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Auswahl der wissenschaftlichen Literatur zum Thema „Non structural protein NS1“
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Zeitschriftenartikel zum Thema "Non structural protein NS1"
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Marc, Daniel. „Influenza virus non-structural protein NS1: interferon antagonism and beyond“. Journal of General Virology 95, Nr. 12 (01.12.2014): 2594–611. http://dx.doi.org/10.1099/vir.0.069542-0.
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Most viruses express one or several proteins that counter the antiviral defences of the host cell. This is the task of non-structural protein NS1 in influenza viruses. Absent in the viral particle, but highly expressed in the infected cell, NS1 dramatically inhibits cellular gene expression and prevents the activation of key players in the IFN system. In addition, NS1 selectively enhances the translation of viral mRNAs and may regulate the synthesis of viral RNAs. Our knowledge of the virus and of NS1 has increased dramatically during the last 15 years. The atomic structure of NS1 has been determined, many cellular partners have been identified and its multiple activities have been studied in depth. This review presents our current knowledge, and attempts to establish relationships between the RNA sequence, the structure of the protein, its ligands, its activities and the pathogenicity of the virus. A better understanding of NS1 could help in elaborating novel antiviral strategies, based on either live vaccines with altered NS1 or on small-compound inhibitors of NS1.
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Beddingfield, Brandon J., Jessica N. Hartnett, Russell B. Wilson, Peter C. Kulakosky, Kristian G. Andersen, Refugio Robles-Sikisaka, Nathan D. Grubaugh et al. „Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay“. Viruses 13, Nr. 9 (05.09.2021): 1771. http://dx.doi.org/10.3390/v13091771.
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Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.
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Sankar, S. Gowri, K. J. Dhanajeyan, R. Paramasivan, V. Thenmozhi, B. K. Tyagi und S. John Vennison. „High-Level Expression of Functionally Active Dengue-2 Non-Structural Antigen 1 Production inEscherichia coli“. BioMed Research International 2013 (2013): 1–6. http://dx.doi.org/10.1155/2013/343195.
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Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein inE. coliby using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing anyin vitroposttranslational modification process.
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Wan, Shu-Wen, Miao-Huei Cheng und Yan-Hua Lee. „Rab27-dependent extracellular vesicle releasing participates in dengue virus non-structural protein 1 secretion“. Journal of Immunology 210, Nr. 1_Supplement (01.05.2023): 236.04. http://dx.doi.org/10.4049/jimmunol.210.supp.236.04.
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Abstract Dengue disease is one of important arthropod-borne disease in the world and causes around 20,000 deaths annually. Dengue virus (DENV) non-structural protein 1 (NS1) is the major secreted non-structural protein from infected cells and highly associated with the pathogenesis of severe dengue. Several studies have shown that the secreted NS1 contribute to increase of vascular permeability, coagulopathy, activation of immune cells as well as inflammation. These findings strongly indicate the pathological roles of secreted NS1; however, the secretory pathway is not fully clear. Accumulating evidence have shown that virus-associated extracellular vesicles (EVs) not only contain viral components including viral proteins, genetic material or virion particle but also host components. Rab27 is one of several GTPases essential to regulate EV releasing by affecting docking of multivesicular body (MVB) to the target plasma membrane. Here, we show that NS1 can be exported from DENV-infected cells by EVs. NS1 as well as EV biogenesis-associated proteins ALG-2-interacting protein X (ALIX) and neutral sphingomyelinase2 (nSMase2) co-accumulate in extracellular vesicles. We next determine the topology of NS1 and demonstrate that NS1 is associated at the surface of EVs. In addition, the secretion of DENV NS1 is regulated by Rab27. The EV biogenesis and secretion inhibitor, Tipifarnib treatment decreases the levels of NS1 and nSMase 2 but not the levels of ALIX in the EVs. Taken together, this study not only provide the new insights of mechanisms in DENV NS1 secretion through Rab27-mediated vesicle trafficking but also valuable therapeutic targets to dengue disease. Supported by grants from Ministry of Science and Technology of Taiwan (MOST 111-2320-B-006 -051 -MY3)
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Susilowati Andajani. „Structural Non-Structural Protein-1 Virus Dengue of Risk Factors on Functional Interference Hepar“. Medico Legal Update 20, Nr. 2 (22.05.2020): 796–800. http://dx.doi.org/10.37506/mlu.v20i2.1213.
