protein starter diets.

    Science.gov (United States)

    Kazemi-Bonchenari, M; Falahati, R; Poorhamdollah, M; Heidari, S R; Pezeshki, A

    2018-01-17

    The objective was to evaluate interactions between starter protein (180 vs. 200 g/kg, DM basis) and a mixture of essential oils (EOs; containing thymol, eugenol, vanillin, limonene and guaiacol) on growth, metabolic and ruminal functions of Holstein dairy calves. In a completely randomized 2 × 2 factorial design, 48 calves, 3 days old (averaging BW 42.7 ± 1.9 kg), were allocated into groups fed the following diets: (i) 180 g/kg CP with no EO (180P-NEO); (ii) 180 g/kg CP with EO (180P-EO); (iii) 200 g/kg CP with no EO (200P-NEO); and (iv) 200 g/kg CP with EO (200P-EO). The EO was supplemented as 1 g/kg of starter DM. Calves were fed ad libitum starter diet and were weaned at day 59 of age, but diets continued until day 80. There were no interactive effects of CP and EO on intake and growth. Pre-weaning feed efficiency tended to be increased for 200P-EO (p = .09). Average daily gain and feed efficiency during pre-weaning period as well as weaning weight were increased (p calves-fed EO (p = .05) and was lower in those fed 180P vs. 200P (p feed efficiency of dairy calves, irrespective of dietary protein content. © 2018 Blackwell Verlag GmbH.

  7. FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System

    Directory of Open Access Journals (Sweden)

    Jörg Willenborg

    2016-07-01

    Full Text Available Streptococcus (S. suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system.

  8. Essential AOP

    DEFF Research Database (Denmark)

    De Fraine, Bruno; Ernst, Erik; Südholt, Mario

    2010-01-01

    Aspect-oriented programming (AOP) has produced interesting language designs, but also ad hoc semantics that needs clarification. We contribute to this clarification with a calculus that models essential AOP, both simpler and more general than existing formalizations. In AOP, advice may intercept...

  9. Highcharts essentials

    CERN Document Server

    Shahid, Bilal

    2014-01-01

    If you are a web developer with a basic knowledge of HTML, CSS, and JavaScript and want to quickly get started with this web charting technology, this is the book for you. This book will also serve as an essential guide to those who have probably used a similar library and are now looking at migrating to Highcharts.

  10. Swift essentials

    CERN Document Server

    Blewitt, Alex

    2014-01-01

    Whether you are a seasoned Objective-C developer or new to the Xcode platform, Swift Essentials will provide you with all you need to know to get started with the language. Prior experience with iOS development is not necessary, but will be helpful to get the most out of the book.

  11. Role of the plant-specific endoplasmic reticulum stress-inducible gene TIN1 in the formation of pollen surface structure in Arabidopsis thaliana

    KAUST Repository

    Iwata, Yuji; Nishino, Tsuneyo; Iwano, Megumi; Takayama, Seiji; Koizumi, Nozomu

    2012-01-01

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional activation of ER-resident molecular chaperones and folding enzymes to maintain cellular homeostasis. This process is known as the ER stress response or the unfolded protein response. We have identified tunicamycin induced 1 (TIN1), a plant-specific ER stress-inducible Arabidopsis thaliana gene. The TIN1 protein is localized in the ER; however, its molecular function has yet to be clarified. In this study, we performed functional analysis of TIN1 in planta. RT-PCR analysis showed that TIN1 is highly expressed in pollen. Analysis using the β-glucuronidase reporter gene demonstrated that the TIN1 promoter is active throughout pollen development, peaking at the time of flowering and in an ovule of an open flower. Although a T-DNA insertion mutant of TIN1 grows normally under ambient laboratory conditions, abnormal pollen surface morphology was observed under a scanning electron microscope. Based on the current and previous observations, a possible physiological function of TIN1 during pollen development is discussed. © 2012 The Japanese Society for Plant Cell and Molecular Biology.

  12. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters.

    Science.gov (United States)

    Ramesh, Sunita A; Tyerman, Stephen D; Xu, Bo; Bose, Jayakumar; Kaur, Satwinder; Conn, Vanessa; Domingos, Patricia; Ullah, Sana; Wege, Stefanie; Shabala, Sergey; Feijó, José A; Ryan, Peter R; Gilliham, Matthew; Gillham, Matthew

    2015-07-29

    The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.

  13. Hibiscus Chlorotic Ringspot Virus Coat Protein Is Essential for Cell-to-Cell and Long-Distance Movement but Not for Viral RNA Replication

    Science.gov (United States)

    Niu, Shengniao; Gil-Salas, Francisco M.; Tewary, Sunil Kumar; Samales, Ashwin Kuppusamy; Johnson, John; Swaminathan, Kunchithapadam; Wong, Sek-Man

    2014-01-01

    Hibiscus chlorotic ringspot virus (HCRSV) is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP) functions on virus replication and movement in kenaf (Hibiscus cannabinus L.), two HCRSV mutants, designated as p2590 (A to G) in which the first start codon ATG was replaced with GTG and p2776 (C to G) in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G) were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G) was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G) inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro. PMID:25402344

  14. Hibiscus chlorotic ringspot virus coat protein is essential for cell-to-cell and long-distance movement but not for viral RNA replication.

    Directory of Open Access Journals (Sweden)

    Shengniao Niu

    Full Text Available Hibiscus chlorotic ringspot virus (HCRSV is a member of the genus Carmovirus in the family Tombusviridae. In order to study its coat protein (CP functions on virus replication and movement in kenaf (Hibiscus cannabinus L., two HCRSV mutants, designated as p2590 (A to G in which the first start codon ATG was replaced with GTG and p2776 (C to G in which proline 63 was replaced with alanine, were constructed. In vitro transcripts of p2590 (A to G were able to replicate to a similar level as wild type without CP expression in kenaf protoplasts. However, its cell-to-cell movement was not detected in the inoculated kenaf cotyledons. Structurally the proline 63 in subunit C acts as a kink for β-annulus formation during virion assembly. Progeny of transcripts derived from p2776 (C to G was able to move from cell-to-cell in inoculated cotyledons but its long-distance movement was not detected. Virions were not observed in partially purified mutant virus samples isolated from 2776 (C to G inoculated cotyledons. Removal of the N-terminal 77 amino acids of HCRSV CP by trypsin digestion of purified wild type HCRSV virions resulted in only T = 1 empty virus-like particles. Taken together, HCRSV CP is dispensable for viral RNA replication but essential for cell-to-cell movement, and virion is required for the virus systemic movement. The proline 63 is crucial for HCRSV virion assembly in kenaf plants and the N-terminal 77 amino acids including the β-annulus domain is required in T = 3 assembly in vitro.

  15. Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

    Science.gov (United States)

    Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

    2017-02-22

    The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

  16. Driver mutations (JAK2V617F, MPLW515L/K or CALR, pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera

    Directory of Open Access Journals (Sweden)

    Federico Lussana

    2017-02-01

    Full Text Available Abstract Background The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3 and high-sensitivity C-reactive protein (hs-CRP are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. Methods We evaluated 305 with essential thrombocythemia (ET and 172 polycythemia vera (PV patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. Results PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. Conclusions These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

  17. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

    2017-04-01

    Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

  18. Linux Essentials

    CERN Document Server

    Smith, Roderick W

    2012-01-01

    A unique, full-color introduction to Linux fundamentals Serving as a low-cost, secure alternative to expensive operating systems, Linux is a UNIX-based, open source operating system. Full-color and concise, this beginner's guide takes a learning-by-doing approach to understanding the essentials of Linux. Each chapter begins by clearly identifying what you will learn in the chapter, followed by a straightforward discussion of concepts that leads you right into hands-on tutorials. Chapters conclude with additional exercises and review questions, allowing you to reinforce and measure your underst

  19. Essential SQLAlchemy

    CERN Document Server

    Copeland, Rick

    2008-01-01

    Essential SQLAlchemy introduces a high-level open-source code library that makes it easier for Python programmers to access relational databases such as Oracle, DB2, MySQL, PostgreSQL, and SQLite. SQLAlchemy has become increasingly popular since its release, but it still lacks good offline documentation. This practical book fills the gap, and because a developer wrote it, you get an objective look at SQLAlchemy's tools rather than an advocate's description of all the "cool" features. SQLAlchemy includes both a database server-independent SQL expression language and an object-relational mappe

  20. Prezi essentials

    CERN Document Server

    Sinclair, Domi

    2014-01-01

    If you want to learn Prezi, and specifically design within Prezi, this is the book for you. Perhaps you already know a bit about Prezi but have never used it, or perhaps you have used Prezi before but want to learn how to incorporate your own custom design elements. In either case, this book will get you up and running quickly. It would be helpful to have a bit of familiarity with basic design concepts and the use of Prezi, but prior experience is not essential.

  1. Essential astrophysics

    CERN Document Server

    Lang, Kenneth R

    2013-01-01

    Essential Astrophysics is a book to learn or teach from, as well as a fundamental reference volume for anyone interested in astronomy and astrophysics. It presents astrophysics from basic principles without requiring any previous study of astronomy or astrophysics. It serves as a comprehensive introductory text, which takes the student through the field of astrophysics in lecture-sized chapters of basic physical principles applied to the cosmos. This one-semester overview will be enjoyed by undergraduate students with an interest in the physical sciences, such as astronomy, chemistry, engineering or physics, as well as by any curious student interested in learning about our celestial science. The mathematics required for understanding the text is on the level of simple algebra, for that is all that is needed to describe the fundamental principles. The text is of sufficient breadth and depth to prepare the interested student for more advanced specialized courses in the future. Astronomical examples are provide...

  2. Alteration of the ATG start codon of the A* protein of bacteriophage [phi]X174 into an ATT codon yields a viable phage indicating that A* protein is not essential for [phi]X174 reproduction

    NARCIS (Netherlands)

    Baas, P.D.; Liewerink, H.; Teeffelen, H.A.A.M. van; Mansfeld, A.D.M. van; Boom, J.M. van; Jansz, H.S.

    1987-01-01

    Bacteriophage X174 gene A encodes two proteins: the gene A protein and the smaller A* protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling

  3. The α' subunit of β-conglycinin and the A1-5 subunits of glycinin are not essential for many hypolipidemic actions of dietary soy proteins in rats.

    Science.gov (United States)

    Chen, Qixuan; Wood, Carla; Gagnon, Christine; Cober, Elroy R; Frégeau-Reid, Judith A; Gleddie, Stephen; Xiao, Chao Wu

    2014-08-01

    This study examined the effects of dietary soy protein (SP) lacking different storage protein subunits and isoflavones (ISF) on the abdominal fat, blood lipids, thyroid hormones, and enzymatic activities in rats. Weanling Sprague-Dawley rats (8 males and 8 females/group) were fed diets containing either 20 % casein without or with supplemental isoflavones or alcohol-washed SP isolate or SP concentrates (SPC) prepared from 6 different soy bean lines for 8 weeks. Feeding of diets containing SPC regardless of their subunit compositions significantly lowered relative liver weights, blood total, free, and LDL cholesterol in both genders (P Soy isoflavones were mainly responsible for the hypocholesterolemic effects and increased plasma free T3, whereas reduction in FFA, abdominal fat, liver weight and increased plasma total T3 were the effects of the soy proteins. Neither the α' subunit of β-conglycinin nor the A1-5 subunits of glycinin are essential for the hypolipidemic properties of soy proteins.

  4. An overview of insights gained and lessons learned from U.S. plant-specific PRA studies

    International Nuclear Information System (INIS)

    Joksimovich, V.

    1985-01-01

    Probabilistic Risk Assessment (PRA) has been under development for over twenty years, but it has reached the level of widespread use only in the aftermath of the TMI accident. Over thirty PRAs have now been completed in the U.S. PRAs have been in the mainstream of many licensing decisions because the NRC recognizes that they provide independent and comprehensive plant safety audit. Some difficulties have been experienced leading to interpretive and intercomparison studies. Numerous global and plant-specific insights have been derived. A new application termed risk management is clearly emerging. (orig./HP)

  5. CaMKII-MEDIATED PHOSPHORYLATION OF THE BOMBYX MORI LIPID STORAGE DROPLET PROTEIN-1 (BmLsd1), AN INSECT PAT FAMILY PROTEIN, IS ESSENTIAL FOR SILKMOTH SEX PHEROMONE BIOSYNTHESIS

    Science.gov (United States)

    The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

  6. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1 from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

    Directory of Open Access Journals (Sweden)

    Musiyenko Alla

    2002-04-01

    Full Text Available Abstract Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes.

  7. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    KAUST Repository

    Benedet, Mattia

    2016-08-16

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

  8. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    KAUST Repository

    Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra; Deho, Gianni

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in gamma-Proteobacteria. LptBFG constitute the IMABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable Delta lptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptF(SupC)). In complementation tests, lptF(SupC) mutants suppress lethality of both Delta lptC and lptC conditional expressionmutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

  9. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Directory of Open Access Journals (Sweden)

    Sylvaine Boissinot

    Full Text Available Cucurbit aphid-borne yellows virus (CABYV is a polerovirus (Luteoviridae family with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT. Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT* incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  10. Both structural and non-structural forms of the readthrough protein of cucurbit aphid-borne yellows virus are essential for efficient systemic infection of plants.

    Science.gov (United States)

    Boissinot, Sylvaine; Erdinger, Monique; Monsion, Baptiste; Ziegler-Graff, Véronique; Brault, Véronique

    2014-01-01

    Cucurbit aphid-borne yellows virus (CABYV) is a polerovirus (Luteoviridae family) with a capsid composed of the major coat protein and a minor component referred to as the readthrough protein (RT). Two forms of the RT were reported: a full-length protein of 74 kDa detected in infected plants and a truncated form of 55 kDa (RT*) incorporated into virions. Both forms were detected in CABYV-infected plants. To clarify the specific roles of each protein in the viral cycle, we generated by deletion a polerovirus mutant able to synthesize only the RT* which is incorporated into the particle. This mutant was unable to move systemically from inoculated leaves inferring that the C-terminal half of the RT is required for efficient long-distance transport of CABYV. Among a collection of CABYV mutants bearing point mutations in the central domain of the RT, we obtained a mutant impaired in the correct processing of the RT which does not produce the RT*. This mutant accumulated very poorly in upper non-inoculated leaves, suggesting that the RT* has a functional role in long-distance movement of CABYV. Taken together, these results infer that both RT proteins are required for an efficient CABYV movement.

  11. Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

    Science.gov (United States)

    Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

    2018-05-22

    Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

  12. Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2–7 Helicase to Reveal Essential Features of Structure and Function

    Science.gov (United States)

    Miller, Justin M.; Enemark, Eric J.

    2015-01-01

    In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2–7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2–7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2–7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2–7 ring. PMID:26539061

  13. Insight into PreImplantation Factor (PIF* mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP through essential RIKP [corrected] binding site.

    Directory of Open Access Journals (Sweden)

    Eytan R Barnea

    Full Text Available Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised.FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control. Murine embryo (d10 lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS. In silico evaluation examined binding of PIF to critical targets, using mutation analysis.PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90, co-chaperone, BAG-3. Remarkably, PIF targets a common RIKP [corrected] site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented.Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a

  14. Bromodomain proteins GTE9 and GTE11 are essential for specific BT2-mediated sugar and ABA responses in Arabidopsis thaliana.

    Science.gov (United States)

    Misra, Anjali; McKnight, Thomas D; Mandadi, Kranthi K

    2018-03-01

    Global Transcription Factor Group E proteins GTE9 and GTE11 interact with BT2 to mediate ABA and sugar responses in Arabidopsis thaliana. BT2 is a BTB-domain protein that regulates responses to various hormone, stress and metabolic conditions in Arabidopsis thaliana. Loss of BT2 results in plants that are hypersensitive to inhibition of germination by abscisic acid (ABA) and sugars. Conversely, overexpression of BT2 results in resistance to ABA and sugars. Here, we report the roles of BT2-interacting partners GTE9 and GTE11, bromodomain and extraterminal-domain proteins of Global Transcription Factor Group E, in BT2-mediated responses to sugars and hormones. Loss-of-function mutants, gte9-1 and gte11-1, mimicked the bt2-1-null mutant responses; germination of all three mutants was hypersensitive to inhibition by glucose and ABA. Loss of either GTE9 or GTE11 in a BT2 over-expressing line blocked resistance to sugars and ABA, indicating that both GTE9 and GTE11 were required for BT2 function. Co-immunoprecipitation of BT2 and GTE9 suggested that these proteins physically interact in vivo, and presumably function together to mediate responses to ABA and sugar signals.

