Academic literature on the topic 'Non-structural proteins'
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Journal articles on the topic "Non-structural proteins"
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Zeidler, Julianna, Lorena Fernandes-Siqueira, Glauce Barbosa, and Andrea Da Poian. "Non-Canonical Roles of Dengue Virus Non-Structural Proteins." Viruses 9, no. 3 (March 13, 2017): 42. http://dx.doi.org/10.3390/v9030042.
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Petric, M., R. H. Yolken, E. J. Dubovi, M. Wiskerchen, and M. S. Collett. "Baculovirus expression of pestivirus non-structural proteins." Journal of General Virology 73, no. 7 (July 1, 1992): 1867–71. http://dx.doi.org/10.1099/0022-1317-73-7-1867.
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Hu, Liya, Sue E. Crawford, Joseph M. Hyser, Mary K. Estes, and BV Venkataram Prasad. "Rotavirus non-structural proteins: structure and function." Current Opinion in Virology 2, no. 4 (August 2012): 380–88. http://dx.doi.org/10.1016/j.coviro.2012.06.003.
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Tam, Donald, Ana C. Lorenzo-Leal, Luis Ricardo Hernández, and Horacio Bach. "Targeting SARS-CoV-2 Non-Structural Proteins." International Journal of Molecular Sciences 24, no. 16 (August 20, 2023): 13002. http://dx.doi.org/10.3390/ijms241613002.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These NSPs are found within open reading frame 1a (ORF1a) and open reading frame 1ab (ORF1ab) of the SARS-CoV-2 genome and encode NSP1 to NSP11 and NSP12 to NSP16, respectively. This study aimed to collect the available literature regarding NSP inhibitors. In addition, we searched the natural product database looking for similar structures. The results showed that similar structures could be tested as potential inhibitors of the NSPs.
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Rohaim, Mohammed A., Rania F. El Naggar, Emily Clayton, and Muhammad Munir. "Structural and functional insights into non-structural proteins of coronaviruses." Microbial Pathogenesis 150 (January 2021): 104641. http://dx.doi.org/10.1016/j.micpath.2020.104641.
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Pressman, E. K. "Heterocomplexes of tick-borne encephalitis structural and non-structural proteins." FEBS Letters 333, no. 3 (November 1, 1993): 268–70. http://dx.doi.org/10.1016/0014-5793(93)80667-j.
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Essen, Lars-Oliver. "Structural genomics of “non-standard” proteins: a chance for membrane proteins?" Gene Function & Disease 3, no. 12 (October 2002): 39–48. http://dx.doi.org/10.1002/1438-826x(200210)3:1/2<39::aid-gnfd39>3.0.co;2-6.
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Kaščáková, Barbora, Zdenek Franta, Zdeno Gardian, Ivana Kutá Smatanová, and Roman Tůma. "Molecular biology and structural study of avian orthoreovirus non-structural proteins." Acta Crystallographica Section A Foundations and Advances 77, a2 (August 14, 2021): C853. http://dx.doi.org/10.1107/s0108767321088450.
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Joachimiak, Andrzej, Changsoo Chang, Michael Endres, Robert Jedrzejczak, Youngchang Kim, Natalia Maltseva, Karolina Michalska, et al. "Crystallography of SARS-CoV-2 non-structural proteins." Acta Crystallographica Section A Foundations and Advances 76, a1 (August 2, 2020): a217. http://dx.doi.org/10.1107/s0108767320097858.
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Yan, Jing, Jianlin Cheng, Lukasz Kurgan, and Vladimir N. Uversky. "Structural and functional analysis of “non-smelly” proteins." Cellular and Molecular Life Sciences 77, no. 12 (September 5, 2019): 2423–40. http://dx.doi.org/10.1007/s00018-019-03292-1.
Full textDissertations / Theses on the topic "Non-structural proteins"
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Zwart, Lizahn. "Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
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Shaw, Andrew Edward. "The Role of Non-Structural Proteins in Bluetongue Virus Replication." Thesis, University College London (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519459.
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Xiao, Yibei [Verfasser]. "Structural and functional studies on coronavirus non-structural proteins 7/8 and 5 / Yibei Xiao." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2014. http://d-nb.info/1050905830/34.
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Horscroft, Nigel John. "Orbivirus non-structural protein NS2 : its role in virus replication." Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:9b550db6-dd9d-4127-941f-93eab2b6e038.
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Young, Natalie Jane. "Bovine Viral Diarrhoea Virus Subunit Vaccines : Contribution of Non-Structural Proteins." Thesis, Royal Veterinary College (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498686.
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Rainsford, Edward. "Functional studies on the rotavirus non-structural proteins NSP5 and NSP6." Thesis, University of Warwick, 2005. http://wrap.warwick.ac.uk/53876/.
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The rotavirus replication cycle has not been fully characterised, one vital stage of virus replication involves large cytoplasmic occlusion bodies termed viroplasms. These are the sites of synthesis and replication of dsRNA, packaging of viral RNA into newly synthesized cores and the formation of double-shelled previrions. The detailed mechanism by which these events occur is poorly understood but is thought to be mediated by the non-structural proteins localised to these structures. Rotavirus gene segment 11 expresses two proteins NSP5 and NSP6 which are found in alternate open reading frames. NSP5 exists is several isoforms which differ on their level of phosphorylation. It has been shown to be essential for virus replication and localises to the viroplasms. The smaller NSP6 protein is the most uncharacterised of all of the rotavirus proteins. It has however been shown to interact with NSP5 and has been tentatively suggested to be localised to the viroplasms. To further investigate these two proteins the pET expression system was utilised to obtain purified protein which was subsequently used to generate mono specific polyclonal antisera. Studies into the function and localisation of these proteins found that both localised to the viroplasms and their relative distributions within these structures were defined. NSP6 was found to be expressed at a low level throughout the course of a rotavirus infection and in contrast to other non-structural proteins, to have a high rate of turnover. The RNA binding ability of both NSP5 and NSP6 was investigated using quantitative filter binding assays and these showed both have sequence independent nucleic acid binding ability. Studies were also conducted into the mechanism of NSP6 expression from the second open reading frame of gene 11, the results obtained being consistent with a leaky scanning mechanism of expression.