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The causes of hepatic damage to DHF are mixed. Dengue virus can infect the liver and cause damage.Histopathology of the liver in cases of fatal dengue shows that hepatocytes and Kupffer cells can be thetarget of viral replication and there is involvement of apoptotic mechanism. This study aims to determinethe association between dengue virus NS1 protein and degree of clinical manifestation of dengue infectionwith hepatic dysfunction. Blood samples after taking the serum, were examined for dengue virus NS1protein and SGOT and SGPT examinations at the Surabaya Health Laboratory Center. Data analysis usedChi Square, Fisher's Exact, Anava and Dunnet (a = 0.05). The results shows that the correlated variablesignificantly with dengue hemorrhagic manifestation degree was dengue virus NS protein1 (p = 0.047),with correlation strength of 26.8%. Variables significantly correlated with liver dysfunction were degreeof clinical manifestation of dengue fever (p = 0.037) with correlation strength of 33.5% and dengue virusNS protein1 (p = 0.023) with correlation strength of 29.3%. There was a significant relationship betweenthe presence of dengue virus NS1 protein and the incidence of DHF. There was a significant relationshipbetween the presence of dengue virus NS1 protein with hepatic dysfunction (SGOT and SGPT) in denguevirus infection.
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Warner, Nikole L., Susan B. Core und Kathryn M. Frietze. „Unbiased Identification of Dengue Virus Non-Structural Protein 1 Peptides for Use in Vaccine Design“. Vaccines 10, Nr. 12 (27.11.2022): 2028. http://dx.doi.org/10.3390/vaccines10122028.
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Dengue virus (DENV) is a global health problem, with over half of the world’s population at risk for infection. Despite this, there is only one licensed vaccine available to prevent infection and safety concerns limit immunization to only a subset of individuals. Most dengue virus vaccine efforts attempt to evoke broadly neutralizing antibodies against structural proteins. However, eliciting antibodies to block the activity of viral proteins involved in pathogenesis could be a useful complementary approach. Studies suggest that non-structural protein 1, which participates in disruption of the endothelial barrier and is hypothesized to play a significant role in the progression to severe dengue, could be a promising target for vaccine efforts. Here, we used an unbiased approach to identify peptide epitopes of dengue virus non-structural protein 1 that could evoke antibodies that bind to NS1 from all 4 serotypes and also bind to DENV-infected cells. DENV-2 NS1 peptides were generated such that 35 overlapping 15 amino acid peptides represented the entire NS1 protein. These peptides were each chemically conjugated to bacteriophage virus-like particles (VLP) and used to immunize mice. Sera were then screened for IgG to cognate peptide as well as binding to recombinant hexameric NS1 from all four DENV serotypes as well as binding to DENV-2 infected cells by microscopy. From these data, we identified several peptides that were able to elicit antibodies that could bind to infected cells as well as DENV NS1. These peptides and their homologues in the corresponding NS1 of other DENV serotypes could be used as potential immunogens to elicit binding antibodies to NS1. Future studies will investigate the functional and protective capacities of antibodies elicited by these immunogens against DENV NS1.
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Cunha, Andreia E. S., Rui J. S. Loureiro, Carlos J. V. Simões und Rui M. M. Brito. „Unveiling New Druggable Pockets in Influenza Non-Structural Protein 1: NS1–Host Interactions as Antiviral Targets for Flu“. International Journal of Molecular Sciences 24, Nr. 3 (03.02.2023): 2977. http://dx.doi.org/10.3390/ijms24032977.