  15. The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

    Directory of Open Access Journals (Sweden)

    Alexandra Steffens

    2015-07-01

    Full Text Available Members of the highly conserved class of BEACH domain containing proteins (BDCPs have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1, associates to mRNA processing bodies (P-bodies, and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

  16. The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase

    NARCIS (Netherlands)

    Ortiz de Orué Lucana, Darío; Bogel, Gabriele; Zou, Peijian; Groves, Matthew R

    2009-01-01

    HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2

  17. Autophagy Proteins ATG5 and ATG7 Are Essential for the Maintenance of Human CD34+ Hematopoietic Stem-Progenitor Cells

    NARCIS (Netherlands)

    Gomez Puerto, Maria Catalina; Folkerts, Hendrik; Wierenga, Albertus T J; Schepers, Koen; Schuringa, Jan Jacob; Coffer, Paul J.; Vellenga, Edo

    2016-01-01

    Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can be considered to be a general cellular housekeeping process, it has become clear that it may also

  18. ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in E.coli.

    NARCIS (Netherlands)

    Loiseau, L.; Gerez, C.; Bekker, M.; Ollagnier-de Choudens, S.; Py, B.; Sanakis, Y.; Teixeira De Mattos, M.J.; Fontecave, M.; Barras, F.

    2007-01-01

    Understanding the biogenesis of iron-sulfur (Fe-S) proteins is relevant to many fields, including bioenergetics, gene regulation, and cancer research. Several multiprotein complexes assisting Fe-S assembly have been identified in both prokaryotes and eukaryotes. Here, we identify in Escherichia coli

  19. A novel Geobacteraceae-specific outer membrane protein J (OmpJ is essential for electron transport to Fe (III and Mn (IV oxides in Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Schiffer Marianne

    2005-07-01

    Full Text Available Abstract Background Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III reduction in the Geobacter species that are the predominant Fe(III reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. Results When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J, was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe (III and insoluble Mn (IV oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal

  20. A Small Stem Loop Structure Of The Ebola Virus Trailer Is Essential For Replication And Interacts With Heat Shock Protein A8

    Science.gov (United States)

    2016-12-02

    Nucleic Acids Research , 2016 1–15 doi: 10.1093/nar/gkw825 A small stem -loop structure of the Ebola virus trailer is essential for replication and...is a single- stranded RNA that is linked to a stem -loop, as found in the region of the replication promoter element of the EBOV genomic leader (18...Kuhn4, Gustavo Palacios3, Sheli R. Radoshitzky3, Stuart F. J. Le Grice1,* and Reed F. Johnson2,* 1RT Biochemistry Section, Basic Research Laboratory

  1. HDA2 and HDA3 are related proteins that interact with and are essential for the activity of the yeast histone deacetylase HDA1

    OpenAIRE

    Wu, Jiansheng; Carmen, Andrew A.; Kobayashi, Ryuji; Suka, Noriyuki; Grunstein, Michael

    2001-01-01

    Histone deacetylase HDA1, the prototype for the class II mammalian deacetylases, is likely the catalytic subunit of the HDA1-containing complex that is involved in TUP1-specific repression and global deacetylation in yeast. Although the class I RPD3-like enzymatic complexes have been well characterized, little is known about the identity and interactions of the factors that associate to form the HDA1 complex. In this paper, we identify related HDA2 and HDA3 proteins that ...

  2. Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.

    Science.gov (United States)

    Gopal, Suhasini R; Chen, Daniel H-C; Chou, Shih-Wei; Zang, Jingjing; Neuhauss, Stephan C F; Stepanyan, Ruben; McDermott, Brian M; Alagramam, Kumar N

    2015-07-15

    Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype

  3. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    International Nuclear Information System (INIS)

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-01-01

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20 NLS mutant gene and examined polysome profile of cells that had been transfected with the S20 NLS gene. As a result, we observed the formation of recombinant 40S carried S20 NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20 NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20 NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20 NLS is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20 NLS . • Cytoplasm-retained S20 NLS is crucial for creating a functional small subunit

  4. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    Energy Technology Data Exchange (ETDEWEB)

    Tai, Lin-Ru [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China); Wu, Jing-Ying; Kirby, Ralph [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China)

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  5. The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

    Science.gov (United States)

    Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

    2012-11-01

    Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

  6. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

    Science.gov (United States)

    Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

    2016-08-01

    Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation. © FASEB.

  7. Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; Only the NTD interaction is essential for lymphoblastoid cell growth

    International Nuclear Information System (INIS)

    Calderwood, Michael A.; Lee, Sungwook; Holthaus, Amy M.; Blacklow, Stephen C.; Kieff, Elliott; Johannsen, Eric

    2011-01-01

    Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the WΦP motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128-204) and EBNA3C(aa211-233). The EBNA3A results are consistent with an earlier report (aa125-222), but the EBNA3C domain is unexpectedly small and includes a 'WTP' sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP → STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.

  8. The plant-specific impact of different pressurization rates in the probabilistic estimation of containment failure modes

    International Nuclear Information System (INIS)

    Ahn, Kwang Il; Yang, Joon Eon; Ha, Jae Joo

    2003-01-01

    The explicit consideration of different pressurization rates in estimating the probabilities of containment failure modes has a profound effect on the confidence of containment performance evaluation that is so critical for risk assessment of nuclear power plants. Except for the sophisticated NUREG-1150 study, many of the recent containment performance analyses (through level 2 PSAs or IPE back-end analyses) did not take into account an explicit distinction between slow and fast pressurization in their analyses. A careful investigation of both approaches shows that many of the approaches adopted in the recent containment performance analyses exactly correspond to the NUREG-1150 approach for the prediction of containment failure mode probabilities in the presence of fast pressurization. As a result, it was expected that the existing containment performance analysis results would be subjected to greater or less conservatism in light of the ultimate failure mode of the containment. The main purpose of this paper is to assess potential conservatism of a plant-specific containment performance analysis result in light of containment failure mode probabilities

  9. Plant-specific Histone Deacetylases HDT½ Regulate GIBBERELLIN 2-OXIDASE 2 Expression to Control Arabidopsis Root Meristem Cell Number

    KAUST Repository

    Li, Huchen

    2017-08-31

    Root growth is modulated by environmental factors and depends on cell production in the root meristem (RM). New cells in the meristem are generated by stem cells and transit-amplifying cells, which together determine RM cell number. Transcription factors and chromatin-remodelling factors have been implicated in regulating the switch from stem cells to transit-amplifying cells. Here we show that two Arabidopsis thaliana paralogs encoding plant-specific histone deacetylases, HDT1 and HDT2, regulate a second switch from transit-amplifying cells to expanding cells. Knockdown of HDT½ (hdt1,2i) results in an earlier switch and causes a reduced RM cell number. Our data show that HDT½ negatively regulate the acetylation level of the C19-GIBBERELLIN 2-OXIDASE 2 (GA2ox2) locus and repress the expression of GA2ox2 in the RM and elongation zone. Overexpression of GA2ox2 in the RM phenocopies the hdt1,2i phenotype. Conversely, knockout of GA2ox2 partially rescues the root growth defect of hdt1,2i. These results suggest that by repressing the expression of GA2ox2, HDT½ likely fine-tune gibberellin metabolism and they are crucial for regulating the switch from cell division to expansion to determine RM cell number. We propose that HDT½ function as part of a mechanism that modulates root growth in response to environmental factors.

  10. The orphan germinant receptor protein GerXAO (but not GerX3b) is essential for L-alanine induced germination in Clostridium botulinum Group II.

    Science.gov (United States)

    Brunt, Jason; Carter, Andrew T; Pye, Hannah V; Peck, Michael W

    2018-05-04

    Clostridium botulinum is an anaerobic spore forming bacterium that produces the potent botulinum neurotoxin that causes a severe and fatal neuro-paralytic disease of humans and animals (botulism). C. botulinum Group II is a psychrotrophic saccharolytic bacterium that forms spores of moderate heat resistance and is a particular hazard in minimally heated chilled foods. Spore germination is a fundamental process that allows the spore to transition to a vegetative cell and typically involves a germinant receptor (GR) that responds to environmental signals. Analysis of C. botulinum Group II genomes shows they contain a single GR cluster (gerX3b), and an additional single gerA subunit (gerXAO). Spores of C. botulinum Group II strain Eklund 17B germinated in response to the addition of L-alanine, but did not germinate following the addition of exogenous Ca 2+ -DPA. Insertional inactivation experiments in this strain unexpectedly revealed that the orphan GR GerXAO is essential for L-alanine stimulated germination. GerX3bA and GerX3bC affected the germination rate but were unable to induce germination in the absence of GerXAO. No role could be identified for GerX3bB. This is the first study to identify the functional germination receptor of C. botulinum Group II.

  11. RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation.

    Science.gov (United States)

    Nakayama, Kei; Ohashi, Rie; Shinoda, Yo; Yamazaki, Maya; Abe, Manabu; Fujikawa, Akihiro; Shigenobu, Shuji; Futatsugi, Akira; Noda, Masaharu; Mikoshiba, Katsuhiko; Furuichi, Teiichi; Sakimura, Kenji; Shiina, Nobuyuki

    2017-11-21

    Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation.

  12. Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

    Science.gov (United States)

    Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

    2014-11-28

    Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

  13. The PP4R1 sub-unit of protein phosphatase PP4 is essential for inhibition of NF-κB by merkel polyomavirus small tumour antigen.

    Science.gov (United States)

    Abdul-Sada, Hussein; Müller, Marietta; Mehta, Rajni; Toth, Rachel; Arthur, J Simon C; Whitehouse, Adrian; Macdonald, Andrew

    2017-04-11

    Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with a high metastatic potential. The majority of MCC cases are caused by the Merkel cell polyomavirus (MCPyV), through expression of the virus-encoded tumour antigens. Whilst mechanisms attributing tumour antigen expression to transformation are being uncovered, little is known of the mechanisms by which MCPyV persists in the host. We previously identified the MCPyV small T antigen (tAg) as a novel inhibitor of nuclear factor kappa B (NF-kB) signalling and a modulator of the host anti-viral response. Here we demonstrate that regulation of NF-kB activation involves a previously undocumented interaction between tAg and regulatory sub-unit 1 of protein phosphatase 4 (PP4R1). Formation of a complex with PP4R1 and PP4c is required to bridge MCPyV tAg to the NEMO adaptor protein, allowing deactivation of the NF-kB pathway. Mutations in MCPyV tAg that fail to interact with components of this complex, or siRNA depletion of PP4R1, prevents tAg-mediated inhibition of NF-kB and pro-inflammatory cytokine production. Comparison of tAg binding partners from other human polyomavirus demonstrates that interactions with NEMO and PP4R1 are unique to MCPyV. Collectively, these data identify PP4R1 as a novel target for virus subversion of the host anti-viral response.

  14. The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora.

    Science.gov (United States)

    Nowrousian, Minou; Cebula, Patricia

    2005-11-03

    The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development), a gene encoding a putative lectin homolog. Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Deltatap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

  15. The gene for a lectin-like protein is transcriptionally activated during sexual development, but is not essential for fruiting body formation in the filamentous fungus Sordaria macrospora

    Directory of Open Access Journals (Sweden)

    Cebula Patricia

    2005-11-01

    Full Text Available Abstract Background The filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies called perithecia that protect the developing ascospores and ensure their proper discharge. In previous microarray analyses, several genes have been identified that are downregulated in sterile mutants compared to the wild type. Among these genes was tap1 (transcript associated with perithecial development, a gene encoding a putative lectin homolog. Results Analysis of tap1 transcript levels in the wild type under conditions allowing only vegetative growth compared to conditions that lead to fruiting body development showed that tap1 is not only downregulated in developmental mutants but is also upregulated in the wild type during fruiting body development. We have cloned and sequenced a 3.2 kb fragment of genomic DNA containing the tap1 open reading frame and adjoining sequences. The genomic region comprising tap1 is syntenic to its homologous region in the closely related filamentous fungus Neurospora crassa. To determine whether tap1 is involved in fruiting body development in S. macrospora, a knockout construct was generated in which the tap1 open reading frame was replaced by the hygromycin B resistance gene hph under the control of fungal regulatory regions. Transformation of the S. macrospora wild type with this construct resulted in a tap1 deletion strain where tap1 had been replaced by the hph cassette. The knockout strain displayed no phenotypic differences under conditions of vegetative growth and sexual development when compared to the wild type. Double mutants carrying the Δtap1 allele in several developmental mutant backgrounds were phenotypically similar to the corresponding developmental mutant strains. Conclusion The tap1 transcript is strongly upregulated during sexual development in S. macrospora; however, analysis of a tap1 knockout strain shows that tap1 is not essential for fruiting body formation in S. macrospora.

  16. Calcium, essential for health

    Science.gov (United States)

    Martínez de Victoria, Emilio

    2016-07-12

    Calcium (Ca) is the most abundant mineral element in our body. It accounts for about 2% of body weight. The functions of calcium are: a) functions skeletal and b) regulatory functions. Bone consists of a protein matrix that mineralizes mainly with calcium (the most abundant), phosphate and magnesium, for it is essential an adequate dietary intake of Ca, phosphorus and vitamin D. The ionic Ca (Ca2+) is essential to maintain and / or perform different specialized functions of, virtually, all body cells cellular. Because of its important functions Ca2+ must be closely regulated, keeping plasma concentrations within narrow ranges. For this reason there is an accurate response against hypocalcemia or hypercalcemia in which the parathormone, calcitriol, calcitonin and vitamin K are involved. Ca intakes in the Spanish population are low in a significant percentage of the older adult’s population, especially in women. The main source of Ca in the diet is milk and milk derivatives. Green leafy vegetables, fruits and legumes can be important sources of Ca in a Mediterranean dietary pattern. The bioavailability of dietary Ca depends on physiological and dietary factors. Physiological include age, physiological status (gestation and lactation) Ca and vitamin D status and disease. Several studies relate Ca intake in the diet and various diseases, such as osteoporosis, cancer, cardiovascular disease and obesity.

  17. Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth.

    Science.gov (United States)

    Lim, Nicholas R; Shohayeb, Belal; Zaytseva, Olga; Mitchell, Naomi; Millard, S Sean; Ng, Dominic C H; Quinn, Leonie M

    2017-07-11

    The second most commonly mutated gene in primary microcephaly (MCPH) patients is wd40-repeat protein 62 (wdr62), but the relative contribution of WDR62 function to the growth of major brain lineages is unknown. Here, we use Drosophila models to dissect lineage-specific WDR62 function(s). Interestingly, although neural stem cell (neuroblast)-specific depletion of WDR62 significantly decreased neuroblast number, brain size was unchanged. In contrast, glial lineage-specific WDR62 depletion significantly decreased brain volume. Moreover, loss of function in glia not only decreased the glial population but also non-autonomously caused neuroblast loss. We further demonstrated that WDR62 controls brain growth through lineage-specific interactions with master mitotic signaling kinase, AURKA. Depletion of AURKA in neuroblasts drives brain overgrowth, which was suppressed by WDR62 co-depletion. In contrast, glial-specific depletion of AURKA significantly decreased brain volume, which was further decreased by WDR62 co-depletion. Thus, dissecting relative contributions of MCPH factors to individual neural lineages will be critical for understanding complex diseases such as microcephaly. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  18. Shelf-life extension of refrigerated sea bass slices wrapped with fish protein isolate/fish skin gelatin-ZnO nanocomposite film incorporated with basil leaf essential oil.

    Science.gov (United States)

    Arfat, Yasir Ali; Benjakul, Soottawat; Vongkamjan, Kitiya; Sumpavapol, Punnanee; Yarnpakdee, Suthasinee

    2015-10-01

    Microbiological, chemical and sensory changes of sea bass slices wrapped with fish protein isolate (FPI)/fish skin gelatin (FSG) films incorporated with 3 % ZnO nanoparticles (ZnONP) (w/w, based on protein content) and 100 % basil leaf essential oil (BEO) (w/w, based on protein content) during storage of 12 days at 4 °C were investigated. Sea bass slices wrapped with FPI/FSG-ZnONP-BEO film had the lowest growth of psychrophilic bacteria, lactic acid bacteria and spoilage microorganisms including Pseudomonas , H2S-producing bacteria and Enterobacteriaceae throughout storage of 12 days in comparison with those wrapped with FPI/FSG-BEO, FPI/FSG-ZnONP, FPI/FSG film, polypropylene film (PP film) and the control (without wrapping), respectively (P < 0.05). Lowered increases in pH, total volatile base, peroxide value and TBARS value were found in FPI/FSG-ZnO-BEO film wrapped samples, compared with others (P < 0.05). Sensory evaluation revealed that shelf-life of sea bass slices was longest for samples wrapped with FPI/FSG-ZnONP-BEO film (12 days), as compared to the control (6 days) (P < 0.05).