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Chung, Keun-Taik. "Interactions and functions of rotavirus non-structural proteins NSP1 and NSP3." Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414314.
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Ponnusamy, Rajesh [Verfasser]. "Crystallographic and biochemical investigations on coronarvirus replication proteins - non-structural proteins 8 and 9 / Rajesh Ponnusamy." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2010. http://d-nb.info/1004773706/34.
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Tai, Hung, and 戴雄. "The role of the non-structural protein, NS1, in influenza virus replication." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660303.
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Motari, Edwin Mwamba. "Structural Studies of Oligosaccharides Attached to Proteins Expressed in Different Organisms and PEGylation of a non-Glycosylated Protein." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1274803162.
Full textBooks on the topic "Non-structural proteins"
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Agency, International Atomic Energy, and Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture., eds. The use of non-structural proteins of foot and mouth disease virus (FMDV) to differentiate between vaccinated and infected animals. Vienna: International Atomic Energy Agency, 2007.
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Thompson, Catherine Isabelle. Protein interaction studies on the rotavirus non-structural protein NSP1. [s.l.]: typescript, 1999.
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Evans, Johanna. Characterisation of the NS1 and the NS2 non-structural protein genes of human respiratory syncytial virus (HRSV). [s.l.]: typescript, 1994.
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Garces, Robert G. Structural analysis of three proteins affecting global transcription levels: Using X-ray crystallography to elucidate functions of non-enzymatic proteins. 2006.
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Harrison, Pauline. Metalloproteins, Part 2: Metal Proteins With Non-Redox Roles (Topics in Molecular and Structural Biology). Verlag Chemie, 1985.
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Meurig Thomas, John. Architects of Structural Biology. Oxford University Press, 2020. http://dx.doi.org/10.1093/oso/9780198854500.001.0001.
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Designed for the non-specialist, the explanations and illustrations used here describe the work, personalities, collaborations, and idiosyncrasies of four of the most distinguished Nobel Laureates of the twentieth century. They exploited a discovery made over a century ago about the nature of X-rays, and thereby created a new branch of science. This enabled them to elucidate, in atomic detail, the structure and mode of action of molecules of the living world: enzymes, vitamins, and viruses, as well as antibiotics. Perutz and Kendrew, from their pioneering work using X-ray diffraction on haemoglobin and myoglobin, the proteins that transport and store oxygen in all animals, led them to establish in 1962 one of the most successful research centres ever—the Laboratory of Molecular Biology (LMB) in Cambridge. Medicines discovered there are used worldwide to treat leukaemia, arthritis, and other diseases. Their work also led to the creation in the United States of the Protein Data Bank that guides scientists in understanding the misfolding of proteins, which cause Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative diseases. This book is first a memoir of these scientists and their contemporaries, many of them friends of the author. Second, it is an insight into the great excitement associated with structural molecular biology, which directly informs our understanding of ourselves. Third, it describes how two renowned research centres in the United Kingdom—the LMB and the Davy-Faraday Research Laboratory—achieved iconic status. It also highlights the importance of the popularization of science, of which Bragg, Perutz, and Kendrew, as well as Dorothy Hodgkin (who solved the structures of penicillin and vitamin B12) were experts.
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Bensimon, David, Vincent Croquette, Jean-François Allemand, Xavier Michalet, and Terence Strick. Single-Molecule Studies of Nucleic Acids and Their Proteins. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198530923.001.0001.
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This book presents a comprehensive overview of the foundations of single-molecule studies, based on manipulation of the molecules and observation of these with fluorescent probes. It first discusses the forces present at the single-molecule scale, the methods to manipulate them, and their pros and cons. It goes on to present an introduction to single-molecule fluorescent studies based on a quantum description of absorption and emission of radiation due to Einstein. Various considerations in the study of single molecules are introduced (including signal to noise, non-radiative decay, triplet states, etc.) and some novel super-resolution methods are sketched. The elastic and dynamic properties of polymers, their relation to experiments on DNA and RNA, and the structural transitions observed in those molecules upon stretching, twisting, and unzipping are presented. The use of these single-molecule approaches for the investigation of DNA–protein interactions is highlighted via the study of DNA and RNA polymerases, helicases, and topoisomerases. Beyond the confirmation of expected mechanisms (e.g., the relaxation of DNA torsion by topoisomerases in quantized steps) and the discovery of unexpected ones (e.g., strand-switching by helicases, DNA scrunching by RNA polymerases, and chiral discrimination by bacterial topoII), these approaches have also fostered novel (third generation) sequencing technologies.
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Meng, X. J. Hepatitis E virus. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198570028.003.0048.
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Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.
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Cartwright, Kent. Shakespeare and the Comedy of Enchantment. Oxford University Press, 2021. http://dx.doi.org/10.1093/oso/9780198868897.001.0001.
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Shakespeare and the Comedy of Enchantment explores the encounter between comedy’s rationalizing dimensions and those extra-rational aspects that elude demystification and exert affective power, an encounter between what is explicable and what is inexplicable. In the context of modernist disenchantment, Shakespeare’s comedies showcase the play of wonder and doubt, leaving behind a sense of residual re-enchantment. The argument thus broadens the perspective of studies that align early modern comedy with developments in science and jurisprudence. As the comic action advances, elements of mystery accrue—uncanny coincidences; magical sympathies; inexplicable repetitions; psychic influences; and wonders, fears, and doubts about the meaning of events—all of whose effects linger after reason has apparently answered the play’s questions, leaving an aura of wonder and wondering. Comic enchantment works through certain devices, tropes, and motifs explored in the chapters: magical clowns who introduce non-realistic stop-time moments that alter the action; structural repetitions that suggest mysteriously converging destinies and opaque but providential outcomes; places with differing characteristics that frame encounters between the regulatory and the protean drives in human existence; desires, thoughts, and utterances that manifest comically monstrous realities, including objects and individuals; characters who return from the dead, facilitated by the desires of the living; play-endings that traffic in harmony and dissonance, yet which can make possible the irrational action of forgiveness. These matters are discussed with extensive reference to Renaissance and modern theories of comedy, and with comparisons to Italian and Tudor comedy.