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Influenza viruses are responsible for significant morbidity and mortality worldwide in winter seasonal outbreaks and in flu pandemics. Influenza viruses have a high rate of evolution, requiring annual vaccine updates and severely diminishing the effectiveness of the available antivirals. Identifying novel viral targets and developing new effective antivirals is an urgent need. One of the most promising new targets for influenza antiviral therapy is non-structural protein 1 (NS1), a highly conserved protein exclusively expressed in virus-infected cells that mediates essential functions in virus replication and pathogenesis. Interaction of NS1 with the host proteins PI3K and TRIM25 is paramount for NS1’s role in infection and pathogenesis by promoting viral replication through the inhibition of apoptosis and suppressing interferon production, respectively. We, therefore, conducted an analysis of the druggability of this viral protein by performing molecular dynamics simulations on full-length NS1 coupled with ligand pocket detection. We identified several druggable pockets that are partially conserved throughout most of the simulation time. Moreover, we found out that some of these druggable pockets co-localize with the most stable binding regions of the protein–protein interaction (PPI) sites of NS1 with PI3K and TRIM25, which suggests that these NS1 druggable pockets are promising new targets for antiviral development.
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Rahim, Md, Ludger Klewes, Ali Zahedi-Amiri, Sabine Mai und Kevin Coombs. „Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation“. Viruses 10, Nr. 12 (19.12.2018): 731. http://dx.doi.org/10.3390/v10120731.
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Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.
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Dubrow, Alyssa, Sirong Lin, Nowlan Savage, Qingliang Shen und Jae-Hyun Cho. „Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK“. Viruses 12, Nr. 3 (20.03.2020): 338. http://dx.doi.org/10.3390/v12030338.
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The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85β subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85β, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85β. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85β in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.
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Asyura, Muhammad Mikail Athif Zhafir, Ahmad Fauzi und Fakhru Adlan Ayub. „Potential of Peptide-Based Non-Structural Protein 1 (NS1) Inhibitor in Obstructing Dengue Virus (DENV) Replication“. Green Medical Journal 3, Nr. 1 (29.04.2021): 1–12. http://dx.doi.org/10.33096/gmj.v3i1.71.
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Introduction: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue
Methods: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue
Conclusion: The review established promising results of using peptide-based intervention on NS1. Further in vivo and randomized controlled trials are advised to solidify the applicability and biosafety of the intervention
Dissertationen zum Thema "Non structural protein NS1"
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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
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Tai, Hung, und 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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Evans, Johanna. „Characterisation of the NS1 and the NS2 non-structural protein genes of human respiratory syncytial virus (HRSV)“. Thesis, University of Warwick, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283482.
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Thompson, Catherine Isabelle. „Protein interaction studies on the rotavirus non-structural protein NSP1“. Thesis, University of Warwick, 1999. http://wrap.warwick.ac.uk/80266/.
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Rotavirus encodes six structural and six non-structural proteins. In contrast to the structural proteins, the functional roles of the non-structural proteins are not well defined beyond a realisation that they must have a role in the viral replication cycle. A fuller understanding of the replication cycle must therefore rest on determining the specific roles played by the non-structural proteins. Non-structural protein NSP1 shows high levels of sequence divergence. A generally well conserved cysteine-rich region at the amino-terminus may form a zinc finger structure. It has been shown to possess non-specific RNA-binding activity, and has been found associated with the smallest of three replication intermediates (RIs) found in infected cells, together with the viral proteins VP1, VP3 and NSP3. VP2 and VP6 are added sequentially to the pre-core RI to form the core RI and single-shelled RI respectively. The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined. The intermolecular interactions that occur between the components of the RIs have not been defined. Protein-protein interactions between NSP1 and VP1, VP2, VP3, and NSP3, from the UKtc strain of bovine rotavirus, were investigated using a variety of approaches, the first of which was the yeast two-hybrid system. In this assay a self-interaction of NSP1 was not detected. Protein-protein interactions between NSPl and VPl, VP2, VP3, and NSP3, were also not detected. Both the full-length protein and a truncated NSPl, consisting of only the amino terminal third of the protein, were tested. A direct self-interaction of NSP3 was shown and quantified. Radio-immunoprecipitation analysis of in vitro translated viral proteins using specific anti-NSP1 serum was also employed. However, it failed to detect direct protein-protein interactions between NSP1 and VPI, VP2, and VP3. Immunoprecipitation of UKtc rotavirus-infected celllysates with anti-NSP1 serum showed the co-precipitation of viral proteins VPl, VP2, VP3NP4, VP6 and NSP3, with NSP1. It was proposed that NSP1 formed a previously unrecognised complex with these proteins. Immunoprecipitation of nuclease-treated infected cell lysates showed a reduction in the co-precipitation of VP2, VP3NP4 and NSP3 with NSP1. No reduction in the co-precipitation of VP6 was seen. The association of the complex proteins may be mediated by RNA binding. Immunoprecipitation with an anti-VP6 monoclonal antibody reciprocally precipitated small amounts of NSP1, VP2, VP3/VP4, and NSP3, with VP6.