  19. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32

    Directory of Open Access Journals (Sweden)

    Sugiyama Hideaki

    2011-05-01

    Full Text Available Abstract Background Elevated numbers of regulatory T cells (Tregs have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32, also known as Glycoprotein A Repetitions Predominant (GARP, has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  20. BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

    Science.gov (United States)

    Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

    2007-01-01

    The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

  1. The assembly of the plant urease activation complex and the essential role of the urease accessory protein G (UreG) in delivery of nickel to urease.

    Science.gov (United States)

    Myrach, Till; Zhu, Anting; Witte, Claus-Peter

    2017-09-01

    Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta , we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential H X H metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first H X H motif is highly beneficial and up to the second H X H motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane*

    Science.gov (United States)

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-01-01

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

  3. X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Yimin Zhong

    Full Text Available Damage to the retinal pigment epithelium (RPE is an early event in the pathogenesis of age-related macular degeneration (AMD. X-box binding protein 1 (XBP1 is a key transcription factor that regulates endoplasmic reticulum (ER homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

  4. Oxidized CaMKII (Ca2+/Calmodulin-Dependent Protein Kinase II) Is Essential for Ventricular Arrhythmia in a Mouse Model of Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Wang, Qiongling; Quick, Ann P; Cao, Shuyi; Reynolds, Julia; Chiang, David Y; Beavers, David; Li, Na; Wang, Guoliang; Rodney, George G; Anderson, Mark E; Wehrens, Xander H T

    2018-04-01

    Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca 2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is vital for normal Ca 2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca 2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca 2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy. 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx :MM-VV mice revealed normalization of intracellular Ca 2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47 phox normalized elevated ox-CaMKII, repaired intracellular Ca 2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca 2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy. © 2018 American Heart Association, Inc.

  5. Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

    Science.gov (United States)

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-04-11

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

  6. Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

    Science.gov (United States)

    Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

    2016-02-01

    Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

  7. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

    Science.gov (United States)

    Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

    2011-05-26

    Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  8. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    Science.gov (United States)

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  9. Evaluating the influence of plant-specific physiological parameterizations on the partitioning of land surface energy fluxes

    Science.gov (United States)

    Sulis, Mauro; Langensiepen, Matthias; Shrestha, Prabhakar; Schickling, Anke; Simmer, Clemens; Kollet, Stefan

    2015-04-01

    Vegetation has a significant influence on the partitioning of radiative forcing, the spatial and temporal variability of soil water and soil temperature. Therefore plant physiological properties play a key role in mediating and amplifying interactions and feedback mechanisms in the soil-vegetation-atmosphere continuum. Because of the direct impact on latent heat fluxes, these properties may also influence weather generating processes, such as the evolution of the atmospheric boundary layer (ABL). In land surface models, plant physiological properties are usually obtained from literature synthesis by unifying several plant/crop species in predefined vegetation classes. In this work, crop-specific physiological characteristics, retrieved from detailed field measurements, are included in the bio-physical parameterization of the Community Land Model (CLM), which is a component of the Terrestrial Systems Modeling Platform (TerrSysMP). The measured set of parameters for two typical European mid-latitudinal crops (sugar beet and winter wheat) is validated using eddy covariance measurements (sensible heat and latent heat) over multiple years from three measurement sites located in the North Rhine-Westphalia region, Germany. We found clear improvements of CLM simulations, when using the crop-specific physiological characteristics of the plants instead of the generic crop type when compared to the measurements. In particular, the increase of latent heat fluxes in conjunction with decreased sensible heat fluxes as simulated by the two new crop-specific parameter sets leads to an improved quantification of the diurnal energy partitioning. These findings are cross-validated using estimates of gross primary production extracted from net ecosystem exchange measurements. This independent analysis reveals that the better agreement between observed and simulated latent heat using the plant-specific physiological properties largely stems from an improved simulation of the

  10. Phosphoinositide metabolism links cGMP-dependent protein kinase G to essential Ca²⁺ signals at key decision points in the life cycle of malaria parasites.

    Directory of Open Access Journals (Sweden)

    Mathieu Brochet

    2014-03-01

    Full Text Available Many critical events in the Plasmodium life cycle rely on the controlled release of Ca²⁺ from intracellular stores to activate stage-specific Ca²⁺-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca²⁺ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca²⁺ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca²⁺ effectors, PKG emerges as a unifying factor to control multiple cellular Ca²⁺ signals essential for malaria parasite development and transmission.

  11. cAMP response element binding protein1 is essential for activation of steroyl co-enzyme a desaturase 1 (Scd1 in mouse lung type II epithelial cells.

    Directory of Open Access Journals (Sweden)

    Nisha Antony

    Full Text Available Cyclic AMP Response Element-Binding Protein 1 (Creb1 is a transcription factor that mediates cyclic adenosine 3', 5'-monophosphate (cAMP signalling in many tissues. Creb1(-/- mice die at birth due to respiratory failure and previous genome-wide microarray analysis of E17.5 Creb1(-/- fetal mouse lung identified important Creb1-regulated gene targets during lung development. The lipogenic enzymes stearoyl-CoA desaturase 1 (Scd1 and fatty acid synthase (Fasn showed highly reduced gene expression in Creb1(-/- lungs. We therefore hypothesized that Creb1 plays a crucial role in the transcriptional regulation of genes involved in pulmonary lipid biosynthetic pathways during lung development. In this study we confirmed that Scd1 and Fasn mRNA levels were down regulated in the E17.5 Creb1(-/- mouse lung while the lipogenic-associated transcription factors SrebpF1, C/ebpα and Pparγ were increased. In vivo studies using germline (Creb1(-/- and lung epithelial-specific (Creb1(EpiΔ/Δ Creb1 knockout mice showed strongly reduced Scd1, but not Fasn gene expression and protein levels in lung epithelial cells. In vitro studies using mouse MLE-15 epithelial cells showed that forskolin-mediated activation of Creb1 increased both Scd1 gene expression and protein synthesis. Additionally, MLE15 cells transfected with a dominant-negative ACreb vector blocked forskolin-mediated stimulation of Scd1 gene expression. Lipid profiling in MLE15 cells showed that dominant-negative ACreb suppressed forskolin-induced desaturation of ether linked lipids to produce plasmalogens, as well as levels of phosphatidylethanolamine, ceramide and lysophosphatidylcholine. Taken together these results demonstrate that Creb1 is essential for the induction and maintenance of Scd1 in developing fetal mouse lung epithelial cells.

  12. Treatment of Essential Tremor

    Science.gov (United States)

    ... for PATIENTS and their FAMILIES TREATMENT OF ESSENTIAL TREMOR This fact sheet is provided to help you understand which therapies help treat essential tremor. Neurologists from the American Academy of Neurology are ...

  13. Failure cause and failure rate evaluation on pumps of BWR plants in PSA. Hypothesis testing for typical or plant specific failure rate of pumps

    International Nuclear Information System (INIS)

    Sanada, Takahiro; Nakamura, Makoto

    2009-01-01

    In support of domestic nuclear industry effort to gather and analyze failure data of components concerning nuclear power plants, Nuclear Information Archives (NUCIA) are published for useful information to help PSA. This report focuses on NUCIA pertaining to pumps in domestic nuclear power plants, and provides the reliable estimation on failure rate of pumps resulting from failure cause analysis and hypothesis testing of classified and plant specific failure rate of pumps for improving quality in PSA. The classified and plant specific failure rate of pumps are estimated by analyzing individual domestic nuclear power plant's data of 26 Boiling Water Reactors (BWRs) concerning functionally structurally classified pump failures reported from beginning of commercial operation to March 31, 2007. (author)

  14. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

    2009-10-30

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

  15. C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

    Science.gov (United States)

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

    2009-01-01

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

  16. Tenebrio molitor Gram-negative-binding protein 3 (TmGNBP3) is essential for inducing downstream antifungal Tenecin 1 gene expression against infection with Beauveria bassiana JEF-007.

    Science.gov (United States)

    Yang, Yi-Ting; Lee, Mi Rong; Lee, Se Jin; Kim, Sihyeon; Nai, Yu-Shin; Kim, Jae Su

    2017-05-23

    The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, β-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  17. The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

    KAUST Repository

    Cui, Peng

    2016-02-18

    © 2016 American Society of Plant Biologists. All rights reserved. Nonsense-mediated decay (NMD) is a posttranscriptional surveillance mechanism in eukaryotes that recognizes and degrades transcripts with premature translation-termination codons. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2 (FRY2; also known as C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 [CPL1]) plays multiple roles in RNA processing in Arabidopsis thaliana. Here, we found that FRY2/CPL1 interacts with two NMD factors, eIF4AIII and UPF3, and is involved in the dephosphorylation of eIF4AIII. This dephosphorylation retains eIF4AIII in the nucleus and limits its accumulation in the cytoplasm. By analyzing RNA-seq data combined with quantitative RT-PCR validation, we found that a subset of alternatively spliced transcripts and 59-extended mRNAs with NMD-eliciting features accumulated in the fry2-1 mutant, cycloheximidetreated wild type, and upf3 mutant plants, indicating that FRY2 is essential for the degradation of these NMD transcripts.

  18. Oral hygiene grade and quality of life in children with chemotherapy-related oral mucositis: a randomized study on the impact of a fluoride toothpaste with salivary enzymes, essential oils, proteins and colostrum extract versus a fluoride toothpaste without menthol.

    Science.gov (United States)

    Bardellini, E; Amadori, F; Majorana, A

    2016-11-01

    The aim of this study was to assess the impact of the use of a fluoride toothpaste (Bioxtra ® , Biopharm, Milan, Italy) with salivary enzymes, essential oils, proteins and colostrum extract versus a fluoride toothpaste without menthol on the oral hygiene grade and on the quality of life (QoL) of children with oral mucositis (OM) grade 1 or 2 receiving chemotherapy for Acute Lymphoblastic Leukaemia (ALL). Patients between 6 and 14 years with OM were randomly assigned to two groups, group A (Bioxtra ® toothpaste) and group B (fluoride toothpaste without menthol). The patients were instructed to brush their teeth at least twice a day using a soft toothbrush with a small head. Oral hygiene grade was assessed using the simplified oral hygiene index (OHI-s); quality of life was assessed using the short form of the Oral Health Impact Profile (OHIP-14) questionnaires. The patients were evaluated on day 1(diagnosis of OM-T0) and on day 8 (T1). Statistical analysis was performed. A total of 64 patients were enrolled. A significant difference (P oral hygiene grade. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

    KAUST Repository

    Cui, Peng; Chen, Tao; Qin, Tao; Ding, Feng; Wang, Zhenyu; Chen, Hao; Xiong, Liming

    2016-01-01

    © 2016 American Society of Plant Biologists. All rights reserved. Nonsense-mediated decay (NMD) is a posttranscriptional surveillance mechanism in eukaryotes that recognizes and degrades transcripts with premature translation-termination codons. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2 (FRY2; also known as C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 [CPL1]) plays multiple roles in RNA processing in Arabidopsis thaliana. Here, we found that FRY2/CPL1 interacts with two NMD factors, eIF4AIII and UPF3, and is involved in the dephosphorylation of eIF4AIII. This dephosphorylation retains eIF4AIII in the nucleus and limits its accumulation in the cytoplasm. By analyzing RNA-seq data combined with quantitative RT-PCR validation, we found that a subset of alternatively spliced transcripts and 59-extended mRNAs with NMD-eliciting features accumulated in the fry2-1 mutant, cycloheximidetreated wild type, and upf3 mutant plants, indicating that FRY2 is essential for the degradation of these NMD transcripts.

  20. Heterogeneity of soil surface ammonium concentration and other characteristics, related to plant specific variability in a Mediterranean-type ecosystem

    International Nuclear Information System (INIS)

    Cruz, Cristina; Bio, Ana M.F.; Jullioti, Aldo; Tavares, Alice; Dias, Teresa; Martins-Loucao, Maria Amelia

    2008-01-01

    Heterogeneity and dynamics of eight soil surface characteristics essential for plants-ammonium and nitrate concentrations, water content, temperature, pH, organic matter, nitrification and ammonification rates-were studied in a Mediterranean-type ecosystem on four occasions over a year. Soil properties varied seasonally and were influenced by plant species. Nitrate and ammonium were present in the soil at similar concentrations throughout the year. The positive correlation between them at the time of greatest plant development indicates that ammonium is a readily available nitrogen source in Mediterranean-type ecosystems. The results presented here suggest that plant cover significantly affects soil surface characteristics. - In Mediterranean-type ecosystems ammonium is present in the soil throughout the year and its concentration is dependent on plant cover

  1. Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

    Science.gov (United States)

    Meiyalaghan, Sathiyamoorthy; Thomson, Susan J; Fiers, Mark W E J; Barrell, Philippa J; Latimer, Julie M; Mohan, Sara; Jones, E Eirian; Conner, Anthony J; Jacobs, Jeanne M E

    2014-01-02

    -down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

  2. Electric circuits essentials

    CERN Document Server

    REA, Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Electric Circuits I includes units, notation, resistive circuits, experimental laws, transient circuits, network theorems, techniques of circuit analysis, sinusoidal analysis, polyph

  3. Calculus III essentials

    CERN Document Server

    REA, Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Calculus III includes vector analysis, real valued functions, partial differentiation, multiple integrations, vector fields, and infinite series.

  4. Statistics I essentials

    CERN Document Server

    Milewski, Emil G

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Statistics I covers include frequency distributions, numerical methods of describing data, measures of variability, parameters of distributions, probability theory, and distributions.

  5. Pre-calculus essentials

    CERN Document Server

    Woodward, Ernest

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Pre-Calculus reviews sets, numbers, operations and properties, coordinate geometry, fundamental algebraic topics, solving equations and inequalities, functions, trigonometry, exponents

  6. Transport phenomena II essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Transport Phenomena II covers forced convention, temperature distribution, free convection, diffusitivity and the mechanism of mass transfer, convective mass transfer, concentration

  7. Differential equations I essentials

    CERN Document Server

    REA, Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Differential Equations I covers first- and second-order equations, series solutions, higher-order linear equations, and the Laplace transform.

  8. Heat transfer II essentials

    CERN Document Server

    REA, The Editors of

    1988-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Heat Transfer II reviews correlations for forced convection, free convection, heat exchangers, radiation heat transfer, and boiling and condensation.

  9. Numerical analysis II essentials

    CERN Document Server

    REA, The Editors of; Staff of Research Education Association

    1989-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Numerical Analysis II covers simultaneous linear systems and matrix methods, differential equations, Fourier transformations, partial differential equations, and Monte Carlo methods.

  10. Algebra & trigonometry II essentials

    CERN Document Server

    REA, Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Algebra & Trigonometry II includes logarithms, sequences and series, permutations, combinations and probability, vectors, matrices, determinants and systems of equations, mathematica

  11. Modern algebra essentials

    CERN Document Server

    Lutfiyya, Lutfi A

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Modern Algebra includes set theory, operations, relations, basic properties of the integers, group theory, and ring theory.

  12. Business statistics I essentials

    CERN Document Server

    Clark, Louise

    2014-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Business Statistics I includes descriptive statistics, introduction to probability, probability distributions, sampling and sampling distributions, interval estimation, and hypothesis t

  13. Computer science I essentials

    CERN Document Server

    Raus, Randall

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Computer Science I includes fundamental computer concepts, number representations, Boolean algebra, switching circuits, and computer architecture.

  14. Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

    Science.gov (United States)

    Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

    2002-10-01

    A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

  15. Essential Bacillus subtilis genes

    DEFF Research Database (Denmark)

    Kobayashi, K.; Ehrlich, S.D.; Albertini, A.

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were...... predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related...... to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from...

  16. Whey protein isolate/cellulose nanofibre/TiO2 nanoparticle/rosemary essential oil nanocomposite film: Its effect on microbial and sensory quality of lamb meat and growth of common foodborne pathogenic bacteria during refrigeration.