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Tennant, Neil. Core Logic. Oxford University Press, 2017. http://dx.doi.org/10.1093/oso/9780198777892.001.0001.
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Core Logic has unusual philosophical, proof-theoretic, metalogical, computational, and revision-theoretic virtues. It is an elegant kernel lying deep within Classical Logic, a canon for constructive and relevant deduction furnishing faithful formalizations of informal constructive mathematical proofs. Its classicized extension provides likewise for non-constructive mathematical reasoning. Confining one’s search to core proofs affords automated reasoners great gains in efficiency. All logico-semantical paradoxes involve only core reasoning. Core proofs are in normal form, and relevant in a highly exigent ‘vocabulary-sharing’ sense never attained before. Essential advances on the traditional Gentzenian treatment are that core natural deductions are isomorphic to their corresponding sequent proofs, and make do without the structural rules of Cut and Thinning. This ensures relevance of premises to conclusions of proofs, without loss of logical completeness. Every core proof converts any verifications of its premises into a verification of its conclusion. Core Logic makes one reassess the dogma of ‘unrestricted’ transitivity of deduction, because any core ‘restriction’ of transitivity ensures a more than compensatory payoff of epistemic gain: A core proof of A from X and one of B from {A}∪Y effectively determine a proof of B or of absurdity from some subset of X∪Y. The primitive introduction and elimination rules governing the logical operators in Core Logic are subtly different from Gentzen’s. They are obtained by smoothly extrapolating protean rules for determining truth values of sentences under interpretations. Core rules are inviolable: One needs all of them in order to revise beliefs rationally in light of new evidence.
Book chapters on the topic "Non-structural proteins"
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Heddad, Mounia, and Iwona Adamska. "Non-Structural Putative Chlorophyll-Binding Proteins of Arabidopsis Thaliana." In Photosynthesis: Mechanisms and Effects, 389–92. Dordrecht: Springer Netherlands, 1998. http://dx.doi.org/10.1007/978-94-011-3953-3_92.
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Ponnusamy, Rajesh, Jeroen R. Mesters, John Ziebuhr, Ralf Moll, and Rolf Hilgenfeld. "Non Structural Proteins 8 and 9 of Human Coronavirus 229E." In Advances in Experimental Medicine and Biology, 49–54. Boston, MA: Springer US, 2006. http://dx.doi.org/10.1007/978-0-387-33012-9_7.
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Pathak, Amita, Bhumika Singh, Dheeraj Kumar Chaurasia, and B. Jayaram. "Molecular Simulation–Driven Drug Repurposing for the Identification of Inhibitors Against Non-Structural Proteins of SARS-CoV-2." In Methods in Pharmacology and Toxicology, 683–713. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/7653_2020_61.
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ElHefnawi, Mahmoud M., Aliaa A. Youssif, Atef Z. Ghalwash, and Wessam H. El Behaidy. "An Integrated Methodology for Mining Promiscuous Proteins: A Case Study of an Integrative Bioinformatics Approach for Hepatitis C Virus Non-structural 5a Protein." In Advances in Experimental Medicine and Biology, 299–305. New York, NY: Springer New York, 2010. http://dx.doi.org/10.1007/978-1-4419-5913-3_34.
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Tadros, Monier Habib, and Gerhart Drews. "Pigment-Proteins of Antenna Complexes from Purple Non-Sulfur Bacteria: Localization in the Membrane, Alignments of Primary Structure and Structural Predictions." In Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, 181–92. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4757-0893-6_21.
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Legname, Giuseppe, Gabriele Giachin, and Federico Benetti. "Structural Studies of Prion Proteins and Prions." In Non-fibrillar Amyloidogenic Protein Assemblies - Common Cytotoxins Underlying Degenerative Diseases, 289–317. Dordrecht: Springer Netherlands, 2011. http://dx.doi.org/10.1007/978-94-007-2774-8_9.
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Frydman-Marom, Anat, Yaron Bram, and Ehud Gazit. "Preparation and Structural Characterization of Pre-fibrillar Assemblies of Amyloidogenic Proteins." In Non-fibrillar Amyloidogenic Protein Assemblies - Common Cytotoxins Underlying Degenerative Diseases, 61–102. Dordrecht: Springer Netherlands, 2011. http://dx.doi.org/10.1007/978-94-007-2774-8_3.
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Avbelj, Franc. "Solvation and Electrostatics as Determinants of Local Structural Order in Unfolded Peptides and Proteins." In Protein and Peptide Folding, Misfolding, and Non-Folding, 131–58. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2012. http://dx.doi.org/10.1002/9781118183373.ch5.
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Alcon-LePoder, S., P. Sivard, M. T. Drouet, A. Talarmin, C. Rice, and M. Flamand. "Secretion of Flaviviral Non-Structural Protein NS1: from Diagnosis to Pathogenesis." In Novartis Foundation Symposia, 233–50. Chichester, UK: John Wiley & Sons, Ltd, 2008. http://dx.doi.org/10.1002/0470058005.ch17.
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Kim, Jay W., and Rahul Singh. "Residue Contexts: Non-sequential Protein Structure Alignment Using Structural and Biochemical Features." In Bioinformatics Research and Applications, 77–88. Berlin, Heidelberg: Springer Berlin Heidelberg, 2010. http://dx.doi.org/10.1007/978-3-642-13078-6_10.
Full textConference papers on the topic "Non-structural proteins"
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Ismail, Pam. "Plant protein functionalization: Exploring cold plasma." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/dyhy9832.