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Whittington, Christi Leigh. „Molecular Dynamics of the RNA Binding Cavity of Influenza A Non-structural Protein 1 (NS1) RNA Binding Domain“. Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4256.
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Molecular dynamics simulations were performed on the influenza A non-structural protein 1 (NS1) RNA binding domain (RBD), a homodimer. Fourteen simulations were performed at 298K, nine ionized with 0.1M KCl and five with no ions. Several analysis techniques were employed to study RBD residue flexibility. The focus of the study was the RNA binding cavity formed by side chains of helix 2 (chain A) and helix 2’ (chain B) and cavity intermonomeric salt bridges. Opening of the salt bridges D29–R46’ and D29’–R46 was observed in several of the trajectories. The RNA binding cavity has large flexibility, where the dimension and shape change during the dynamics. One pair of residues surrounding the cavity and necessary for RNA binding, residues R38 and R38’, have motions during the simulations which cover the top of the cavity. There is correlation between the salt bridge breaking, flexibility of R38 and R38’, and the cavity size and shape changes. Possible RBD small molecule drug targets are these two salt bridges and the pair R38 and R38’. Disrupting the events that occur around these areas could possibly inactivate RNA binding function of the domain. These results could have implications in searching for potential molecules that effectively treat influenza A.
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Monastyrskaya, Katherine Valerie. „Characterisation and modification of non-structural protein NS1 of BTV-10 in relation to virus-specified tubule formation“. Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282517.
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To, Thuan. „PDZ Binding Motif of NS1 Proteins of Influenza A Viruses: : A Virulent Factor in the Expression of Interferon-β?“ Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-177337.
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Background: The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective: The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods: The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.
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Lymperopoulos, Konstantinos. „Functional characterisation of the bluetongue virus non-structural protein NS2-protein and RNA-protein interactions“. Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424733.
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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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Lacheiner, Karen. „Tubules composed of non-structural protein NS1 of african horsesickness virus as a system for the immune display of foreign peptides“. Pretoria : [s.n.], 2006. http://upetd.up.ac.za/thesis/available/etd-07092008-103908.
Der volle Inhalt der QuelleBücher zum Thema "Non structural protein NS1"
1
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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2
Thompson, Catherine Isabelle. Protein interaction studies on the rotavirus non-structural protein NSP1. [s.l.]: typescript, 1999.
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3
Vaheri, Antti, James N. Mills, Christina F. Spiropoulou und Brian Hjelle. Hantaviruses. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198570028.003.0035.