    Science.gov (United States)

    Alizadeh Sani, Mahmood; Ehsani, Ali; Hashemi, Mohammad

    2017-06-19

    The use of biodegradable nanocomposite films in active packaging is of great importance since they can have a controlled release of antimicrobial compounds. This study was conducted to evaluate the efficacy of whey protein isolate (WPI)/cellulose nanofibre (CNF) nanocomposite films containing 1.0% (w/w) titanium dioxide (TiO 2 ) and 2.0% (w/v) rosemary essential oil (REO) in preserving the microbial and sensory quality of lamb meat during the storage at 4±1°C. Initially, the best concentration of each compound to be added to the film was determined by micro-dilution and disc diffusion methods. The microbial and sensory properties of lamb meat were controlled in two groups (control and treatment) over 15days of storage. Then, the samples were analysed for total viable count (TVC), Pseudomonas spp. count, Enterobacteriaceae count, Lactic acid bacteria (LAB) count, inoculated Staphylococcus aureus count, Listeria monocytogenes count, and Escherichia coli O 157 :H 7 count. Microbial analysis and nine-point hedonic scale was applied for the sensory analysis. Results indicated that the use of nanocomposite films significantly reduced the bacterial counts of treatment group. Higher inhibition effect was observed on Gram-positive bacteria than on Gram-negative bacteria (Psensory evaluations also showed that the use of nanocomposite films significantly increased the shelf life of treated meat (15days) compared to the control meat (6days). Based on the results of this study, the edible nanocomposite films were effective in preserving the microbial and sensory qualities of lamb meat; therefore, this application is recommended in meat especially red meat. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Plasmid AZOBR_p1-borne fabG gene for putative 3-oxoacyl-[acyl-carrier protein] reductase is essential for proper assembly and work of the dual flagellar system in the alphaproteobacterium Azospirillum brasilense Sp245.

    Science.gov (United States)

    Filip'echeva, Yulia A; Shelud'ko, Andrei V; Prilipov, Alexei G; Burygin, Gennady L; Telesheva, Elizaveta M; Yevstigneyeva, Stella S; Chernyshova, Marina P; Petrova, Lilia P; Katsy, Elena I

    2018-02-01

    Azospirillum brasilense can swim and swarm owing to the activity of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf), respectively. Experimental data on the regulation of the Fla and Laf assembly in azospirilla are scarce. Here, the coding sequence (CDS) AZOBR_p1160043 (fabG1) for a putative 3-oxoacyl-[acyl-carrier protein (ACP)] reductase was found essential for the construction of both types of flagella. In an immotile leaky Fla - Laf - fabG1::Omegon-Km mutant, Sp245.1610, defects in flagellation and motility were fully complemented by expressing the CDS AZOBR_p1160043 from plasmid pRK415. When pRK415 with the cloned CDS AZOBR_p1160045 (fliC) for a putative 65.2 kDa Sp245 Fla flagellin was transferred into the Sp245.1610 cells, the bacteria also became able to assemble a motile single flagellum. Some cells, however, had unusual swimming behavior, probably because of the side location of the organelle. Although the assembly of Laf was not restored in Sp245.1610 (pRK415-p1160045), this strain was somewhat capable of swarming motility. We propose that the putative 3-oxoacyl-[ACP] reductase encoded by the CDS AZOBR_p1160043 plays a role in correct flagellar location in the cell envelope and (or) in flagellar modification(s), which are also required for the inducible construction of Laf and for proper swimming and swarming motility of A. brasilense Sp245.

  18. Physics I essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Physics I includes vectors and scalars, one-dimensional motion, plane motion, dynamics of a particle, work and energy, conservation of energy, dynamics of systems of particles, rotation

  19. Electronics II essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Electronics II covers operational amplifiers, feedback and frequency compensation of OP amps, multivibrators, logic gates and families, Boolean algebra, registers, counters, arithmet

  20. Thermodynamics I essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Thermodynamics I includes review of properties and states of a pure substance, work and heat, energy and the first law of thermodynamics, entropy and the second law of thermodynamics

  1. C programming language essentials

    CERN Document Server

    Ackermann, Ernest C

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. C Programming Language discusses fundamental notions, data types and objects, expressions, statements, declarations, function and program structure, the preprocessor, and the standar

  2. Electronics I essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Electronics I covers fundamentals of semiconductor devices, junction diodes, bipolar junction transistors, power supplies, multitransistor circuits, small signals, low-frequency anal

  3. Thermodynamics II essentials

    CERN Document Server

    REA, The Editors of

    2013-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Thermodynamics II includes review of thermodynamic relations, power and refrigeration cycles, mixtures and solutions, chemical reactions, chemical equilibrium, and flow through nozzl

  4. Group theory I essentials

    CERN Document Server

    Milewski, Emil G

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Group Theory I includes sets and mapping, groupoids and semi-groups, groups, isomorphisms and homomorphisms, cyclic groups, the Sylow theorems, and finite p-groups.

  5. Boolean algebra essentials

    CERN Document Server

    Solomon, Alan D

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Boolean Algebra includes set theory, sentential calculus, fundamental ideas of Boolean algebras, lattices, rings and Boolean algebras, the structure of a Boolean algebra, and Boolean

  6. Laplace transforms essentials

    CERN Document Server

    Shafii-Mousavi, Morteza

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Laplace Transforms includes the Laplace transform, the inverse Laplace transform, special functions and properties, applications to ordinary linear differential equations, Fourier tr

  7. Physical chemistry II essentials

    CERN Document Server

    REA, The Editors of

    1992-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Physical Chemistry II includes reaction mechanisms, theoretical approaches to chemical kinetics, gravitational work, electrical and magnetic work, surface work, kinetic theory, collisional and transport properties of gases, statistical mechanics, matter and waves, quantum mechanics, and rotations and vibrations of atoms and molecules.

  8. Statistics II essentials

    CERN Document Server

    Milewski, Emil G

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Statistics II discusses sampling theory, statistical inference, independent and dependent variables, correlation theory, experimental design, count data, chi-square test, and time se

  9. Algebra & trigonometry I essentials

    CERN Document Server

    REA, Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Algebra & Trigonometry I includes sets and set operations, number systems and fundamental algebraic laws and operations, exponents and radicals, polynomials and rational expressions, eq

  10. Geometry I essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Geometry I includes methods of proof, points, lines, planes, angles, congruent angles and line segments, triangles, parallelism, quadrilaterals, geometric inequalities, and geometric

  11. Transport phenomena I essentials

    CERN Document Server

    REA, The Editors of

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Transport Phenomena I includes viscosity, flow of Newtonian fluids, velocity distribution in laminar flow, velocity distributions with more than one independent variable, thermal con

  12. Data structures II essentials

    CERN Document Server

    Smolarski, Dennis C

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Data Structures II includes sets, trees, advanced sorting, elementary graph theory, hashing, memory management and garbage collection, and appendices on recursion vs. iteration, alge

  13. Computer science II essentials

    CERN Document Server

    Raus, Randall

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Computer Science II includes organization of a computer, memory and input/output, coding, data structures, and program development. Also included is an overview of the most commonly

  14. Data structures I essentials

    CERN Document Server

    Smolarski, Dennis C

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Data Structures I includes scalar variables, arrays and records, elementary sorting, searching, linked lists, queues, and appendices of binary notation and subprogram parameter passi

  15. Set theory essentials

    CERN Document Server

    Milewski, Emil G

    2012-01-01

    REA's Essentials provide quick and easy access to critical information in a variety of different fields, ranging from the most basic to the most advanced. As its name implies, these concise, comprehensive study guides summarize the essentials of the field covered. Essentials are helpful when preparing for exams, doing homework and will remain a lasting reference source for students, teachers, and professionals. Set Theory includes elementary logic, sets, relations, functions, denumerable and non-denumerable sets, cardinal numbers, Cantor's theorem, axiom of choice, and order relations.

  16. Roitt's essential immunology

    National Research Council Canada - National Science Library

    Delves, Peter J; Roitt, Ivan M

    2011-01-01

    ... of the immune system, the hallmark easy-reading style of Roitt's Essential Immunology clearly explains the key principles needed by medical and health sciences students, from the basis of immunity to clinical applications...

  17. Benign Essential Blepharospasm

    Science.gov (United States)

    ... the same for many years; and, in rare cases, improve spontaneously. Clinical Trials Throughout the U.S. and Worldwide NINDS Clinical Trials Related ... Definition Benign essential blepharospasm (BEB) is a progressive neurological ...

  18. Marketingmanagement : De essentie

    NARCIS (Netherlands)

    Kotler, P.J.; Keller, K.; Robben, H.S.J.

    2007-01-01

    'Marketingmanagement, de essentie' biedt een volledige introductie in modern marketingmanagement. De nieuwste concepten en onderzoeksresultaten komen aan bod. Zo wordt veel aandacht besteed aan holistische marketing en is de impact van technologische ontwikkelingen op hedendaagse marketing in deze

  19. Marketing management : De essentie

    NARCIS (Netherlands)

    Kotler, P.J.; Keller, K.; Robben, H.S.J.

    2010-01-01

    'Marketingmanagement, de essentie' biedt een volledige introductie in modern marketingmanagement. De nieuwste concepten en onderzoeksresultaten komen aan bod. Zo wordt veel aandacht besteed aan holistische marketing en is de impact van technologische ontwikkelingen op hedendaagse marketing in deze

  20. Essential software architecture

    CERN Document Server

    Gorton, Ian

    2011-01-01

    Job titles like ""Technical Architect"" and ""Chief Architect"" nowadays abound in software industry, yet many people suspect that ""architecture"" is one of the most overused and least understood terms in professional software development. Gorton's book tries to resolve this dilemma. It concisely describes the essential elements of knowledge and key skills required to be a software architect. The explanations encompass the essentials of architecture thinking, practices, and supporting technologies. They range from a general understanding of structure and quality attributes through technical i

  1. Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans.

    Science.gov (United States)

    Mercx, Sébastien; Smargiasso, Nicolas; Chaumont, François; De Pauw, Edwin; Boutry, Marc; Navarre, Catherine

    2017-01-01

    Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N -glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker ( bar ), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells.

  2. Python essential reference

    CERN Document Server

    Beazley, David M

    2009-01-01

    Python Essential Reference is the definitive reference guide to the Python programming language — the one authoritative handbook that reliably untangles and explains both the core Python language and the most essential parts of the Python library. Designed for the professional programmer, the book is concise, to the point, and highly accessible. It also includes detailed information on the Python library and many advanced subjects that is not available in either the official Python documentation or any other single reference source. Thoroughly updated to reflect the significant new programming language features and library modules that have been introduced in Python 2.6 and Python 3, the fourth edition of Python Essential Reference is the definitive guide for programmers who need to modernize existing Python code or who are planning an eventual migration to Python 3. Programmers starting a new Python project will find detailed coverage of contemporary Python programming idioms.

  3. Essential travel medicine

    CERN Document Server

    Zuckerman, Jane N; Leggat, Peter

    2015-01-01

    This 1st edition of Essential Travel Medicine provides an excellent concise introduction to the specialty of Travel Medicine. This core text will enable health care practitioners particularly those new to the clinical practice of Travel Medicine, to gain a fundamental understanding of the diverse and complex issues which can potentially affect the health of the many millions of people who undertake international travel. Jane N Zuckerman is joined by Gary W Brunette from CDC and Peter A Leggat from Australia as Editors. Leading international specialists in their fields have contributed authoritative chapters reflecting current knowledge to facilitate best clinical practice in the different aspects of travel medicine. The aim of Essential Travel Medicine is to provide a comprehensive guide to Travel Medicine as well as a fundamental knowledge base to support international undergraduate and postgraduate specialty training programmes in the discipline of Travel Medicine. The 1st edition of Essential Travel ...

  4. Physics Essentials For Dummies

    CERN Document Server

    Holzner, Steven

    2010-01-01

    For students who just need to know the vital concepts of physics, whether as a refresher, for exam prep, or as a reference, Physics Essentials For Dummies is a must-have guide. Free of ramp-up and ancillary material, Physics Essentials For Dummies contains content focused on key topics only. It provides discrete explanations of critical concepts taught in an introductory physics course, from force and motion to momentum and kinetics. This guide is also a perfect reference for parents who need to review critical physics concepts as they help high school students with homework assignments, as we

  5. Essentials of Computational Electromagnetics

    CERN Document Server

    Sheng, Xin-Qing

    2012-01-01

    Essentials of Computational Electromagnetics provides an in-depth introduction of the three main full-wave numerical methods in computational electromagnetics (CEM); namely, the method of moment (MoM), the finite element method (FEM), and the finite-difference time-domain (FDTD) method. Numerous monographs can be found addressing one of the above three methods. However, few give a broad general overview of essentials embodied in these methods, or were published too early to include recent advances. Furthermore, many existing monographs only present the final numerical results without specifyin

  6. Essential Palatal Myoclonus

    Directory of Open Access Journals (Sweden)

    Bhuwan Raj Pandey

    2017-06-01

    Full Text Available Introduction: Palatal myoclonus is a rare condition presenting with clicking sound in ear or muscle tremor in pharynx. There are two varieties: essential and symptomatic. Various treatment options exists ranging from watchful observation to botulinum toxin injection. We have not found any reported case of palatal myoclonus from our country. Here we present a case of essential palatal myoclonus managed with clonazepam. Case report: A young female presented in Ear Nose and Throat clinic with complain of auditory click and spontaneous rhythmic movement of throat muscles for eight months. On examination, there was involuntary, rhythmic contraction of bilateral soft-palate, uvula, and base of tongue. Neurological, eye, and peripheral examination were normal. A diagnosis of essential palatal myoclonus was made. It was managed successfully with clonazepam; patient was still on low dose clonazepam at the time of making this report. Conclusion: Essential palatal myoclonus can be clinically diagnosed and managed even in settings where MRI is not available or affordable.

  7. The 2003 essential. AREVA

    International Nuclear Information System (INIS)

    2004-07-01

    This document presents the essential activities of the Areva Group, a world nuclear industry leader. This group proposes technological solutions to produce the nuclear energy and to transport the electric power. It develops connection systems for the telecommunication, the computers and the automotive industry. Key data on the program management, the sustainable development activities and the different divisions are provided. (A.L.B.)

  8. Essentials of Risk Theory

    NARCIS (Netherlands)

    Roeser, S.; Hillerbrand, R.; Sandin, P.; Peterson, M.B.

    2012-01-01

    Risk has become one of the main topics in fields as diverse as engineering, medicine and economics, and it is also studied by social scientists, psychologists and legal scholars. This Springer Essentials version offers an overview of the in-depth handbook and highlights some of the main points

  9. Essential trichomegaly: case report

    Directory of Open Access Journals (Sweden)

    Julia Dutra Rossetto

    2013-02-01

    Full Text Available The present study reports two cases of symptomatic essential trichomegaly. Trichomegaly may develop in various diseases, including anorexia nervosa, hypothyroidism, pregnancy, pretibial myxedema, systemic lupus erythematosus, vernal keratoconjunctivitis, and uveitis. The exact incidence trichomegaly is unknown, and the condition remains sporadically reported. Two cases of symptomatic trichomegaly without any associated systemic disorder are presented in this paper.

  10. Genetics Home Reference: essential tremor

    Science.gov (United States)

    ... Facebook Twitter Home Health Conditions Essential tremor Essential tremor Printable PDF Open All Close All Enable Javascript to view the expand/collapse boxes. Description Essential tremor is a movement disorder that causes involuntary, rhythmic ...

  11. Essentials of artificial intelligence

    CERN Document Server

    Ginsberg, Matt

    1993-01-01

    Since its publication, Essentials of Artificial Intelligence has beenadopted at numerous universities and colleges offering introductory AIcourses at the graduate and undergraduate levels. Based on the author'scourse at Stanford University, the book is an integrated, cohesiveintroduction to the field. The author has a fresh, entertaining writingstyle that combines clear presentations with humor and AI anecdotes. At thesame time, as an active AI researcher, he presents the materialauthoritatively and with insight that reflects a contemporary, first hand

  12. Moodle administration essentials

    CERN Document Server

    Henrick, Gavin

    2015-01-01

    If you are an experienced system administrator and know how to manage servers and set up web environments but now want to explore Moodle, this book is perfect for you. You'll get to grips with the basics and learn to manage Moodle quickly, focusing on essential tasks. Having prior knowledge of virtual learning environments would be beneficial, but is not mandatory to make the most of this book.