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While plant protein is gaining traction, functionality limitations is hindering its market growth. Improving plant protein functionality will enable successful utilization in various food applications, including meat alternatives. There are several reports on plant protein functionality and applications, but much is still not known about the effect of different processing and modifications on the structural and associated functional changes. Cold plasma, a non-thermal processing technique, is being explored as a novel means for protein functionalization. Cold plasma technology involves the exposure of plasma, a partially ionized gas to proteins. The reactive species, generated by cold plasma can induce several chemical reactions including oxidation, bond cleavage, and/or polymerization. This presentation will demonstrate the effect of various cold plasma treatments on pea and other plant protein structural and functional properties. Protein isolates are subjected to several cold plasma treatment conditions. Reactive species and changes due to potential chemical reactions are monitored. Specifically, changes in the protein tertiary, secondary and primary structure will be evaluated, and chemical reactions will be elucidated. The impact of structural change on protein functionality will be highlighted. This research will provide for the first time a controlled evaluation of the impact of cold plasma on protein structural and functional characteristics. Cold plasma treatment may lead to the production of a viable plant protein ingredient with functional properties that are comparable or better than those of traditional protein ingredients.
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Stoicescu, Ramona, Razvan-Alexandru Stoicescu, Codrin Gheorghe, Adina Honcea, and Iulian Bratu. "CONSIDERATIONS ON SARS-COV-2 DIAGNOSIS IN THE LABORATORY OF UNIVERSITY EMERGENCY CLINICAL HOSPITAL OF CONSTANTA." In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/07.
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Coronaviruses are members of the Coronaviridae family. They are enveloped, non-segmented, positive-sense, single-stranded RNA viruses. Their genome size is about 30 kilobases (kb) which consist, at the 5’ end, of non-structural open reading frames (ORFs: ORF1a, ORF 1b) which code for 16 non structural proteins, and at the 3’ end the genes which code for four structural proteins including membrane (M), envelope (E), spike (S), and nucleocapsid (N) proteins. Due to the rapid spread of COVID-19, a reliable detection method is needed for patient diagnosis especially in the early stages of the disease. WHO has recommended nucleic acid amplification tests such as real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay detects three SARS-CoV-2 RNA targets: the envelope (E) gene, the nucleocapsid (N) gene and a region of the open reading frame (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene from SARS-CoV-2 virus isolate Wuhan-Hu-1. Our study was made in the first 3 months of the year 2021 using the real-time RT PCR results obtained in the Cellular Biology ward of the University Emergency Clinical Hospital. In our lab we are testing the inpatients from the hospital wards (Neurology, Pediatrics, Surgery, Internal medicine, ICU, Cardiology, etc.); we are also testing the outpatients from Dialysis and Oncology, 2 days prior to their therapy; we also test the health care personnel. The number of tests we performed was: in January 1456, with 399 positive results (27.4%), 33 deaths; in February 1273 tests, 221 positive (17.36%), 16 deaths; in March 1471 tests, 373 positive (25.36%), 37 deceased.
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Polack, B., A. Duperray, and R. Berthier. "PLATELET AND ENDOTHELIAL CELL CYTOADHESINS ARE BIOSYNTHESIZED AND PROCESSED VIA SIMILAR PATHWAYS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642815.
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The cytoadhesin family represents a group of heterodimeric adhesion receptors with common structural, fonctional and immunochemical properties. Platelet and endothelial cell (EC) GPIIbIII a related proteins exemplify two members of this family. In the present study the biosynthesis and processing of EC GPIIbIII a were examined and compared with that of platelet GPIIbIIIa to verify whether the diversity of these cytoadhesins was related to post translational events.Endothelial cells of human umbilical cord vein origin were metabolicaly labeled with 35S-methionine. The newly synthesized proteins were analyzed and immunoprecipitated with polyclonal antibodies against the purified platelet GPIIbllla complex and the isolated GPIIb and GPIIIa. Under non-reducing conditions three bands were detected at 135 kD, 125 kD and 90kD with the anti GPIIbIIIa. Samples obtained from pulse chase experiments and analysed under non reducing conditions indicated that the 135 kD band derived from the 125 kD band. Under reducing conditions the 135 kD generated two bands at 118 kD and 25 kD. The 125 KD band also gave a 118 kD band and the 90 kD band shifted to 100 kD. These results indicated that the mature form of 135 kD is composed of two polypeptidic chains which derive from a common single chain precursor similar to that observed in the megakaryocyte. The mature protein and the precursor molecule were not recognised by anti GPIIb antibodies. Also immunoprecipitated by polyclonal anti GPIIIa antibodies was a single chain protein of 100kD. In addition, endoglycosidase treatement showed that both EC GPIIIa and platelet GPIIIa were glycosylated protein of the high mannose type.These results indicate that although structural differences exists between platelet and endothelial cell GPIIbllla, the two membrane glycoproteins have a similar cellular transit.
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Agard, David A., Yashushi Hiraoka, and John W. Sedat. "Three-dimensional Optical Microscopy of Biological Specimens." In Signal Recovery and Synthesis. Washington, D.C.: Optica Publishing Group, 1986. http://dx.doi.org/10.1364/srs.1986.thd1.
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The ability to analyze biological specimens in three dimensions represents one of the major achievements of modern structural biology. For all but the simplest repeating structures, three-dimensional analysis is a crucial prerequisite for understanding complex biological assemblies. Near atomic resolution analysis of of crystalline proteins, nucleic acids, and viruses by X-ray crystallography approaches the routine. The current frontier focuses on the three dimensional analysis of non-crystalline, non-symmetric biological structures of cellular dimension. Electron microscope tomography and three dimensional optical microscopy are perhaps the most powerful methods for these studies.
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Rawal, Atul, Kristen L. Rhinehardt, and Ram V. Mohan. "Mechanical Properties of Spider Silk for Use As a Biomaterial: Molecular Dynamics Investigations." In ASME 2020 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2020. http://dx.doi.org/10.1115/imece2020-23951.