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Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent- and insectivore-borne zoonotic viruses. Several hantaviruses are human pathogens, some with 10-35% mortality, and cause two diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Hantaviruses are enveloped and have a three-segmented, single-stranded, negative-sense RNA genome. The L gene encodes an RNA-dependent RNA polymerase, the M gene encodes two glycoproteins (Gn and Gc), and the S gene encodes a nucleocapsid protein. In addition, the S genes of some hantaviruses have an NSs open reading frame that can act as an interferon antagonist. Similarities between phylogenies have suggested ancient codivergence of the viruses and their hosts to many authors, but increasing evidence for frequent, recent host switching and local adaptation has led to questioning of this model. Infected rodents establish persistent infections with little or no effect on the host. Humans are infected from aerosols of rodent excreta, direct contact of broken skin or mucous membranes with infectious virus, or rodent bite. One hantavirus, Andes virus, is unique in that it is known to be transmitted from person-to-person. HFRS and HCPS, although primarily affecting kidneys and lungs, respectively, share a number of clinical features, such as capillary leakage, TNF-, and thrombocytopenia; notably, hemorrhages and alterations in renal function also occur in HCPS and cardiac and pulmonary involvement are not rare in HFRS. Of the four structural proteins, both in humoral and cellular immunity, the nucleocapsid protein appears to be the principal immunogen. Cytotoxic T-lymphocyte responses are seen in both HFRS and HCPS and may be important for both protective immunity and pathogenesis. Diagnosis is mainly based on detection of IgM antibodies although viral RNA (vRNA) may be readily, although not invariably, detected in blood, urine and saliva. For sero/genotyping neutralization tests/RNA sequencing are required. Formalin-inactivated vaccines have been widely used in China and Korea but not outside Asia. Hantaviruses are prime examples of emerging and re-emerging infections and, given the limited number of rodents and insectivores thus far studied, it is likely that many new hantaviruses will be detected in the near future.
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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 und 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.
Buchteile zum Thema "Non structural protein NS1"
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Alcon-LePoder, S., P. Sivard, M. T. Drouet, A. Talarmin, C. Rice und 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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Castillo, Juan Manuel, Adán Hernández-Acosta, César Pacheco, Edgar Quezada-Ruiz, Fernando Rodríguez-León, Raymundo Cruz und Juan Ernesto Ludert. „Exploring the Protein-Protein Interactions, Traffic, and Functions of the Dengue Virus Non-structural 1 (NS1) Protein in the Vector Mosquito“. In The Latin American Studies Book Series, 255–71. Cham: Springer Nature Switzerland, 2024. http://dx.doi.org/10.1007/978-3-031-68419-7_12.
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Boireau, P., M. F. Madelaine, D. Saulnier, J. Laporte und J. F. Vautherot. „Identification, Expression in E. coli and Insect Cells of the Non-Structural Protein NS2 Encoded by mRNA2 of Bovine Coronavirus (BCV)“. In Coronaviruses, 69–74. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4615-2996-5_11.
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Legname, Giuseppe, Gabriele Giachin und 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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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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Vitale, Rosa Maria, Giuseppina Andreotti, Pietro Amodeo und Andrea Motta. „Structural Elements Regulating Interactions in the Early Stages of Fibrillogenesis: A Human Calcitonin Model System“. In Protein and Peptide Folding, Misfolding, and Non-Folding, 351–88. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2012. http://dx.doi.org/10.1002/9781118183373.ch12.
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Frydman-Marom, Anat, Yaron Bram und 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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Kim, Jay W., und 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.
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Schwarz, Birgit, Edward Routledge und Stuart Siddell. „Characterization of the MHV-JHM Non-Structural Protein Encoded by mRNA 2“. In Advances in Experimental Medicine and Biology, 317–24. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4684-5823-7_43.
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Jans, David A., und Alexander J. Martin. „Nucleocytoplasmic Trafficking of Dengue Non-structural Protein 5 as a Target for Antivirals“. In Advances in Experimental Medicine and Biology, 199–213. Singapore: Springer Singapore, 2018. http://dx.doi.org/10.1007/978-981-10-8727-1_15.
Der volle Inhalt der QuelleKonferenzberichte zum Thema "Non structural protein NS1"
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Nazarenko, E. A., und E. S. Starodubova. „CELL-SYNTHESIZED NON-STRUCTURAL PROTEIN 1 OF TICK-BORNE ENCEPHALITIS VIRUS IS ASSOCIATED WITH EXOSOMES AND INCREASE THEIR SECRETION“. In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-257.
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Recent studies propose the importance of extracellular vesicles (EVs) in the pathogenesis of tick-borne encephalitis virus (TBEV), but the involvement of individual viral proteins on them has not yet been studied. In this work, it was shown that the expression of TBEV nonstructural protein 1 (NS1) in cells increases the secretion of EVs. It has been established that NS1 is found in the fraction of exosomes obtained by precipitation, as well as by chromatography of cell culture media.