  13. Essentials of Endodontic Microsurgery

    Science.gov (United States)

    2010-04-01

    Holtzman DJ, et al. Quality of root-end preparations using ultrasonic and rotary instrumentation in cadavers. J Endod 2000;26:281. 39. Peters CI...00-00-2010 to 00-00-2010 4. TITLE AND SUBTITLE Essentials of Endodontic Microsurgery 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT... Endodontic Program,Harvard School of Dental Medicine,Boston,MA 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND

  14. Process Improvement Essentials

    CERN Document Server

    Persse, James R

    2006-01-01

    Process Improvement Essentials combines the foundation needed to understand process improvement theory with the best practices to help individuals implement process improvement initiatives in their organization. The three leading programs: ISO 9001:2000, CMMI, and Six Sigma--amidst the buzz and hype--tend to get lumped together under a common label. This book delivers a combined guide to all three programs, compares their applicability, and then sets the foundation for further exploration.

  15. IPv6 Essentials

    CERN Document Server

    Hagen, Silvia

    2006-01-01

    IPv6 Essentials, Second Edition provides a succinct, in-depth tour of all the new features and functions in IPv6. It guides you through everything you need to know to get started, including how to configure IPv6 on hosts and routers and which applications currently support IPv6. Aimed at system and network administrators, engineers, network designers, and IT managers, this book will help you understand, plan for, design, and integrate IPv6 into your current IPv4 infrastructure

  16. Android application security essentials

    CERN Document Server

    Rai, Pragati

    2013-01-01

    Android Application Security Essentials is packed with examples, screenshots, illustrations, and real world use cases to secure your apps the right way.If you are looking for guidance and detailed instructions on how to secure app data, then this book is for you. Developers, architects, managers, and technologists who wish to enhance their knowledge of Android security will find this book interesting. Some prior knowledge of development on the Android stack is desirable but not required.

  17. Rake task management essentials

    CERN Document Server

    Koleshko, Andrey

    2014-01-01

    A step-by-step and interactive approach explaining the Rake essentials along with code examples and advanced features. If you are a developer who is acquainted with the Ruby language and want to speed up writing the code concerned with files, then this book is for you. To start reading this book, basic Ruby knowledge is required; however, a huge amount of experience with the language is not necessary.

  18. A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

    Science.gov (United States)

    Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

    2009-11-01

    Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance

  19. Samii's essentials in neurosurgery

    International Nuclear Information System (INIS)

    Ramina, Ricardo; Pontifical Catholic Univ. of Parana, Curitiba; Pires Aguiar, Paulo Henrique; Sao Paulo Univ.; Hospital Santa Paula, Sao Paulo; Tatagiba, Marcos

    2008-01-01

    'Samii's Essentials in Neurosurgery' contains selected papers written by internationally recognized contributors who were trained by Professor Madjid Samii in Hannover, Germany. The main topics deal with cutting-edge technology in neurosurgery, skull-base surgery, and specific peripheral nerve, spine, and vascular surgeries. The texts and a wealth of illustrations review and reinforce guidelines on the diagnosis and management of situations that readers are likely to encounter in everyday practice. This book will be of great interest to neurosurgeons, neurologists, ENT surgeons, neuroradiologists, and neurophysiotherapists. (orig.)

  20. Geometry essentials for dummies

    CERN Document Server

    Ryan, Mark

    2011-01-01

    Just the critical concepts you need to score high in geometry This practical, friendly guide focuses on critical concepts taught in a typical geometry course, from the properties of triangles, parallelograms, circles, and cylinders, to the skills and strategies you need to write geometry proofs. Geometry Essentials For Dummies is perfect for cramming or doing homework, or as a reference for parents helping kids study for exams. Get down to the basics - get a handle on the basics of geometry, from lines, segments, and angles, to vertices, altitudes, and diagonals Conque

  1. Essentials of cloud computing

    CERN Document Server

    Chandrasekaran, K

    2014-01-01

    ForewordPrefaceComputing ParadigmsLearning ObjectivesPreambleHigh-Performance ComputingParallel ComputingDistributed ComputingCluster ComputingGrid ComputingCloud ComputingBiocomputingMobile ComputingQuantum ComputingOptical ComputingNanocomputingNetwork ComputingSummaryReview PointsReview QuestionsFurther ReadingCloud Computing FundamentalsLearning ObjectivesPreambleMotivation for Cloud ComputingThe Need for Cloud ComputingDefining Cloud ComputingNIST Definition of Cloud ComputingCloud Computing Is a ServiceCloud Computing Is a Platform5-4-3 Principles of Cloud computingFive Essential Charact

  2. Autodesk Maya 2014 essentials

    CERN Document Server

    Naas, Paul

    2013-01-01

    The premiere book on getting started with Maya 2014 Whether you're just beginning, or migrating from another 3D application, this step-by-step guide is what you need to get a good working knowledge of Autodesk Maya 2014. Beautifully illustrated with full-color examples and screenshots, Autodesk Maya 2014 Essentials explains the basics of Maya as well as modeling, texturing, animating, setting a scene, and creating visual effects. You'll absorb important concepts and techniques, and learn how to confidently use Maya tools the way professionals do. Each chapter includes fun and cha

  3. Essential dynamics and relativity

    CERN Document Server

    O'Donnell, Peter J

    2014-01-01

    Essential Dynamics & Relativity provides students with an introduction to the core aspects of dynamics and special relativity. The author reiterates important ideas and terms throughout and covers concepts that are often missing from other textbooks at this level. He also places each topic within the wider constructs of the theory, without jumping from topic to topic to illustrate a point.The first section of the book focuses on dynamics, discussing the basic aspects of single particle motion and analyzing the motion of multi-particle systems. The book also explains the dynamical behavior of b

  4. 3D Animation Essentials

    CERN Document Server

    Beane, Andy

    2012-01-01

    The essential fundamentals of 3D animation for aspiring 3D artists 3D is everywhere--video games, movie and television special effects, mobile devices, etc. Many aspiring artists and animators have grown up with 3D and computers, and naturally gravitate to this field as their area of interest. Bringing a blend of studio and classroom experience to offer you thorough coverage of the 3D animation industry, this must-have book shows you what it takes to create compelling and realistic 3D imagery. Serves as the first step to understanding the language of 3D and computer graphics (CG)Covers 3D anim

  5. Twisted network programming essentials

    CERN Document Server

    Fettig, Abe

    2005-01-01

    Twisted Network Programming Essentials from O'Reilly is a task-oriented look at this new open source, Python-based technology. The book begins with recommendations for various plug-ins and add-ons to enhance the basic package as installed. It then details Twisted's collection simple network protocols, and helper utilities. The book also includes projects that let you try out the Twisted framework for yourself. For example, you'll find examples of using Twisted to build web services applications using the REST architecture, using XML-RPC, and using SOAP. Written for developers who want to s

  6. The essential David Bohm

    CERN Document Server

    Nichol, Lee

    2002-01-01

    There are few scientists of the twentieth century whose life's work has created more excitement and controversy than that of physicist David Bohm (1917-1992). For the first time in a single volume, The Essential David Bohm offers a comprehensive overview of Bohm's original works from a non-technical perspective. Including three chapters of previously unpublished material, and a forward by the Dalai Lama, each reading has been selected to highlight some aspect of the implicate order process, and to provide an introduction to one of the most provocative thinkers of our time.

  7. Microsoft Windows Security Essentials

    CERN Document Server

    Gibson, Darril

    2011-01-01

    Windows security concepts and technologies for IT beginners IT security can be a complex topic, especially for those new to the field of IT. This full-color book, with a focus on the Microsoft Technology Associate (MTA) program, offers a clear and easy-to-understand approach to Windows security risks and attacks for newcomers to the world of IT. By paring down to just the essentials, beginners gain a solid foundation of security concepts upon which more advanced topics and technologies can be built. This straightforward guide begins each chapter by laying out a list of topics to be discussed,

  8. Microsoft Windows networking essentials

    CERN Document Server

    Gibson, Darril

    2011-01-01

    The core concepts and technologies of Windows networking Networking can be a complex topic, especially for those new to the field of IT. This focused, full-color book takes a unique approach to teaching Windows networking to beginners by stripping down a network to its bare basics, thereby making each topic clear and easy to understand. Focusing on the new Microsoft Technology Associate (MTA) program, this book pares down to just the essentials, showing beginners how to gain a solid foundation for understanding networking concepts upon which more advanced topics and technologies can be built.

  9. Cisco Networking Essentials

    CERN Document Server

    McMillan, Troy

    2011-01-01

    An engaging approach for anyone beginning a career in networking As the world leader of networking products and services, Cisco products are constantly growing in demand. Yet, few books are aimed at those who are beginning a career in IT--until now. Cisco Networking Essentials provides a solid foundation on the Cisco networking products and services with thorough coverage of fundamental networking concepts. Author Troy McMillan applies his years of classroom instruction to effectively present high-level topics in easy-to-understand terms for beginners. With this indispensable full-color resour

  10. French essentials for dummies

    CERN Document Server

    Lawless, Laura K

    2011-01-01

    Just the core concepts you need to write and speak French correctly If you have some knowledge of French and want to polish your skills, French Essentials For Dummies focuses on just the core concepts you need to communicate effectively. From conjugating verbs to understanding tenses, this easy-to-follow guide lets you skip the suffering and score high at exam time. French 101 - get the lowdown on the basics, from expressing dates and times to identifying parts of speech Gender matters - see how a noun's gender determines the articles, adjectives, and pronouns y

  11. Surface chemistry essentials

    CERN Document Server

    Birdi, K S

    2013-01-01

    Surface chemistry plays an important role in everyday life, as the basis for many phenomena as well as technological applications. Common examples range from soap bubbles, foam, and raindrops to cosmetics, paint, adhesives, and pharmaceuticals. Additional areas that rely on surface chemistry include modern nanotechnology, medical diagnostics, and drug delivery. There is extensive literature on this subject, but most chemistry books only devote one or two chapters to it. Surface Chemistry Essentials fills a need for a reference that brings together the fundamental aspects of surface chemistry w

  12. Cisco networking essentials

    CERN Document Server

    McMillan, Troy

    2015-01-01

    Start a career in networking Cisco Networking Essentials, 2nd Edition provides the latest for those beginning a career in networking. This book provides the fundamentals of networking and leads you through the concepts, processes, and skills you need to master fundamental networking concepts. Thinking of taking the CCENT Cisco Certified Entry Networking Technician ICND1 Exam 100-101? This book has you covered! With coverage of important topics and objectives, each chapter outlines main points and provides clear, engaging discussion that will give you a sound understanding of core topics and c

  13. RabbitMQ essentials

    CERN Document Server

    Dossot, David

    2014-01-01

    This book is a quick and concise introduction to RabbitMQ. Follow the unique case study of Clever Coney Media as they progressively discover how to fully utilize RabbitMQ, containing clever examples and detailed explanations.Whether you are someone who develops enterprise messaging products professionally or a hobbyist who is already familiar with open source Message Queuing software and you are looking for a new challenge, then this is the book for you. Although you should be familiar with Java, Ruby, and Python to get the most out of the examples, RabbitMQ Essentials will give you the push y

  14. Advanced methods for a probabilistic safety analysis of fires. Development of advanced methods for performing as far as possible realistic plant specific fire risk analysis (fire PSA)

    International Nuclear Information System (INIS)

    Hofer, E.; Roewekamp, M.; Tuerschmann, M.

    2003-07-01

    In the frame of the research project RS 1112 'Development of Methods for a Recent Probabilistic Safety Analysis, Particularly Level 2' funded by the German Federal Ministry of Economics and Technology (BMWi), advanced methods, in particular for performing as far as possible realistic plant specific fire risk analyses (fire PSA), should be developed. The present Technical Report gives an overview on the methodologies developed in this context for assessing the fire hazard. In the context of developing advanced methodologies for fire PSA, a probabilistic dynamics analysis with a fire simulation code including an uncertainty and sensitivity study has been performed for an exemplary scenario of a cable fire induced by an electric cabinet inside the containment of a modern Konvoi type German nuclear power plant taking into consideration the effects of fire detection and fire extinguishing means. With the present study, it was possible for the first time to determine the probabilities of specified fire effects from a class of fire events by means of probabilistic dynamics supplemented by uncertainty and sensitivity analyses. The analysis applies a deterministic dynamics model, consisting of a dynamic fire simulation code and a model of countermeasures, considering effects of the stochastics (so-called aleatory uncertainties) as well as uncertainties in the state of knowledge (so-called epistemic uncertainties). By this means, probability assessments including uncertainties are provided to be used within the PSA. (orig.) [de

  15. Risk-based technical specifications: Development and application of an approach to the generation of a plant specific real-time risk model

    International Nuclear Information System (INIS)

    Puglia, B.; Gallagher, D.; Amico, P.; Atefi, B.

    1992-10-01

    This report describes a process developed to convert an existing PRA into a model amenable to real time, risk-based technical specification calculations. In earlier studies (culminating in NUREG/CR-5742), several risk-based approaches to technical specification were evaluated. A real-time approach using a plant specific PRA capable of modeling plant configurations as they change was identified as the most comprehensive approach to control plant risk. A master fault tree logic model representative of-all of the core damage sequences was developed. Portions of the system fault trees were modularized and supercomponents comprised of component failures with similar effects were developed to reduce the size of the model and, quantification times. Modifications to the master fault tree logic were made to properly model the effect of maintenance and recovery actions. Fault trees representing several actuation systems not modeled in detail in the existing PRA were added to the master fault tree logic. This process was applied to the Surry NUREG-1150 Level 1 PRA. The master logic mode was confirmed. The model was then used to evaluate frequency associated with several plant configurations using the IRRAS code. For all cases analyzed computational time was less than three minutes. This document Volume 2, contains appendices A, B, and C. These provide, respectively: Surry Technical Specifications Model Database, Surry Technical Specifications Model, and a list of supercomponents used in the Surry Technical Specifications Model

  16. Essential numerical computer methods

    CERN Document Server

    Johnson, Michael L

    2010-01-01

    The use of computers and computational methods has become ubiquitous in biological and biomedical research. During the last 2 decades most basic algorithms have not changed, but what has is the huge increase in computer speed and ease of use, along with the corresponding orders of magnitude decrease in cost. A general perception exists that the only applications of computers and computer methods in biological and biomedical research are either basic statistical analysis or the searching of DNA sequence data bases. While these are important applications they only scratch the surface of the current and potential applications of computers and computer methods in biomedical research. The various chapters within this volume include a wide variety of applications that extend far beyond this limited perception. As part of the Reliable Lab Solutions series, Essential Numerical Computer Methods brings together chapters from volumes 210, 240, 321, 383, 384, 454, and 467 of Methods in Enzymology. These chapters provide ...

  17. DSP Architecture Design Essentials

    CERN Document Server

    Marković, Dejan

    2012-01-01

    In DSP Architecture Design Essentials, authors Dejan Marković and Robert W. Brodersen cover a key subject for the successful realization of DSP algorithms for communications, multimedia, and healthcare applications. The book addresses the need for DSP architecture design that maps advanced DSP algorithms to hardware in the most power- and area-efficient way. The key feature of this text is a design methodology based on a high-level design model that leads to hardware implementation with minimum power and area. The methodology includes algorithm-level considerations such as automated word-length reduction and intrinsic data properties that can be leveraged to reduce hardware complexity. From a high-level data-flow graph model, an architecture exploration methodology based on linear programming is used to create an array of architectural solutions tailored to the underlying hardware technology. The book is supplemented with online material: bibliography, design examples, CAD tutorials and custom software.

  18. Are Archetypes Essential?

    Science.gov (United States)

    Colman, Warren

    2018-06-01

    This paper distinguishes between Jung's theoretical discourse regarding the archetypes and his phenomenological account of numinous experience. For this author, the initial attraction of 'my Jung' came from both the vivid Romanticism of his descriptions of the anima and the apparent 'ground of being' offered by his theory of archetypes. However, the essentialism inherent to archetypal theory in general and the anima in particular has necessitated a re-evaluation of Jung's theory in terms of emergence theory. My own version of this emphasises the role of symbols in the constitution of affect through collective human action in the world. In this reconfiguration, the visceral energy of numinous experience is retained while the problematic theory of archetypes is no longer needed. © 2018, The Society of Analytical Psychology.