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Abstract Even though silkworm are the most dominant type of silk fibers used for commercial applications, spider silk has a definitive role in biomedical applications due to its biocompatibility and excellent mechanical properties as biomaterials. In recent years, recombinant production of the silk proteins at a larger scale has found new interest. Spider silk composites with a combination of a variety of other biomaterials have also been used to improve properties such as bio-compatibility, mechanical strength and controlled degradation. [1] A major constituent of spider silk fibers, are spidroin proteins. These are made up of repetitive segments flanked by conserved non-repetitive domains. The fiber proteins consist of a light chain and a heavy chain that are connected via a single disulfide bond. [2] Present paper employed steered molecular dynamics (SMD) as the principal method of investigating the mechanical properties of these nanoscale spider silk protein 3LR2, with a residual count of 134 amino acids. [3]. SMD simulations were performed by pulling on β-chain of the protein in the x-direction, while holding the other fixed. The focus of this paper is to investigate the mechanical properties of the nanoscale spider silk proteins with lengths of about 4.5nm in a folded state, leading to understanding of their feasibility in bio-printing of a composite spider silk biomaterial with a blend of various other biomaterials such as collagen. An in-depth insight into the fraying and tensile deformation and structural properties of the spider silk proteins are of innovative significance for a multitude of biomedical engineering applications. A calculated Gibbs free energy value of 18.59 kCal/mol via umbrella sampling corresponds with a complete separation of a single chain from a spider silk protein in case of fraying. Force needed for complete separation of the chain from the spider silk protein is analyzed, and discussed in this paper. It is found that the protein molecule undergoes a tensile stretch at strain rates of ≅ 11.65. An elastic modulus of 20.136 GPa, calculated via simple SMD simulations by subjecting the silk β-chain to a tensile stretch is also presented.
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Lykotrafitis, George, and He Li. "Two-Component Coarse-Grain Model for Erythrocyte Membrane." In ASME 2011 International Mechanical Engineering Congress and Exposition. ASMEDC, 2011. http://dx.doi.org/10.1115/imece2011-62133.
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Biological membranes are vital components of living cells as they function to maintain the structural integrity of the cells. Red blood cell (RBC) membrane comprises the lipid bilayer and the cytoskeleton network. The lipid bilayer consists of phospholipids, integral membrane proteins, peripheral proteins and cholesterol. It behaves as a 2D fluid. The cytoskeleton is a network of spectrin tetramers linked at the actin junctions. It is connected to the lipid bilayer primarily via Band-3 and ankyrin proteins. In this paper, we introduce a coarse-grained model with high computational efficiency for simulating a variety of dynamic and topological problems involving erythrocyte membranes. Coarse-grained agents are used to represent a cluster of lipid molecules and proteins with a diameter on the order of lipid bilayer thickness and carry both translational and rotational freedom. The membrane cytoskeleton is modeled as a canonical exagonal network of entropic springs that behave as Worm-Like-Chains (WLC). By simultaneously invoking these characteristics, the proposed model facilitates simulations that span large length-scales (∼ μm) and time-scales (∼ ms). The behavior of the model under shearing at different rates is studied. At low strain rates, the resulted shear stress is mainly due to the spectrin network and it shows the characteristic non-linear behavior of entropic networks, while the viscosity of the fluid-like lipid bilayer contributes to the resulting shear stress at higher strain rates. The apparent ease of this model in combining the spectrin network with the lipid bilayer presents a major advantage over conventional continuum methods such as finite element or finite difference methods for cell membranes.
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Lammers, Steven, Tosin Feyintola, Kendall Hunter, Emily Gibson, Tim Lei, Phil Kao, H. Jerry Qi, Craig Lanning, Robin Shandas, and Kurt Stenmark. "Microstructural Changes in Collagen and Elastin and Their Impact on the Mechanics of the Pulmonary Artery in Hypertension." In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-53958.
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In pulmonary arteries (PA), mechanical function is largely driven by the underlying microstructure of the structural proteins collagen and elastin, which reside within the extracellular matrix (ECM) of the arterial tissue. It has long been established that much of the mechanical non-linearity associated with arterial tissue is the result of collagen mechanics. Arterial collagen is arranged within the vascular wall as tortuous fibrils with a bulk fiber orientation of roughly helical configuration. When arterial tissue is deformed, these collagen fibers become straightened in the direction of applied load. At some critical deformation, termed the transition stretch (λTrans), collagen fibers begin to carry load, thus significantly altering material stiffness. This in turn gives rise to the non-linear force-stretch (F-λ) response typical of these tissues, Figure 1. We have recently found that λTrans is significantly reduced in the hypoxia-induced pulmonary hypertensive (PH) rat model. We therefore propose that this model constitutes an ideal system to study the effect of collagen microstructure on the mechanics of arterial tissues in response to PH vascular remodeling. We hypothesize that quantitative characterization of collagen microstructure will predict pulmonary artery (PA) λTrans within this model system. By directly relating collagen microstructural changes to bulk tissue mechanics in response to PH-induced vascular remodeling we can better understand how changes in collagen structure impact pulmonary hemodynamic capacitance, a major component of cardiac load and contributing factor to right heart failure.
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Vermeer, C., BA M. Soute, and MM W. Ulrich. "IN VITRO CARBOXYLATION OF EXOGENOUS PROTEIN SUBSTRATES BY VITAMIN K-DEPENDENT CARBOXYLASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643994.