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Sun, Bin, Pengying Yue, Jingjing Li, Hu Han, Jinfeng Ouyang, Yingyue Cai, Yongbo Yang und Yi Li. „Notice of Retraction: Expression and Purification of Non-Structural Protein NS1 of Human Bocavirus and Generation of Its Antibody“. In 2011 5th International Conference on Bioinformatics and Biomedical Engineering. IEEE, 2011. http://dx.doi.org/10.1109/icbbe.2011.5781326.
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Ramu, Shyrar Tanussiya, Dinuka Ariyaratne, Chandima Jeewandara, Deshni Jayathilaka, Laksiri Gomes, Ananda Wijewickrama, Graham Ogg und Gathsaurie Neelika Malavige. „Identifying an Immunodominant Epitope of Dengue Non-Structural Protein 1 (NS1) in Acute Secondary Dengue Infections for Vaccine Development or Development of Treatment Modalities“. In 2020 From Innovation to Impact (FITI). IEEE, 2020. http://dx.doi.org/10.1109/fiti52050.2020.9424907.
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Alipanah, Morteza, John A. Lednicky, J. Glenn Morris und Z. Hugh Fan. „A Point-Of-Care Device Integrating Sample Preparation With Isothermal Amplification for Detection of Mayaro Virus“. In ASME 2023 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2023. http://dx.doi.org/10.1115/imece2023-114292.
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Abstract Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed the first reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of MAYV. We designed six LAMP primers targeting MAYV’s non-structural protein (NS1) gene and determined the visual limit of detection of at least 10 viral genome equivalents (GEs) per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. A 30 min. RT-LAMP assay was integrated with valve-enabled sample preparation device wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting. Colorimetric detection was achieved by naked eye or a smartphone. The functions of our platform were demonstrated using purified RNA and cultured viruses for their potential usage. We have used the device for detection of cultured MAYV in 50 min.
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Ascone, I., G. Nobili, M. Benfatto und A. Congiu-Castellano. „XAS Characterization of the Zn Site of Non-structural Protein 3 (NS3) from Hepatitis C Virus“. In X-RAY ABSORPTION FINE STRUCTURE - XAFS13: 13th International Conference. AIP, 2007. http://dx.doi.org/10.1063/1.2644511.
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Salem, Saeed, und 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 und 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.
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Makino, Kyosuke, Kazuhiko Misawa, Terumasa Ito und Hironori Ito. „Non-thermal protein structural change by THz pulsed light irradiation“. In Terahertz, RF, Millimeter, and Submillimeter-Wave Technology and Applications XVII, herausgegeben von Laurence P. Sadwick und Tianxin Yang. SPIE, 2024. http://dx.doi.org/10.1117/12.3007091.
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Ismail, N. F., Khuan Y. Lee, L. N. Ismail, A. F. Abdul Rahim, N. S. Mohamad Hadis und A. R. M. Radzol. „Structural, Electrical and Raman Characterization of AgNP-coated Porous Silicon SERS Substrate for Detection of Dengue NS1 Protein“. In 2022 IEEE-EMBS Conference on Biomedical Engineering and Sciences (IECBES). IEEE, 2022. http://dx.doi.org/10.1109/iecbes54088.2022.10079329.
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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.
Berichte der Organisationen zum Thema "Non structural protein NS1"
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Gafny, Ron, A. L. N. Rao und 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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Elbaum, Michael, und Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, März 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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Shomer, Ilan, Louise Wicker, Uzi Merin und William L. Kerr. Interactions of Cloud Proteins, Pectins and Pectinesterases in Flocculation of Citrus Cloud. United States Department of Agriculture, Februar 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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Christopher, David A., und Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, Mai 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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Morrison, Mark, Joshuah Miron, Edward A. Bayer und 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, März 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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Tzfira, Tzvi, Michael Elbaum und Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, Dezember 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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Lurie, Susan, John Labavitch, Ruth Ben-Arie und Ken Shackel. Woolliness in Peaches and Nectarines. United States Department of Agriculture, 1995. http://dx.doi.org/10.32747/1995.7570557.bard.
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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.