  19. Essential French grammar

    CERN Document Server

    Thacker, Mike

    2014-01-01

    Essential French Grammar is an innovative reference grammar and workbook for intermediate and advanced undergraduate students of French (CEFR levels B2 to C1). Its clear explanations of grammar are supported by contemporary examples and lively cartoon drawings.  Each chapter contains: * real-life language examples in French, with English translations * a 'key points' box and tables that summarise grammar concepts * a variety of exercises to reinforce learning * a contemporary primary source or literary extract to illustrate grammar in context. To aid your understanding, this book also contains a glossary of grammatical terms in French and English, useful verb tables and a key to the exercises. Together, these features all help you to grasp complex points of grammar and develop your French language skills.

  20. Essentials of nonlinear optics

    CERN Document Server

    Murti, Y V G S

    2014-01-01

    Current literature on Nonlinear Optics varies widely in terms of content, style, and coverage of specific topics, relative emphasis of areas and the depth of treatment. While most of these books are excellent resources for the researchers, there is a strong need for books appropriate for presenting the subject at the undergraduate or postgraduate levels in Universities. The need for such a book to serve as a textbook at the level of the bachelors and masters courses was felt by the authors while teaching courses on nonlinear optics to students of both science and engineering during the past two decades. This book has emerged from an attempt to address the requirement of presenting the subject at college level. A one-semester course covering the essentials can effectively be designed based on this.

  1. Essential real analysis

    CERN Document Server

    Field, Michael

    2017-01-01

    This book provides a rigorous introduction to the techniques and results of real analysis, metric spaces and multivariate differentiation, suitable for undergraduate courses. Starting from the very foundations of analysis, it offers a complete first course in real analysis, including topics rarely found in such detail in an undergraduate textbook such as the construction of non-analytic smooth functions, applications of the Euler-Maclaurin formula to estimates, and fractal geometry.  Drawing on the author’s extensive teaching and research experience, the exposition is guided by carefully chosen examples and counter-examples, with the emphasis placed on the key ideas underlying the theory. Much of the content is informed by its applicability: Fourier analysis is developed to the point where it can be rigorously applied to partial differential equations or computation, and the theory of metric spaces includes applications to ordinary differential equations and fractals. Essential Real Analysis will appeal t...

  2. TQM: the essential concepts.

    Science.gov (United States)

    Chambers, D W

    1998-01-01

    This is an introduction to the major concepts in total quality management, a loose collection of management approaches that focus on continuous improvement of processes, guided by routine data collection and adjustment of the processes. Customer focus and involvement of all members of an organization are also characteristics commonly found in TQM. The seventy-five-year history of the movement is sketched from its beginning in statistical work on quality assurance through the many improvements and redefinitions added by American and Japanese thinkers. Essential concepts covered include: control cycles, focus on the process rather than the defects, the GEAR model, importance of the customer, upstream quality, just-in-time, kaizen, and service quality.

  3. Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kitagaki, Hiroshi; Wu, Hong; Shimoi, Hitoshi; Ito, Kiyoshi

    2002-11-01

    The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.

  4. Genome-wide survey and expression analysis of the plant-specific NAC transcription factor family in soybean during development and dehydration stress.

    Science.gov (United States)

    Le, Dung Tien; Nishiyama, Rie; Watanabe, Yasuko; Mochida, Keiichi; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo; Tran, Lam-Son Phan

    2011-08-01

    Plant-specific NAC transcription factors (TFs) play important roles in regulating diverse biological processes, including development, senescence, growth, cell division and responses to environmental stress stimuli. Within the soybean genome, we identified 152 full-length GmNAC TFs, including 11 membrane-bound members. In silico analysis of the GmNACs, together with their Arabidopsis and rice counterparts, revealed similar NAC architecture. Next, we explored the soybean Affymetrix array and Illumina transcriptome sequence data to analyse tissue-specific expression profiles of GmNAC genes. Phylogenetic analysis using stress-related NAC TFs from Arabidopsis and rice as seeding sequences identified 58 of the 152 GmNACs as putative stress-responsive genes, including eight previously reported dehydration-responsive GmNACs. We could design gene-specific primers for quantitative real-time PCR verification of 38 out of 50 newly predicted stress-related genes. Twenty-five and six GmNACs were found to be induced and repressed 2-fold or more, respectively, in soybean roots and/or shoots in response to dehydration. GmNAC085, whose amino acid sequence was 39%; identical to that of well-known SNAC1/ONAC2, was the most induced gene upon dehydration, showing 390-fold and 20-fold induction in shoots and roots, respectively. Our systematic analysis has identified excellent tissue-specific and/or dehydration-responsive candidate GmNAC genes for in-depth characterization and future development of improved drought-tolerant transgenic soybeans.

  5. In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

    Directory of Open Access Journals (Sweden)

    Dref C De Moura

    Full Text Available The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L. plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

  6. Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

    Science.gov (United States)

    Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

    2007-05-01

    Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

  7. Rhetoric and Essentially Contested Arguments

    Science.gov (United States)

    Garver, Eugene

    1978-01-01

    Draws a connection between Gallie's essentially contested concepts and Aristotle's account of rhetorical argument by presenting a definition of Essentially Contested Argument which is used as the connecting term between rhetoric and essentially contested concepts and by demonstrating the value of making this connection. (JF)

  8. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

    Science.gov (United States)

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-01-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  9. Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin*

    Science.gov (United States)

    Stoeckius, Marlon; Erat, Anna; Fujikawa, Tatsuya; Hiromura, Makoto; Koulova, Anna; Otterbein, Leo; Bianchi, Cesario; Tobiasch, Edda; Dagon, Yossi; Sellke, Frank W.; Usheva, Anny

    2012-01-01

    The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition. PMID:22262839

  10. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

  11. Exploring the relationship between fractal features and bacterial essential genes

    International Nuclear Information System (INIS)

    Yu Yong-Ming; Yang Li-Cai; Zhao Lu-Lu; Liu Zhi-Ping; Zhou Qian

    2016-01-01

    Essential genes are indispensable for the survival of an organism in optimal conditions. Rapid and accurate identifications of new essential genes are of great theoretical and practical significance. Exploring features with predictive power is fundamental for this. Here, we calculate six fractal features from primary gene and protein sequences and then explore their relationship with gene essentiality by statistical analysis and machine learning-based methods. The models are applied to all the currently available identified genes in 27 bacteria from the database of essential genes (DEG). It is found that the fractal features of essential genes generally differ from those of non-essential genes. The fractal features are used to ascertain the parameters of two machine learning classifiers: Naïve Bayes and Random Forest. The area under the curve (AUC) of both classifiers show that each fractal feature is satisfactorily discriminative between essential genes and non-essential genes individually. And, although significant correlations exist among fractal features, gene essentiality can also be reliably predicted by various combinations of them. Thus, the fractal features analyzed in our study can be used not only to construct a good essentiality classifier alone, but also to be significant contributors for computational tools identifying essential genes. (paper)

  12. An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells.

    Science.gov (United States)

    Naegelen, Isabelle; Plançon, Sébastien; Nicot, Nathalie; Kaoma, Tony; Muller, Arnaud; Vallar, Laurent; Tschirhart, Eric J; Bréchard, Sabrina

    2015-03-01

    Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. © Society for Leukocyte Biology.

  13. Essential oils and anxiolytic aromatherapy.

    Science.gov (United States)

    Setzer, William N

    2009-09-01

    A number of essential oils are currently in use as aromatherapy agents to relieve anxiety, stress, and depression. Popular anxiolytic oils include lavender (Lavandula angustifolia), rose (Rosa damascena), orange (Citrus sinensis), bergamot (Citrus aurantium), lemon (Citrus limon), sandalwood (Santalum album), clary sage (Salvia sclarea), Roman chamomile (Anthemis nobilis), and rose-scented geranium (Pelargonium spp.). This review discusses the chemical constituents and CNS effects of these aromatherapeutic essential oils, as well as recent studies on additional essential oils with anxiolytic activities.

  14. Essential Oils and Antifungal Activity

    Science.gov (United States)

    Coppola, Raffaele; De Feo, Vincenzo

    2017-01-01

    Since ancient times, folk medicine and agro-food science have benefitted from the use of plant derivatives, such as essential oils, to combat different diseases, as well as to preserve food. In Nature, essential oils play a fundamental role in protecting the plant from biotic and abiotic attacks to which it may be subjected. Many researchers have analyzed in detail the modes of action of essential oils and most of their components. The purpose of this brief review is to describe the properties of essential oils, principally as antifungal agents, and their role in blocking cell communication mechanisms, fungal biofilm formation, and mycotoxin production. PMID:29099084

  15. Yeast Interacting Proteins Database: YNL152W, YMR032W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YNL152W INN1 Essential protein that associates with the contractile actomyosin ring... Bait description Essential protein that associates with the contractile actomyosin ring, required for ingre

  16. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-04

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  17. Characterization equipment essential drawing plan

    International Nuclear Information System (INIS)

    WILSON, G.W.

    1999-01-01

    The purpose of this document is to list the Characterization equipment drawings that are classified as Essential Drawings. Essential Drawings: Are those drawings identified by the facility staff as necessary to directly support the safe operation of the facility or equipment (HNF 1997a). The Characterization equipment drawings identified in this report are deemed essential drawings as defined in HNF-PRO-242, Engineering Drawing Requirements (HNF 1997a). These drawings will be prepared, revised, and maintained per HNF-PRO-440, Engineering Document Change Control (HNF 1997b). All other Characterization equipment drawings not identified in this document will be considered Support drawings until the Characterization Equipment Drawing Evaluation Report is completed

  18. Ubiquitin domain proteins in disease

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

  19. Essential idempotents and simplex codes

    Directory of Open Access Journals (Sweden)

    Gladys Chalom

    2017-01-01

    Full Text Available We define essential idempotents in group algebras and use them to prove that every mininmal abelian non-cyclic code is a repetition code. Also we use them to prove that every minimal abelian code is equivalent to a minimal cyclic code of the same length. Finally, we show that a binary cyclic code is simplex if and only if is of length of the form $n=2^k-1$ and is generated by an essential idempotent.

  20. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  1. Step-wise and lineage-specific diversification of plant RNA polymerase genes and origin of the largest plant-specific subunits.

    Science.gov (United States)

    Wang, Yaqiong; Ma, Hong

    2015-09-01

    Proteins often function as complexes, yet little is known about the evolution of dissimilar subunits of complexes. DNA-directed RNA polymerases (RNAPs) are multisubunit complexes, with distinct eukaryotic types for different classes of transcripts. In addition to Pol I-III, common in eukaryotes, plants have Pol IV and V for epigenetic regulation. Some RNAP subunits are specific to one type, whereas other subunits are shared by multiple types. We have conducted extensive phylogenetic and sequence analyses, and have placed RNAP gene duplication events in land plant history, thereby reconstructing the subunit compositions of the novel RNAPs during land plant evolution. We found that Pol IV/V have experienced step-wise duplication and diversification of various subunits, with increasingly distinctive subunit compositions. Also, lineage-specific duplications have further increased RNAP complexity with distinct copies in different plant families and varying divergence for subunits of different RNAPs. Further, the largest subunits of Pol IV/V probably originated from a gene fusion in the ancestral land plants. We propose a framework of plant RNAP evolution, providing an excellent model for protein complex evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  2. The nucleobase cation symporter 1 of Chlamydomonas reinhardtii and that of the evolutionarily distant Arabidopsis thaliana display parallel function and establish a plant-specific solute transport profile.

    Science.gov (United States)

    Schein, Jessica R; Hunt, Kevin A; Minton, Janet A; Schultes, Neil P; Mourad, George S

    2013-09-01

    The single cell alga Chlamydomonas reinhardtii is capable of importing purines as nitrogen sources. An analysis of the annotated C. reinhardtii genome reveals at least three distinct gene families encoding for known nucleobase transporters. In this study the solute transport and binding properties for the lone C. reinhardtii nucleobase cation symporter 1 (CrNCS1) are determined through heterologous expression in Saccharomyces cerevisiae. CrNCS1 acts as a transporter of adenine, guanine, uracil and allantoin, sharing similar - but not identical - solute recognition specificity with the evolutionary distant NCS1 from Arabidopsis thaliana. The results suggest that the solute specificity for plant NCS1 occurred early in plant evolution and are distinct from solute transport specificities of single cell fungal NCS1 proteins. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Levels of essential and non-essential elements in commercially ...

    African Journals Online (AJOL)

    The focus of this study was to assess the quality of commercially packaged moringa tea brands available in the retail markets in Nigerian cities on the basis of the essential and nonessential elemental content in their tissues. Four different brands of moringa tea comprising two locally processed teas and two imported teas ...

  4. Levels of Essential and Non-Essential Elements in Commercially ...

    African Journals Online (AJOL)

    Enebi Jasper

    INTRODUCTION. Plants have been ... plant metabolism and biosynthesis and act as cofactors for ... plant body. 3 . Some metals are essential nutrients (zinc, iron, copper, and chromium), ... non-destructive analysis, increased total speed, ... oleifera play both a curative and preventive ... maintenance of cardiac rhythm. 16.

  5. Essentials of Career Interest Assessment. Essentials of Psychological Assessment Series.

    Science.gov (United States)

    Prince, Jeffrey P.; Heiser, Lisa J.

    This book is a quick reference source to guide the career professional through the essentials of using the most popular career interest tools. It summarizes important technical aspects of each inventory, and offers step-by-step guidance in the interpretation and use of the various inventories. The chapters are: (1) "Overview"; (2)…

  6. Identification of RNA binding motif proteins essential for cardiovascular development

    NARCIS (Netherlands)

    S. Maragh (Samantha); R.A. Miller (Ronald); S.L. Bessling (Seneca); D.M. McGaughey (David); M.W. Wessels (Marja); B.M. de Graaf (Bianca); E.A. Stone (Eric); A.M. Bertoli Avella (Aida); J.D. Gearhart (John); S. Fisher (Shannon); A.S. McCallion (Andrew)

    2011-01-01

    textabstractBackground: We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis

  7. Identification of RNA binding motif proteins essential for cardiovascular development

    Directory of Open Access Journals (Sweden)

    Bertoli-Avella Aida M

    2011-10-01

    Full Text Available Abstract Background We recently identified Rbm24 as a novel gene expressed during mouse cardiac development. Due to its tightly restricted and persistent expression from formation of the cardiac crescent onwards and later in forming vasculature we posited it to be a key player in cardiogenesis with additional roles in vasculogenesis and angiogenesis. Results To determine the role of this gene in cardiac development, we have identified its zebrafish orthologs (rbm24a and rbm24b, and functionally evaluated them during zebrafish embryogenesis. Consistent with our underlying hypothesis, reduction in expression of either ortholog through injection of morpholino antisense oligonucleotides results in cardiogenic defects including cardiac looping and reduced circulation, leading to increasing pericardial edema over time. Additionally, morphant embryos for either ortholog display incompletely overlapping defects in the forming vasculature of the dorsal aorta (DA, posterior caudal vein (PCV and caudal vein (CV which are the first blood vessels to form in the embryo. Vasculogenesis and early angiogenesis in the trunk were similarly compromised in rbm24 morphant embryos at 48 hours post fertilization (hpf. Subsequent vascular maintenance was impaired in both rbm24 morphants with substantial vessel degradation noted at 72 hpf. Conclusion Taken collectively, our functional data support the hypothesis that rbm24a and rbm24b are key developmental cardiac genes with unequal roles in cardiovascular formation.