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In vivo treatment of experimental animals with vitamin K-antagonists induces the accumulation of non-carboxylated coagulation factor precursors in the liver, where they are tightly bound to vitamin K-dependent carboxylase. If hepatic carboxylase is isolated from warfarin-treated animals, it is obtained therefore almost exclusively in the form of an enzyme/substrate complex. If carboxylase is prepared from non-treated animals, on the other hand, the resulting enzyme is predominantly substrate-free. Small substrates like F L E E L or decarboxylated osteocalcinare carboxylated equally well by both types of carboxylase, but protein substrates(Mr > 30 000) are recognized exclusively by substrate-free carboxylase.Initial attempts to purify carboxylasewere performed with livers from warfarin-treated cows as a starting material. Antibodies against the normal blood coagulation factors crossreact with the hepatic precursor proteins so that the enzyme/substrate complexes could be specifically extracted from detergent-solubilized microsomes by the substrate/antibody interaction. This procedure resulted ina substantial purification of carboxylase, but because its endogenous substrate remained firmly bound, even after it had been carboxylated in vitro, the enzyme system was not suitable for the carboxylation of protein substrates.Therefore a second strategy was developed by which substrate-free carboxylase (from normal livers) was partly purified by sequential extraction of the microsomal membranes with detergents, followed by ammonium sulfate precipitation and size exclusion chromatography.This procedure resulted in a soluble carboxylase complex, still consisting of 7 proteins and phosphatidylcholine. Although further dissociation of the complex resulted in a complete loss of activity, it is not sure if all components play a role in the carboxylation reaction. Exogenous substrates which could be carboxylated by substrate-free carboxylase were: the penta-peptide F L E E L, descarboxyprothrombin from bovine plasma, thermally decarboxylated osteocalcin from bovine bone and non-car-boxy lated coagulaton factor precursors which had been produced by recombinant-DNA techniques in various laboratories. The . efficiency of CO^ incorporation was: 1 mole per 100 moles of F L E E L, 1 mole per 240 moles of descarboxy-prothrombin, 1 mole per mole of decarboxylated osteocalcin and 8 moles per mole of a recombinant factor IX precursor. We assume that the high efficiency with which the recombinant coagulation factor precursors were carboxylated is due to the presence of at least part of their leader sequence. The importance of the aminoacid chain preceding the first carboxylatable Glu residue is demonstrated by the fact that descarboxylated osteocalcin of bovine origin is carboxylated with a relatively high efficiency, whereas descarboxylated osteocalcin from monkey bone is not recognized atal.. Yet the only difference between the two substrates is found in their aminoacids 3 and 4, whereas the first carboxylatable Glu occurs at position 17. It seems, therefore, that the aminoacids 1-16 in bovine osteocalcin mimic to some extent part of the leader sequence in the coagulation factor precursors. Chemical or biochemical modification of decarboxylated osteocalcin might reveal which structural features contribute to its recognition by hepatic carboxylase.The optimal conditions for carboxylation include a high concentration of dithiols (e.g. DTT) and under these conditions disulfide bridges are reduced. Obviously this will lead to a complete destruction of the biological activity of various carboxylated products. Therefore we have searched for a more natural reducing system and it was found that the bacterial thioredoxin/thiore-doxin-reductase system in the presence of 40 uM NADFH was able to replace DTT in the reaction mixtures. Since a comparable system also occurs in calf liver it seems not unlikely that this is the physiological counterpart of the dithiols used in vitro.
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Salem, Saeed, and Mohammed J. Zaki. "ITERATIVE NON-SEQUENTIAL PROTEIN STRUCTURAL ALIGNMENT." In Proceedings of the CSB 2008 Conference. PUBLISHED BY IMPERIAL COLLEGE PRESS AND DISTRIBUTED BY WORLD SCIENTIFIC PUBLISHING CO., 2008. http://dx.doi.org/10.1142/9781848162648_0016.
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Maleki, Mina, Michael Hall, and Luis Rueda. "Using structural domains to predict obligate and non-obligate protein-protein interactions." In 2012 IEEE Symposium on Computational Intelligence in Bioinformatics and Computational Biology (CIBCB). IEEE, 2012. http://dx.doi.org/10.1109/cibcb.2012.6217204.
Full textReports on the topic "Non-structural proteins"
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Shomer, Ilan, Louise Wicker, Uzi Merin, and William L. Kerr. Interactions of Cloud Proteins, Pectins and Pectinesterases in Flocculation of Citrus Cloud. United States Department of Agriculture, February 2002. http://dx.doi.org/10.32747/2002.7580669.bard.
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The overall objective was to understand the cloud flocculation of citrus juice by characterization of the interactions between proteins and pectins, and to determine the role of PE isozymes in catalyzing this phenomenon. Specific objectives were to: 1. identify/characterize cloud-proteins in relation to their coagulable properties and affinity to pectins; 2. to determine structural changes of PME and other proteins induced by cation/pectin interactions; 3. localize cloud proteins, PME and bound protein/pectates in unheated and pasteurized juices; 4. to create "sensitized" pectins and determine their effect on clarification. The original objectives were not changed but the methods and approach were modified due to specific research requirements. Two i postulates were: 1. there is a specific interaction of cloud proteins with de-esterified regions of ! pectin and this contributes to cloud loss; 2. isozymes of pectin-methyl-esterase (PME) vary in efficiency to create sensitized pectins. The appearance of citrus fruit juice is an important quality factor and is determined by the color and turbidity that .are conferred by the suspended particles, i.e., by the cloud and its homogeneity. Under some circumstances the cloud tend to flocculate and the juice clarifies. The accepted approach to explain the clarification is based on pectin demethoxylation by PME that promotes formation of Ca-pectate. Therefore, the juice includes immediate heat-inactivation upon ~ squeezing. Protein coagulation also promotes cloud instability of citrus fruit extracts. However, the clarification mechanism is not fully understood. Information accumulated from several laboratories indicates that clarification is a more complex process than can be explained by a single mechanism. The increasing trend to consume natural-fresh juice emphasizing the importance of the knowledge to assure homogeneity of fresh juice. The research included complementary directions: Conditions that induce cloud-instability of natural- juice [IL]. Evaluate purification schemes of protein [USA]. Identifications of proteins, pectin and neutral sugars ([IL]; Structure of the cloud components using light and electron microscopy and immuno-labeling of PME, high-methoxyl-pectin (HMP) and low-methoxyl-pectin (LMP); Molecular weight of calcium sensitized pectins [US]; Evaluation of the products of PME activity [US]. Fractions and size distribution and cloud components [IL-US]. The optimal pH activity of PME is 7 and the flocculation pH of the cloud is 3-4. Thus, the c roles of PME, proteins and pectins in the cloud instability, were studied in pH ranges of 2- 7. The experiments led to establish firstly repeatable simulate conditions for cloud instability [IL]. Thermostable PME (TS-PE) known to induce cloud instability, but also thermolabile forms of PME (TL-PE) caused clarification, most likely due to the formation and dissolution of inactive :. PE-pectin complexes and displacement of a protective colloid from the cloud surface [US]. Furthermore, elimination of non-PME protein increases TS-PE activity, indicating that non-PME proteins moderate PME activity [US]. Other experiments Concomitantly with the study of the PME activity but promotes the association of cloud-proteins to pectin. Adjusting of the juice pH to f 7 retains the cloud stability and re-adjusting of the pH to 40% DE reacts to immuno-labeling in the cloud fragments, whereas
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Gafny, Ron, A. L. N. Rao, and Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Christopher, David A., and Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, May 2004. http://dx.doi.org/10.32747/2004.7586534.bard.
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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.
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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.
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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.
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Rahimipour, Shai, and David Donovan. Renewable, long-term, antimicrobial surface treatments through dopamine-mediated binding of peptidoglycan hydrolases. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597930.bard.
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There is a need for renewable antimicrobial surface treatments that are semi- permanent, can eradicate both biofilms and planktonic pathogens over long periods of time and that do not select for resistant strains. This proposal describes a dopamine binding technology that is inexpensive, bio-friendly, non-toxic, and uses straight-forward commercially available products. The antimicrobial agents are peptidoglycanhydrolase enzymes that are non-toxic and highly refractory to resistance development. The goal of this project is to create a treatment that will be applicable to a wide variety of surfaces and will convey long-lasting antimicrobial activity. Although the immediate goal is to create staphylolytic surfaces, the technology should be applicable to any pathogen and will thus contribute to no less than 3 BARD priorities: 1) increased animal production by protecting animals from invasive and emerging diseases, 2) Antimicrobial food packaging will improve food safety and security and 3) sustainable bio- energy systems will be supported by coating fermentation vats with antimicrobials that could protect ethanolic fermentations from Lactobacillus contamination that reduces ethanol yields. The dopamine-based modification of surfaces is inspired by the strong adhesion of mussel adhesion proteins to virtually all types of surfaces, including metals, polymers, and inorganic materials. Peptidoglycanhydrolases (PGHs) meet the criteria of a surface bound antimicrobial with their site of action being extracellular peptidoglycan (the structural basis of the bacterial cell wall) that when breached causes osmotic lysis. As a proof of principle, we will develop technology using peptidoglycanhydrolase enzymes that target Staphylococcus aureus, a notoriously contagious and antimicrobial-resistant pathogen. We will test for susceptibility of the coating to a variety of environmental stresses including UV light, abrasive cleaning and dessication. In order to avoid resistance development, we intend to use three unique, synergistic, simultaneous staphylococcal enzyme activities. The hydrolases are modular such that we have created fusion proteins with three lytic activities that are highly refractory to resistance development. It is essential to use multiple simultaneous activities to avoid selecting for antimicrobial resistant strains. This strategy is applicable to both Gram positive and negative pathogens. We anticipate that upon completion of this award the technology will be available for commercialization within the time required to achieve a suitable high volume production scheme for the required enzymes (~1-2 years). We expect the modified surface will remain antimicrobial for several days, and when necessary, the protocol for renewal of the surface will be easily applied in a diverse array of environments, from food processing plants to barnyards.
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Morrison, Mark, Joshuah Miron, Edward A. Bayer, and Raphael Lamed. Molecular Analysis of Cellulosome Organization in Ruminococcus Albus and Fibrobacter Intestinalis for Optimization of Fiber Digestibility in Ruminants. United States Department of Agriculture, March 2004. http://dx.doi.org/10.32747/2004.7586475.bard.
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Improving plant cell wall (fiber) degradation remains one of the highest priority research goals for all ruminant enterprises dependent on forages, hay, silage, or other fibrous byproducts as energy sources, because it governs the provision of energy-yielding nutrients to the host animal. Although the predominant species of microbes responsible for ruminal fiber degradation are culturable, the enzymology and genetics underpinning the process are poorly defined. In that context, there were two broad objectives for this proposal. The first objective was to identify the key cellulosomal components in Ruminococcus albus and to characterize their structural features as well as regulation of their expression, in response to polysaccharides and (or) P AA/PPA. The second objective was to evaluate the similarities in the structure and architecture of cellulosomal components between R. albus and other ruminal and non-ruminal cellulolytic bacteria. The cooperation among the investigators resulted in the identification of two glycoside hydrolases rate-limiting to cellulose degradation by Ruminococcus albus (Cel48A and CeI9B) and our demonstration that these enzymes possess a novel modular architecture specific to this bacterium (Devillard et al. 2004). We have now shown that the novel X-domains in Cel48A and Cel9B represent a new type of carbohydrate binding module, and the enzymes are not part of a ceiluiosome-like complex (CBM37, Xu et al. 2004). Both Cel48A and Cel9B are conditionally expressed in response to P AA/PPA, explaining why cellulose degradation in this bacterium is affected by the availability of these compounds, but additional studies have shown for the first time that neither PAA nor PPA influence xylan degradation by R. albus (Reveneau et al. 2003). Additionally, the R. albus genome sequencing project, led by the PI. Morrison, has supported our identification of many dockerin containing proteins. However, the identification of gene(s) encoding a scaffoldin has been more elusive, and recombinant proteins encoding candidate cohesin modules are now being used in Israel to verify the existence of dockerin-cohesin interactions and cellulosome production by R. albus. The Israeli partners have also conducted virtually all of the studies specific to the second Objective of the proposal. Comparative blotting studies have been conducted using specific antibodies prepare against purified recombinant cohesins and X-domains, derived from cellulosomal scaffoldins of R. flavefaciens 17, a Clostridium thermocellum mutant-preabsorbed antibody preparation, or against CbpC (fimbrial protein) of R. albus 8. The data also suggest that additional cellulolytic bacteria including Fibrobacter succinogenes S85, F. intestinalis DR7 and Butyrivibrio fibrisolvens Dl may also employ cellulosomal modules similar to those of R. flavefaciens 17. Collectively, our work during the grant period has shown that R. albus and other ruminal bacteria employ several novel mechanisms for their adhesion to plant surfaces, and produce both cellulosomal and non-cellulosomal forms of glycoside hydrolases underpinning plant fiber degradation. These improvements in our mechanistic understanding of bacterial adhesion and enzyme regulation now offers the potential to: i) optimize ruminal and hindgut conditions by dietary additives to maximize fiber degradation (e.g. by the addition of select enzymes or PAA/PPA); ii) identify plant-borne influences on adhesion and fiber-degradation, which might be overcome (or improved) by conventional breeding or transgenic plant technologies and; iii) engineer or select microbes with improved adhesion capabilities, cellulosome assembly and fiber degradation. The potential benefits associated with this research proposal are likely to be realized in the medium term (5-10 years).