  8. Trypanosome Letm1 protein is essential for mitochondrial potassium homeostasis

    Czech Academy of Sciences Publication Activity Database

    Hashimi, Hassan; McDonald, Lindsay M.; Stříbrná, Eva; Lukeš, Julius

    2013-01-01

    Roč. 288, č. 37 (2013), s. 26914-26925 ISSN 0021-9258 R&D Projects: GA ČR GAP305/12/2261 Institutional support: RVO:60077344 Keywords : Bioenergetics * Letm1 * Mitochondria * Potassium Transport * Translation * Trypanosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.600, year: 2013

  9. Essential Medicines in a High Income Country: Essential to Whom?

    Science.gov (United States)

    Duong, Mai; Moles, Rebekah J; Chaar, Betty; Chen, Timothy F

    2015-01-01

    To explore the perspectives of a diverse group of stakeholders engaged in medicines decision making around what constitutes an "essential" medicine, and how the Essential Medicines List (EML) concept functions in a high income country context. In-depth qualitative semi-structured interviews were conducted with 32 Australian stakeholders, recognised as decision makers, leaders or advisors in the area of medicines reimbursement or supply chain management. Participants were recruited from government, pharmaceutical industry, pharmaceutical wholesale/distribution companies, medicines non-profit organisations, academic health disciplines, hospitals, and consumer groups. Perspectives on the definition and application of the EML concept in a high income country context were thematically analysed using grounded theory approach. Stakeholders found it challenging to describe the EML concept in the Australian context because many perceived it was generally used in resource scarce settings. Stakeholders were unable to distinguish whether nationally reimbursed medicines were essential medicines in Australia. Despite frequent generic drug shortages and high prices paid by consumers, many struggled to describe how the EML concept applied to Australia. Instead, broad inclusion of consumer needs, such as rare and high cost medicines, and consumer involvement in the decision making process, has led to expansive lists of nationally subsidised medicines. Therefore, improved communication and coordination is needed around shared interests between stakeholders regarding how medicines are prioritised and guaranteed in the supply chain. This study showed that decision-making in Australia around reimbursement of medicines has strayed from the fundamental utilitarian concept of essential medicines. Many stakeholders involved in medicine reimbursement decisions and management of the supply chain did not consider the EML concept in their approach. The wide range of views of what stakeholders

  10. Essential Medicines in a High Income Country: Essential to Whom?

    Directory of Open Access Journals (Sweden)

    Mai Duong

    Full Text Available To explore the perspectives of a diverse group of stakeholders engaged in medicines decision making around what constitutes an "essential" medicine, and how the Essential Medicines List (EML concept functions in a high income country context.In-depth qualitative semi-structured interviews were conducted with 32 Australian stakeholders, recognised as decision makers, leaders or advisors in the area of medicines reimbursement or supply chain management. Participants were recruited from government, pharmaceutical industry, pharmaceutical wholesale/distribution companies, medicines non-profit organisations, academic health disciplines, hospitals, and consumer groups. Perspectives on the definition and application of the EML concept in a high income country context were thematically analysed using grounded theory approach.Stakeholders found it challenging to describe the EML concept in the Australian context because many perceived it was generally used in resource scarce settings. Stakeholders were unable to distinguish whether nationally reimbursed medicines were essential medicines in Australia. Despite frequent generic drug shortages and high prices paid by consumers, many struggled to describe how the EML concept applied to Australia. Instead, broad inclusion of consumer needs, such as rare and high cost medicines, and consumer involvement in the decision making process, has led to expansive lists of nationally subsidised medicines. Therefore, improved communication and coordination is needed around shared interests between stakeholders regarding how medicines are prioritised and guaranteed in the supply chain.This study showed that decision-making in Australia around reimbursement of medicines has strayed from the fundamental utilitarian concept of essential medicines. Many stakeholders involved in medicine reimbursement decisions and management of the supply chain did not consider the EML concept in their approach. The wide range of views of

  11. Tank farms essential drawing plan

    International Nuclear Information System (INIS)

    Domnoske-Rauch, L.A.

    1998-01-01

    The purpose of this document is to define criteria for selecting Essential Drawings, Support Drawings, and Controlled Print File (CPF) drawings and documents for facilities that are part of East and West Tank Farms. Also, the drawings and documents that meet the criteria are compiled separate listings. The Essential Drawing list and the Support Drawing list establish a priority for updating technical baseline drawings. The CPF drawings, denoted by an asterisk (*), defined the drawings and documents that Operations is required to maintain per the TWRS Administration Manual. The Routing Boards in Buildings 272-WA and 272-AW are not part of the CPF

  12. Analgesic Potential of Essential Oils

    Directory of Open Access Journals (Sweden)

    José Ferreira Sarmento-Neto

    2015-12-01

    Full Text Available Pain is an unpleasant sensation associated with a wide range of injuries and diseases, and affects approximately 20% of adults in the world. The discovery of new and more effective drugs that can relieve pain is an important research goal in both the pharmaceutical industry and academia. This review describes studies involving antinociceptive activity of essential oils from 31 plant species. Botanical aspects of aromatic plants, mechanisms of action in pain models and chemical composition profiles of the essential oils are discussed. The data obtained in these studies demonstrate the analgesic potential of this group of natural products for therapeutic purposes.

  13. Drosophila Protein interaction Map (DPiM)

    OpenAIRE

    Guruharsha, K.G.; Obar, Robert A.; Mintseris, Julian; Aishwarya, K.; Krishnan, R.T.; VijayRaghavan, K.; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when,...

  14. Accumulative capabilities of essential nutrient elements in organs of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... Cu > Mn > Zn > B. In conclusion, the ACs for essential nutrient elements differed, with the ... and Fe were quantified using Perkin Elmer Atomic Absorption .... vitamin C, protein and fat ranging from 249.6 to 266 .... Boron determination in plant tissues by ... Accumulation of cadmium and selected elements.

  15. Essential Medicines in National Constitutions

    Science.gov (United States)

    Toebes, Brigit; Hogerzeil, Hans

    2016-01-01

    Abstract A constitutional guarantee of access to essential medicines has been identified as an important indicator of government commitment to the progressive realization of the right to the highest attainable standard of health. The objective of this study was to evaluate provisions on access to essential medicines in national constitutions, to identify comprehensive examples of constitutional text on medicines that can be used as a model for other countries, and to evaluate the evolution of constitutional medicines-related rights since 2008. Relevant articles were selected from an inventory of constitutional texts from WHO member states. References to states’ legal obligations under international human rights law were evaluated. Twenty-two constitutions worldwide now oblige governments to protect and/or to fulfill accessibility of, availability of, and/or quality of medicines. Since 2008, state responsibilities to fulfill access to essential medicines have expanded in five constitutions, been maintained in four constitutions, and have regressed in one constitution. Government commitments to essential medicines are an important foundation of health system equity and are included increasingly in state constitutions. PMID:27781006

  16. Intellectual Workers and Essential Freedoms.

    Science.gov (United States)

    Edley, Christopher Jr.

    2000-01-01

    Journalists and college professors deserve certain privileges, including freedom of speech and academic freedom, but they must adapt their work to increasingly diverse populations. They must confront public mistrust, convince people that these freedoms are worthwhile, and protect essential public rights through what they study and teach and to…

  17. Essential Fluid Dynamics for Scientists

    Science.gov (United States)

    Braithwaite, Jonathan

    2017-12-01

    The book is an introduction to the subject of fluid mechanics, essential for students and researchers in many branches of science. It illustrates its fundamental principles with a variety of examples drawn mainly from astrophysics and geophysics as well as from everyday experience. Prior familiarity with basic thermodynamics and vector calculus is assumed.

  18. Essential oils in broiler nutrition

    NARCIS (Netherlands)

    Lee, Kyung-woo

    2002-01-01

    Dietary antibiotics at low, subtherapeutic levels have been shown to improve growth performance in farm animals. However, there is a trend to look for alternatives to dietary antibiotics, due to occurrence of antibiotic-resistance bacteria. The present thesis explored the essential oils as the

  19. Essentials of supply chain management

    CERN Document Server

    Hugos, Michael H

    2011-01-01

    The latest thinking, strategies, developments, and technologies to stay current in supply chain management Presenting the core concepts and techniques of supply chain management in a clear, concise and easily readable style, the Third Edition of Essentials of Supply Chain Management outlines the most crucial tenets and concepts of supply chain management.

  20. Essentials for the Teacher's Toolbox

    Science.gov (United States)

    Uhler, Jennifer

    2012-01-01

    Every profession has a set of essential tools for carrying out its work. Airplane mechanics cannot repair engines without sophisticated diagnostics, wrenches, and pliers. Surgeons cannot operate without scalpels and clamps. In contrast, teaching has often been perceived as a profession requiring only students, chalk, and a blackboard in order for…

  1. Diversity - Essential quality for agronomy

    Science.gov (United States)

    Diversity is an essential quality for success in agronomy because agronomy touches everyone, everywhere in some way, and food production is crucial for every society around the world. Consider, for example, our membership, which is diverse in almost every way imaginable. This diversity brings differ...

  2. Hyper-V Replica essentials

    CERN Document Server

    Krstevski, Vangel

    2013-01-01

    a in various deployment scenarios.Hyper-V Replica Essentials is for Windows Server administrators who want to improve their system availability and speed up disaster recovery. You will need experience in Hyper-V deployment because Hyper-V Replica is built in the Hyper-V platform.

  3. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  4. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  5. Autophagy is essential for hearing in mice.

    Science.gov (United States)

    Fujimoto, Chisato; Iwasaki, Shinichi; Urata, Shinji; Morishita, Hideaki; Sakamaki, Yuriko; Fujioka, Masato; Kondo, Kenji; Mizushima, Noboru; Yamasoba, Tatsuya

    2017-05-11

    Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.

  6. Kif4 Is Essential for Mouse Oocyte Meiosis.

    Science.gov (United States)

    Camlin, Nicole J; McLaughlin, Eileen A; Holt, Janet E

    2017-01-01

    Progression through the meiotic cell cycle must be strictly regulated in oocytes to generate viable embryos and offspring. During mitosis, the kinesin motor protein Kif4 is indispensable for chromosome condensation and separation, midzone formation and cytokinesis. Additionally, the bioactivity of Kif4 is dependent on phosphorylation via Aurora Kinase B and Cdk1, which regulate Kif4 function throughout mitosis. Here, we examine the role of Kif4 in mammalian oocyte meiosis. Kif4 localized in the cytoplasm throughout meiosis I and II, but was also observed to have a dynamic subcellular distribution, associating with both microtubules and kinetochores at different stages of development. Co-localization and proximity ligation assays revealed that the kinetochore proteins, CENP-C and Ndc80, are potential Kif4 interacting proteins. Functional analysis of Kif4 in oocytes via antisense knock-down demonstrated that this protein was not essential for meiosis I completion. However, Kif4 depleted oocytes displayed enlarged polar bodies and abnormal metaphase II spindles, indicating an essential role for this protein for correct asymmetric cell division in meiosis I. Further investigation of the phosphoregulation of meiotic Kif4 revealed that Aurora Kinase and Cdk activity is critical for Kif4 kinetochore localization and interaction with Ndc80 and CENP-C. Finally, Kif4 protein but not gene expression was found to be upregulated with age, suggesting a role for this protein in the decline of oocyte quality with age.

  7. USP7 is a SUMO deubiquitinase essential for DNA replication

    DEFF Research Database (Denmark)

    Lecona, Emilio; Rodriguez-Acebes, Sara; Specks, Julia

    2016-01-01

    Post-translational modification of proteins by ubiquitin (Ub) and Ub-like modifiers regulates DNA replication. We have previously shown that chromatin around replisomes is rich in SUMO and poor in Ub, whereas mature chromatin exhibits an opposite pattern. How this SUMO-rich, Ub-poor environment...... is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...... to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7...

  8. Essentials of radiation heat transfer

    CERN Document Server

    Balaji

    2014-01-01

    Essentials of Radiation Heat Transfer is a textbook presenting the essential, fundamental information required to gain an understanding of radiation heat transfer and equips the reader with enough knowledge to be able to tackle more challenging problems. All concepts are reinforced by carefully chosen and fully worked examples, and exercise problems are provided at the end of every chapter. In a significant departure from other books on this subject, this book completely dispenses with the network method to solve problems of radiation heat transfer in surfaces. It instead presents the powerful radiosity-irradiation method and shows how this technique can be used to solve problems of radiation in enclosures made of one to any number of surfaces. The network method is not easily scalable. Secondly, the book introduces atmospheric radiation, which is now being considered as a potentially important area, in which engineers can contribute to the technology of remote sensing and atmospheric sciences in general, b...

  9. Communication: essential strategies for success.

    Science.gov (United States)

    O'Connor, Mary

    2013-06-01

    This department highlights change management strategies that may be successful in strategically planning and executing organizational change initiatives. With the goal of presenting practical approaches helpful to nurse leaders advance organizational change, content includes evidence-based projects, tool, and resources that mobilize and sustain organizational change initiatives. In this article, the author discusses strategies for communication for change processes, whether large or small. Intentional planning and development of a communication strategy alongside, not as an afterthought, to change initiatives are essential.

  10. Essentials of nuclear medicine imaging

    CERN Document Server

    Mettler, Fred A. Jr

    2012-01-01

    Essentials of Nuclear Medicine Imaging, by Drs. Fred A Mettler and Milton J Guiberteau, provides the practical and comprehensive guidance you need to master key nuclear imaging techniques. From physics, instrumentation, quality control, and legal requirements to hot topics such as sodium fluoride, radiopharmaceuticals, and recommended pediatric administered doses and guidelines, this sixth edition covers the fundamentals and recent developments in the practice of nuclear medicine.

  11. Essential themes in Personnel economics

    OpenAIRE

    Josheski, Dushko

    2014-01-01

    In this paper are presented essential themes in the subject of personnel economics. In the first part analysis has been conducted on the impact of peer pressure on workplace behaviour. Then again models for compensation structures within firms, and their influence on the utility of work by employees. In the final section of the paper the productivity spillover effect has been analyzed, and the causes of existence of spillovers and their impact on workers’ productivity

  12. Essentials of natural gas microturbines

    CERN Document Server

    Boicea, Valentin A

    2013-01-01

    Addressing a field which, until now, has not been sufficiently investigated, Essentials of Natural Gas Microturbines thoroughly examines several natural gas microturbine technologies suitable not only for distributed generation but also for the automotive industry. An invaluable resource for power systems, electrical, and computer science engineers as well as operations researchers, microturbine operators, policy makers, and other industry professionals, the book: Explains the importance of natural gas microturbines and their use in distributed energy resource (DER) systemsDiscusses the histor

  13. Repelling mosquitoes with essential oils

    Science.gov (United States)

    Riley, L.

    2017-12-01

    Mosquitoes carry diseases than can lead to serious illness and death. According to the World Health Organization, mosquitoes infect over 300 million people a year with Malaria and Dengue Fever, two life threatening diseases vectored by mosquitoes. Although insecticides are the most effective way to control mosquitoes, they are not always environmentally friendly. Therefore, alternative tactics should be considered. In this study, we looked at the repellency of various essential oils on female Aedes aegypti through a series of laboratory assays.

  14. Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization

    DEFF Research Database (Denmark)

    Greve, Krestine; Bravo Munoz, Tanja La Cour; Jensen, Michael Krogh

    2003-01-01

    RING miduler, NAC transkriptionsfatorer, abscisinsyre inducerbar, proteinnedbrydning, subcellulær lo......RING miduler, NAC transkriptionsfatorer, abscisinsyre inducerbar, proteinnedbrydning, subcellulær lo...

  15. Cyber essentials a pocket guide

    CERN Document Server

    Calder, Alan

    2014-01-01

    Every year, thousands of computer systems in the UK are compromised. The majority fall victim to easily preventable cyber attacks, carried out with tools which are freely available on the Internet.   Cyber Essentials is the UK Government's reaction to the proliferation of these attacks. It requires that organisations put basic security measures in place, enabling them to reliably counter the most common tactics employed by cyber criminals. From 1 October 2014, all suppliers bidding for a range of government ICT contracts - in particu

  16. Microsoft Windows Operating System Essentials

    CERN Document Server

    Carpenter, Tom

    2012-01-01

    A full-color guide to key Windows 7 administration concepts and topics Windows 7 is the leading desktop software, yet it can be a difficult concept to grasp, especially for those new to the field of IT. Microsoft Windows Operating System Essentials is an ideal resource for anyone new to computer administration and looking for a career in computers. Delving into areas such as fundamental Windows 7 administration concepts and various desktop OS topics, this full-color book addresses the skills necessary for individuals looking to break into a career in IT. Each chapter begins with a list of topi

  17. Axure RP 6 Prototyping Essentials

    CERN Document Server

    Schwartz, Ezra

    2012-01-01

    Axure RP 6 Prototyping Essentials is a detailed, practical primer on the leading rapid prototyping tool. Short on jargon and high on concepts, real-life scenarios and step-by-step guidance through hands-on examples, this book will show you how to integrate Axure into your UX workflow. This book is written for UX practitioners, business analysts, product managers, and anyone else who is involved in UX projects. The book assumes that you have no or very little familiarity with Axure. It will help you if you are evaluating the tool for an upcoming project or are required to quickly get up to spee

  18. Linear feedback controls the essentials

    CERN Document Server

    Haidekker, Mark A

    2013-01-01

    The design of control systems is at the very core of engineering. Feedback controls are ubiquitous, ranging from simple room thermostats to airplane engine control. Helping to make sense of this wide-ranging field, this book provides a new approach by keeping a tight focus on the essentials with a limited, yet consistent set of examples. Analysis and design methods are explained in terms of theory and practice. The book covers classical, linear feedback controls, and linear approximations are used when needed. In parallel, the book covers time-discrete (digital) control systems and juxtapos

  19. Overview of the essential tools

    International Nuclear Information System (INIS)

    Mount, R.P.