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Shahak, Yosepha, and Donald R. Ort. Physiological Bases for Impaired Photosynthetic Performance of Chilling-Sensitive Fruit Trees. United States Department of Agriculture, May 2001. http://dx.doi.org/10.32747/2001.7575278.bard.
Full textAbstract:
Chilling-sensitivity is an important agricultural problem in both the U.S. and Israel. Most research attention has focused so far on herbaceous crop plants, even though the problem is also acute in the fruit tree industry. Under BARD funding we made substantial progress in identifying the mechanisms involved in the disruption of photosynthesis following a chill in mango. Our investigation with fruit trees has been substantially accelerated by drawing on our knowledge and experience with herbaceous crops. The four original research objectives, focused or discovering the underlying mechanisms of chill-induced inhibition of photosynthesis in fruit trees, and the main achievements are listed below. [1] Separating stomatal from non-stomatal components of chilling on photosynthesis in fruit trees. We found evidence that the dark chill-induced inhibition of photosynthesis in mango was E combination of both stomatal and mesophyll components. [2] Differentiating photo damage from light-induced photo protection of photosystem II (PSII). Dark chilling exacerbate high light photoinhibition, as a result of primary inhibition in the carbor reduction cycle. Nevertheless, in Israeli orchards we observed chronic photoinhibition of PSII photochemistry in the winter. This photo damage was reversible over a few days if sunlight was attenuated with filters or night temperature rose. Practical implications of this finding deserve further investment. Additional achievement was the development of a new biophysical tool to study macro-structural changes of LHCII particles in intact, attached leaves. [3] Determine the role of oxidative stress in the dark-chilling-induced inhibition, with emphasis on oxygen radical scavenging, lipid peroxidation and redox-controlled carbon-cycle enzymes. We found an increase in lipid peroxidation following a dark chill, and partial protective effects or an antioxidant. However, the photoinhibition observed in mango orchards in Israel during the winter did not appear to be a general oxidative stress. [4] Investigate whether chilling interferes with the diurnal and circadian rhythm of gene expression of key photosynthetic proteins as has been shown for chilling-sensitive crop plants. The results indicated that most of the circadian rhythm in photosynthesis was due to reduced lea: internal CO2 concentrations during the subjective night, as a result of rhythmic stomatal closure Chilling-induced interference with circadian timing in mango, does not play the central role in chilling inhibition of photosynthesis that has previously been demonstrated in certain chilling sensitive herbaceous plants. Practical implications of the research achievements are feasible, but require few more years of research.
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Lurie, Susan, John Labavitch, Ruth Ben-Arie, and Ken Shackel. Woolliness in Peaches and Nectarines. United States Department of Agriculture, 1995. http://dx.doi.org/10.32747/1995.7570557.bard.
Full textAbstract:
The overall goal of the research was to understand the processes involved in the development of woolliness in peaches and nectarines. Four specific hypotheses were proposed and in the course of the research evidence was gathered t support two of them and to not support two others. The hypotheses and a summary of the evidence are outlined below. 1. That woolliness arises from an imbalance between the activities of the cell wall pectin degrading enzymes. Using 'Flavortop' nectarines and 'Hermoza' peaches as model systems, storage regimes were manipulated to induce or prevent woolliness. The expression (mRNA abundance), protein content (Western blotting), and activity of polygalacturonase (PG) and pectin esterase (PE) were followed. Expression of the enzymes was not different, but activity and the ratio between PG and PE activities were quite different in fruits developing woolliness or ripening normally. This was also examined by looking at the substrate, the pectin moiety of the cell wall, and i woolly fruit there were more high molecular weight pectins with regions of non-methylated galacturonic acid residues. Taking an in vitro approach it was found a) that PE activity was stable at 0oC while PG activity decreased; b) incubating the calcium pectate fraction of the cell wall with PE extracted from peaches caused the polymers to form a gel characteristic of the visual woolly symptoms in peaches. 2. That continued cell wall synthesis occurs during storage and contributes to structural changes i cell walls and improper dissolution and softening after storage. We tried to adapt our technique of adding 13C-glucose to fruit discs, which was used successfully to follow cell wall synthesis during tomato ripening. However, the difference in sugar content between the two fruits (4% in tomato and 12% in peach) meant that the 13C-glucose was much more diluted within the general metabolite pool. We were unable to see any cell wall synthesis which meant that either the dilution factor was too great, or that synthesis was not occurring. 3. That controlled atmosphere (CA) prevents woolliness by lowering all enzyme activities. CA was found to greatly reduce mRNA abundance of the cell wall enzymes compared to regular air storage. However, their synthesis and activity recovered during ripening after CA storage and did not after regular air storage. Therefore, CA prevented the inhibition of enzyme activation found in regular air storage. 4. That changes in cell wall turgor and membrane function are important events in the development of woolliness. Using a micro pressure probe, turgor was measured in cells of individual 'O'Henry' and 'CalRed' peaches which were woolly or healthy. The relationship between firmness and turgor was the same in both fruit conditions. These data indicate that the development and expression of woolliness are not associated with differences in membrane function, at least with regard to the factors that determine cell turgor pressure. In addition, during the period of the grant additional areas were explored. Encoglucanase, and enzyme metabolizing hemicellulose, was found to be highly expressed air stored, but not in unstored or CA stored fruit. Activity gels showed higher activity in air stored fruit as well. This is the first indication that other components of the cell wall may be involved in woolliness. The role of ethylene in woolliness development was also investigated at it was found a) that woolly fruits had decreased ability to produce ethylene, b) storing fruits in the presence of ethylene delayed the appearance of woolliness. This latter finding has implication for an inexpensive strategy for storing peaches and nectarines.