    1989-01-01

    The essential tools needed for the analysis of HEP experiments are software, networking, data handling, and CPU power. Leaving most software topics to other papers, this paper describes how we can use today's hardware effectively for HEP data analysis. It describes a new but viable model for HEP computing and data handling on a single site, and outlines the challenge of extending this environment to the wider university-based community. Finally it touches on the revolution in software design that may be triggered by the revolution in computing technology. (orig.)

  20. Essentials of Electromagnetics for Engineering

    Science.gov (United States)

    de Wolf, David A.

    2000-11-01

    Essentials of Electromagnetics for Engineering introduces the key physical and engineering principles of electromagnetics. Throughout the book, David de Wolf describes the intermediate steps in mathematical derivations that many other textbooks leave out. He covers in depth the concepts of fields and potentials and then progresses to magnetostatics, Maxwell's equations, electrodynamics and wave propagation, waveguides, transmission lines, and antennas. At each stage, de Wolf stresses the physical principles underlying the mathematical results. He also includes homework exercises, a separate chapter on numerical methods in electromagnetics, and a broad range of worked examples to illustrate important concepts. Solutions manual available.

  1. Redefining the essential trafficking pathway for outer membrane lipoproteins

    OpenAIRE

    Grabowicz, Marcin; Silhavy, Thomas J.

    2017-01-01

    In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one...

  2. Catalogue of requirements for a plant-specific safety inspection of German nuclear power plants taking into account the Fukushima-I (Japan) events; Anforderungskatalog fuer anlagenbezogene Ueberpruefungen deutscher Kernkraftwerke unter Beruecksichtigung der Ereignisse in Fukushima-I (Japan)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2011-03-30

    The catalogue of requirements for a plant-specific safety inspection of German nuclear power plants taking into account the Fukushima-I (Japan) events worked out by the German RSK (reactor safety commission) includes the following inspection topics: natural events like earth quakes, floods, weather-based consequences and possible superposition; civilization-based events like airplane crash, gas release, reactor accident consequences for neighboring units, terroristic impacts, external attacks on computer-based control systems. Further event-independent assumptions have to be considered: station blackout, long-term emergency power supply requirement, failure of auxiliary cooling water supply, efficacy of preventive measures, aggravating boundary conditions for the performance of emergency measures.

  3. Emerging treatments for essential thrombocythemia

    Directory of Open Access Journals (Sweden)

    Okoli S

    2011-12-01

    Full Text Available Steven Okoli, Claire HarrisonDepartment of Haematology, Guy's and St Thomas' NHS Foundation Trust, Great Maze Pond, London, UKAbstract: In 1934, Epstein and Goedel used the term hemorrhagic thrombocythemia to describe a disorder characterized by permanent elevation of a platelet count to more than three times normal, hyperplasia of megakaryocytes, and the tendency for venous thrombosis and spontaneous hemorrhage. Over the last 75 years, and particularly in the past 6 years, major progress has been made in our understanding of essential thrombocythemia (ET and its pathogenesis with the identification of the highly prevalent JAK-2 V617F and other mutations. Current management of this condition is based upon historical data and with treatments that have not changed significantly for nearly two decades. This study discusses this and recent progress, highlighting exciting new data with old and new drugs, as well as which patients in particular should be evaluated for these new therapies.Keywords: essential thrombocythemia, interferon, JAK inhibitor

  4. Economic demand and essential value.

    Science.gov (United States)

    Hursh, Steven R; Silberberg, Alan

    2008-01-01

    The strength of a rat's eating reflex correlates with hunger level when strength is measured by the response frequency that precedes eating (B. F. Skinner, 1932a, 1932b). On the basis of this finding, Skinner argued response frequency could index reflex strength. Subsequent work documented difficulties with this notion because responding was affected not only by the strengthening properties of the reinforcer but also by the rate-shaping effects of the schedule. This article obviates this problem by measuring strength via methods from behavioral economics. This approach uses demand curves to map how reinforcer consumption changes with changes in the "price" different ratio schedules impose. An exponential equation is used to model these demand curves. The value of this exponential's rate constant is used to scale the strength or essential value of a reinforcer, independent of the scalar dimensions of the reinforcer. Essential value determines the consumption level to be expected at particular prices and the response level that will occur to support that consumption. This approach permits comparing reinforcers that differ in kind, contributing toward the goal of scaling reinforcer value. (c) 2008 APA, all rights reserved

  5. Essential Tremor: A Neurodegenerative Disease?

    Directory of Open Access Journals (Sweden)

    Julian Benito-Leon

    2014-07-01

    Full Text Available Background: Essential tremor (ET is one of the most common neurological disorders among adults, and is the most common of the many tremor disorders. It has classically been viewed as a benign monosymptomatic condition, yet over the past decade, a growing body of evidence indicates that ET is a progressive condition that is clinically heterogeneous, as it may be associated with a spectrum of clinical features, with both motor and non‐motor elements. In this review, I will describe the most significant emerging milestones in research which, when taken together, suggest that ET is a neurodegenerative condition.Methods: A PubMed search conducted in June 2014 crossing the terms “essential tremor” (ET and “neurodegenerative” yielded 122 entries, 20 of which included the term “neurodegenerative” in the article title. This was supplemented by articles in the author's files that pertained to this topic.Results/Discussion: There is an open and active dialogue in the medical community as to whether ET is a neurodegenerative disease, with considerable evidence in favor of this. Specifically, ET is a progressive disorder of aging associated with neuronal loss (reduction in Purkinje cells as well as other post‐mortem changes that occur in traditional neurodegenerative disorders. Along with this, advanced neuroimaging techniques are now demonstrating distinct structural changes, several of which are consistent with neuronal loss, in patients with ET. However, further longitudinal clinical and neuroimaging longitudinal studies to assess progression are required.

  6. Combining Shigella Tn-seq data with gold-standard E. coli gene deletion data suggests rare transitions between essential and non-essential gene functionality.

    Science.gov (United States)

    Freed, Nikki E; Bumann, Dirk; Silander, Olin K

    2016-09-06

    Gene essentiality - whether or not a gene is necessary for cell growth - is a fundamental component of gene function. It is not well established how quickly gene essentiality can change, as few studies have compared empirical measures of essentiality between closely related organisms. Here we present the results of a Tn-seq experiment designed to detect essential protein coding genes in the bacterial pathogen Shigella flexneri 2a 2457T on a genome-wide scale. Superficial analysis of this data suggested that 481 protein-coding genes in this Shigella strain are critical for robust cellular growth on rich media. Comparison of this set of genes with a gold-standard data set of essential genes in the closely related Escherichia coli K12 BW25113 revealed that an excessive number of genes appeared essential in Shigella but non-essential in E. coli. Importantly, and in converse to this comparison, we found no genes that were essential in E. coli and non-essential in Shigella, implying that many genes were artefactually inferred as essential in Shigella. Controlling for such artefacts resulted in a much smaller set of discrepant genes. Among these, we identified three sets of functionally related genes, two of which have previously been implicated as critical for Shigella growth, but which are dispensable for E. coli growth. The data presented here highlight the small number of protein coding genes for which we have strong evidence that their essentiality status differs between the closely related bacterial taxa E. coli and Shigella. A set of genes involved in acetate utilization provides a canonical example. These results leave open the possibility of developing strain-specific antibiotic treatments targeting such differentially essential genes, but suggest that such opportunities may be rare in closely related bacteria.

  7. HCVpro: Hepatitis C virus protein interaction database

    KAUST Repository

    Kwofie, Samuel K.; Schaefer, Ulf; Sundararajan, Vijayaraghava Seshadri; Bajic, Vladimir B.; Christoffels, Alan G.

    2011-01-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers

  8. Molecular Basis of Essential Thrombocytosis

    Science.gov (United States)

    2008-06-01

    by a clinically visible journal [Ref. 11]. IX. INDIVIDUALS RECEIVING PAY Jean Wainer, BS Research Support Specialist © 2005 Schattauer GmbH...33 More recently, several techniques have been devised to directly compare cellular proteomes, including isotope -coded affinity tags (ICATs)34 and...derived tryptic peptides with oxygen -18, have been used to study the differential expression of platelet proteins.40 Limitations of proteomic techniques

  9. Protein - Which is Best?

    Science.gov (United States)

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  10. Essential AOP: The A Calculus

    DEFF Research Database (Denmark)

    De Fraine, Bruno; Ernst, Erik; Südholt, Mario

    2012-01-01

    Aspect-oriented programming (AOP) has produced interesting language designs, but also ad hoc semantics that needs clarification. We contribute to this clarification with a calculus that models essential AOP, both simpler and more general than existing formalizations. In AOP, advice may intercept......-oriented code. Two well-known pointcut categories, call and execution, are commonly considered similar.We formally expose their differences, and resolve the associated soundness problem. Our calculus includes type ranges, an intuitive and concise alternative to explicit type variables that allows advice...... to be polymorphic over intercepted methods. We use calculus parameters to cover type safety for a wide design space of other features. Type soundness is verified in Coq....

  11. The PSI family of nuclear proteins is required for growth in arabidopsis.

    Science.gov (United States)

    Stührwohldt, Nils; Hartmann, Jens; Dahlke, Renate I; Oecking, Claudia; Sauter, Margret

    2014-10-01

    PSI1 was identified as a gene that is co-expressed with the phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 in Arabidopsis thaliana. It represents a plant-specific protein family of unknown function with six members in two clades. Clade 1 members PSI1, PSI2 and PSI3 were characterized in this study. All three are nuclear localized. A predicted N-terminal myristoylation site was functionally analyzed. psi1-1 seedlings have shorter roots and hypocotyls. This growth-retarded phenotype was restored by expression of either wildtype PSI1 or PSI1 G2A with a mutated myristate attachment site in the psi1-1 background suggesting that myristate attachment was not essential for PSI1 function. psi2-1 and psi3-1 seedlings have a wildtype phenotype but overexpression of PSI1 or PSI2 promoted seedling growth. PSI2 activity appears to be linked to PSK signaling as psi2-1 and psi2-1 psi3-1 roots are unresponsive to PSK. PSI3 functions in vegetative plant growth synergistic with PSI2. psi3-1 and particularly psi2-1 psi3-1 rosettes are small. Overexpression of PSI3 promoted plant growth indicating that PSI3 is limiting at the vegetative stage. Severe dwarfism of psi2-1 psi3-1 plants results from reduced cell growth and proliferation and premature leaf growth arrest. Plants further display reduced fertility and premature senescence revealing a crucial function of PSI proteins in vegetative growth and reproduction. Psi single and double knock-out plants have less and PSI3ox plants have more starch compared to wt and growth retardation is partially rescued by sucrose. Our studies reveal a crucial function of the nuclear-localized PSI proteins in growth possibly through metabolic control.

  12. Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation

    NARCIS (Netherlands)

    de Kort, B.J.

    2012-01-01

    Proteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive

  13. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  14. Properties and antioxidant activity of fish skin gelatin film incorporated with citrus essential oils.

    Science.gov (United States)

    Tongnuanchan, Phakawat; Benjakul, Soottawat; Prodpran, Thummanoon

    2012-10-01

    Properties of protein-based film from fish skin gelatin incorporated with different citrus essential oils, including bergamot, kaffir lime, lemon and lime (50% based on protein) in the presence of 20% and 30% glycerol were investigated. Films containing 20% glycerol had higher tensile strength (TS) but lower elongation at break (EAB), compared with those prepared with 30% glycerol, regardless of essential oils incorporated (pfish skin. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Acceptance and storage of fresh cheese made with essential oils

    Directory of Open Access Journals (Sweden)

    Joelmir Grassi Presente

    2017-08-01

    Full Text Available The aim of this work was to evaluate the acceptance and conservation of Minas fresh cheese with essential oils added of oregano and ginger in its formulation. The quality of the milk used as raw material was evaluated for pH, acidity, alizarol, total solids, density, and total microbial load. The cheeses produced were characterized as pH, acidity, moisture, lipids, proteins and ashes. The cheeses were also evaluated by sensorial affective tests using hedonic and attitude scales, in order to determine the acceptance and purchase intention by judges. The count of total aerobic mesophilic microorganisms was used to estimate the shelf-life of cheeses. The milk used as raw material is presented within the quality standards required by legislation. The cheeses made with essential oils showed pH and acidity around 6.9 and 0.87%, respectively, 57.6% moisture, 31.3% lipids, 11.4% protein and 0.9% ash. The cheese added essential oil of oregano and the control cheese were those given by the judges the best values for acceptance (7.5 and 7.6, respectively and purchase intention (4.2 and 4.4 respectively. Regarding the estimated shelf-life, the cheeses added essential oil of oregano and ginger had lower overall microbial load values compared to the control (no oil and mixed (two oils addition, presented counts values with up 106 UFC/g only from the 28th day of storage.

  16. Redefining the essential trafficking pathway for outer membrane lipoproteins.

    Science.gov (United States)

    Grabowicz, Marcin; Silhavy, Thomas J

    2017-05-02

    The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

  17. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  18. MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

    Science.gov (United States)

    Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology.

  19. Learning Neuroimaging. 100 essential cases

    International Nuclear Information System (INIS)

    Asis Bravo-Rodriguez, Francisco de; Diaz-Aguilera, Rocio; Hygino da Cruz, Luiz Celso

    2012-01-01

    Neuroradiology is the branch of radiology that comprises both imaging and invasive procedures related to the brain, spine and spinal cord, head, neck, organs of special sense (eyes, ears, nose), cranial and spinal nerves, and cranial, cervical, and spinal vessels. Special training and skills are required to enable the neuroradiologist to function as an expert diagnostic and therapeutic consultant and practitioner. In addition to knowledge of imaging findings, the neuroradiologist is required to learn the fundamentals of structural and functional neuroanatomy, neuropathology, and neuropathophysiology as well as the clinical manifestations of diseases of the brain, spine and spinal cord, head, neck, and organs of special sense. This book is intended as an introduction to neuroradiology and aims to provide the reader with a comprehensive overview of this highly specialized radiological subspecialty. One hundred illustrated cases from clinical practice are presented in a standard way. Each case is supported by representative images and is divided into three parts: a brief summary of the patient's medical history, a discussion of the disease, and a description of the most characteristic imaging features of the disorder. The focus is not only on common neuroradiological entities such as stroke and acute head trauma but also on less frequent disorders that the practitioner should recognize. Learning Neuroimaging: 100 Essential Cases is an ideal resource for neuroradiology and radiology residents, neurology residents, neurosurgery residents, nurses, radiology technicians, and medical students. (orig.)

  20. Learning Neuroimaging. 100 essential cases

    Energy Technology Data Exchange (ETDEWEB)

    Asis Bravo-Rodriguez, Francisco de [Reina Sofia University Hospital, Cordoba (Spain). Diagnostic and Therapeutics Neuroradiology; Diaz-Aguilera, Rocio [Alto Guadalquivir Hospital, Andujar, Jaen (Spain). Dept. of Radiology; Hygino da Cruz, Luiz Celso [Universidade Federal do Rio de Janeiro (Brazil). CDPI and IRM Ressonancia Magnetica

    2012-07-01

    Neuroradiology is the branch of radiology that comprises both imaging and invasive procedures related to the brain, spine and spinal cord, head, neck, organs of special sense (eyes, ears, nose), cranial and spinal nerves, and cranial, cervical, and spinal vessels. Special training and skills are required to enable the neuroradiologist to function as an expert diagnostic and therapeutic consultant and practitioner. In addition to knowledge of imaging findings, the neuroradiologist is required to learn the fundamentals of structural and functional neuroanatomy, neuropathology, and neuropathophysiology as well as the clinical manifestations of diseases of the brain, spine and spinal cord, head, neck, and organs of special sense. This book is intended as an introduction to neuroradiology and aims to provide the reader with a comprehensive overview of this highly specialized radiological subspecialty. One hundred illustrated cases from clinical practice are presented in a standard way. Each case is supported by representative images and is divided into three parts: a brief summary of the patient's medical history, a discussion of the disease, and a description of the most characteristic imaging features of the disorder. The focus is not only on common neuroradiological entities such as stroke and acute head trauma but also on less frequent disorders that the practitioner should recognize. Learning Neuroimaging: 100 Essential Cases is an ideal resource for neuroradiology and radiology residents, neurology residents, neurosurgery residents, nurses, radiology technicians, and medical students. (orig.)